Cancer Biol Med 2021. doi: 10.20892/j.issn.2095-3941.2020.0209

ORIGINAL ARTICLE

LncRNA HIF1A-AS2 accelerates malignant phenotypes of renal carcinoma by modulating miR-30a-5p/SOX4 axis as a ceRNA

Mingwei Chen1, Xuedong Wei1, Xiu Shi2, Le Lu1, Guangbo Zhang1,3, Yuhua Huang1, Jianquan Hou1 1Department of Urology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China;2 Department of Obstetrics and Gynecology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China; 3Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Jiangsu Key Laboratory of Clinical Immunology, Soochow University, Jiangsu Key Laboratory of Gastrointestinal Tumor Immunology, Suzhou 215006, China

ABSTRACT Objective: Several reports have proposed that lncRNAs, as potential biomarkers, participate in the progression and growth of malignant tumors. HIF1A-AS2 is a novel lncRNA and potential biomarker, involved in the genesis and development of carcinomas. However, the molecular mechanism of HIF1A-AS2 in renal carcinoma is unclear. Methods: The relative expression levels of HIF1A-AS2 and miR-30a-5p were detected using RT-qPCR in renal carcinoma tissues and cell lines. Using loss-of-function and overexpression, the biological effects of HIF1A-AS2 and miR-30a-5p in kidney carcinoma progression were characterized. Dual luciferase reporter analysis and Western blot were used to detect the potential mechanism of HIF1A-AS2 in renal carcinomas. Results: HIF1A-AS2 was upregulated in kidney carcinoma tissues when compared with para-carcinoma tissues (P < 0.05). In addition, tumor size, tumor node mestastasis stage and differentiation were identified as being closely associated with HIF1A-AS2 expression (P < 0.05). Knockdown or overexpression of HIF1A-AS2 either restrained or promoted the malignant phenotype and WNT/β-catenin signaling in renal carcinoma cells (P < 0.05). MiR-30a-5p was downregulated in renal cancers and partially reversed HIF1A-AS2 functions in malignant renal tumor cells. HIF1A-AS2 acted as a microRNA sponge that actively regulated the relative expression of SOX4 in sponging miR-30a-5p and subsequently increased the malignant phenotypes of renal carcinomas. HIF1A-AS2 showed a carcinogenic effect and miR-30a-5p acted as an antagonist of the anti- effects in the pathogenesis of renal carcinomas. Conclusions: The HIF1A-AS2-miR-30a-5p-SOX4 axis was associated with the malignant progression and development of renal carcinoma. The relative expression of HIF1A-AS2 was negatively correlated with the expression of miR-30a-5p, and was closely correlated with SOX4 mRNA levels in renal cancers. KEYWORDS HIF1A-AS2; SOX4; miR-30a-5p; kidney carcinoma; ceRNA

3 Introduction with distant . The treatment of renal malignancies therefore represents an ongoing challenge. Various studies have reported possible mechanisms and causes of kidney car- Malignant tumors greatly affect human health1,2. Renal cell cinomas4-11; however, the precise mechanism of RCC remains carcinoma (RCC) accounts for 2%–3% of adult malignant unclear. tumors, and the prognoses of patients with advanced kid- The lncRNA is a novel kind of noncoding RNA, which is at ney carcinomas is very poor, especially for those individuals least 200 nucleotides in length. The lncRNAs may participate in the progression of various diseases, especially malignant 12-17 Correspondence to: Jianquan Hou tumors . The lncRNAs, as important tumor gene regulators, E-mail: [email protected] effect tumor biological behavior, transcriptional regulation, ORCID: https://orcid.org/0000-0002-0961-2973 and post-transcriptional regulation18-22. Evidence suggests that Received: May 4, 2020; accepted: August 21, 2020 lncRNAs play vital roles in malignant carcinomas, and have Available at www.cancerbiomed.org ©2021 Cancer Biology & Medicine. Creative Commons recently been reported to contribute to migration, prolifera- Attribution-NonCommercial 4.0 International License tion, apoptosis, metastasis, and other biological processes23-28. 588 Chen et al. HIF1A-AS2 accelerates RCC via sponging miR-30a-5p

The lncRNA, HIF1A-AS2, has been shown to be involved to subsequently increase the expression of SOX4 at a post- in the progression of various malignancies29-41. For example, transcriptional level. Furthermore, HIF1A-AS2 functioned HIF1A-AS2 exacerbates the growth of gastric cancer29, while it in a ceRNA-dependent manner to sponge miR-30a-5p to accelerates triple-negative breast carcinoma cell invasion and tightly regulate SOX4 expression. HIF1A-AS2 also acted as a proliferation, and has been shown to enhance paclitaxel resist- significant tumor regulator and potential therapeutic target. ance30-33. Abnormal expression of HIF1A-AS2 has also been The HIF1A-AS2-miR-30a-5p-SOX4 axis was involved in the reported to be involved in the progression of bladder can- progression of renal carcinomas, which highlights its possible cer34,35. The expression of HIF1A-AS2 in mesenchymal glio- application in clinical diagnosis and therapy. blastoma is related to hypoxia and sponging to miR-153-3p, which mediates HIF-1α expression and accelerates angiogen- Materials and methods esis from hypoxia in human umbilical vein endothelial cells (HUVECs)36. HIF1A-AS2 sponges miR-129-5p to regulate Patient samples DNMT3A expression and the epithelial-mesenchymal tran- sition (EMT) in colorectal cancer37. The HIF1A-AS2/miR- Our study included kidney carcinoma patients who received 665/IL6 system also regulates osteogenic differentiation38. tumorectomy. We quick-froze the kidney carcinoma tissues HIF1A-AS2 regulates HIF-1A expression through transcrip- and paired normal peritumoral specimens in liquid nitrogen tional competition involving adenosine deaminase acting on after resection. We received written informed consent from RNA39. Downregulated HIF1A-AS2 also affects the pathogen- each patient. Our experimental protocol was approved by esis and progression of preeclampsia40. HIF1A-AS2 sponges the Institutional Ethics Review Board of the First Affiliated miR-33b-5p to regulate SIRT6 in osteosarcomas41. Our study Hospital of Soochow University (Approval No. 2019110). was therefore directed toward investigating the potential role of HIF1A-AS2 in the progression of carcinomas, as well as Cell lines and cell culture assessing its contribution to the progression and development of renal carcinoma. Similar to most malignancies, there are The cells were cultured in an incubator at 37 °C and 5% some therapeutic challenges related to renal cancers, which CO2. The 786-O, ACHN, OS-RC-2, and 293T cell lines were have existed for a long period of time. obtained from the Institute of Cell Biology, Chinese Academy The (miRNAs) are 18–25 nucleotide non-cod- of Sciences, Shanghai, China. A total of 1% antibiotics (100 U/ ing RNAs, which can regulate the tumorigenesis and progres- mL penicillin and 100 μg/mL streptomycin sulfate) and 10% sion of many tumors42-49. The miRNAs also play a role in the fetal bovine serum (FBS) were added into Minimal Essential evolution of species, and miR-30a-5p has been reported to Medium (MEM), Dulbecco’s Modified Eagle Medium play a role in many diseases, including cholangiocarcinoma42; (DMEM), and RPMI 1640. The 786-O, OS-RC-2, ACHN, and it can also act as a negative regulator, contributing to the evo- 293T cells were cultivated in RPMI 1640, MEM, or DMEM. lution of renal carcinomas. Extensive studies have reported that lncRNAs function as Cell transfection miRNAs sponges50-56. We first reported a mutual correlation between HIF1A-AS2 and miR-30a-5p as miRNA sponges in Specific siRNA oligonucleotides were transiently trans- renal carcinomas. These findings increased our knowledge of fected into renal cancer cells using si-HIF1A-AS2 sense the expression patterns of lncRNA-miRNA sponges. (5’-GAGUUGGAGGUGUUGAAGCAAAUAU-3’) and anti- In the present study, HIF1A-AS2 was shown to be highly sense (5’-AUAUUUGCUUCAACACCUCCAACUC-3’). expressed in renal cancer tissues and cells, with miR-30a-5p Si-NC and si-RNA (si-HIF1A-AS2) were purchased from Gene being present only in small amounts. HIF1A-AS2 expression Pharma (Suzhou, China). SOX4-specific siRNAs (si-SOX4, was closely correlated with differentiation across tumor node sc-38412) were purchased from Santa Cruz Biotechnology metastasis (TNM) stages. HIF1A-AS2 was found to facili- (Santa Cruz, CA, USA) and Gene Pharma (Suzhou, China). tate renal cancer progression, while miR-30a-5p suppressed The renal carcinoma cells were cultivated in 6-well plates and this process. Mechanistically, the upregulated expression of transfected using Lipofectamine 3000 Transfection Reagent HIF1A-AS2 may inhibit the relative expression of miR-30a-5p, according to the manufacturer’s instructions (Thermo Fisher Cancer Biol Med Vol 18, No 2 May 2021 589

Scientific, Waltham, MA, USA). The plasmid vectors (pcD- tip was then used to generate clean lines in 6-well plates. Cell NA3.1-HIF1A-AS2, and the negative control) were purchased images were then captured using a digital camera. After 24 h, from Gene Pharma. The cells were cultured for at least 24 h the images of cells were again captured using a digital camera. before transfection, and the transfected cells with the corre- sponding vector were collected after 48 h of transfection. Flow cytometry assay

RT-PCR SiRNAs or plasmid vectors were respectively transfected in kidney carcinoma cells. After 48 h of transfection, cells were Total RNA was extracted from the specimens and renal cells collected and resuspended in fixation fluid, which included 5 using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) based µL annexin V-FITC, 10 µL propidium iodide, and 195 µL cell on the product description. The cDNA was synthesized from suspension. Flow cytometry (Beckman Coulter, San Jose, CA, whole RNA using the Prime Script RT Reagent Kit with USA) was used to detect cell apoptosis. gDNA Eraser (Takara, Dalian, China). SYBR Premix Ex Taq II (Takara) was used to detect the relative expression levels of Western blot analysis HIF1A-AS2 using RT-qPCR and the CFX96 sequence detec- tion system (Bio-Rad, Hercules, CA, USA). Supplementary Total was separated by 10% SDS–PAGE and transferred Table S1 shows the main primer sequences. The endogenous to polyvinylidene difluoride membranes. After blocking in 5% controls were glyceraldehyde 3-phosphate dehydrogenase nonfat milk, the membranes were incubated overnight for (GAPDH) and U6 small nuclear RNA. A relative quantifica- 16 h in 4 °C with the primary antibody. The membranes were tion method (2-ΔΔCt) was used to calculate the expressions, then incubated for 1–2 h with a secondary antibody, and an which were normalized to endogenous controls. enhanced chemiluminescence ECL kit (Beyotime, Shanghai, China) was used to visualize the bands. β-Actin, tubulin, or Cell proliferation assays GAPDH were used as internal standards. The antibodies used are listed in Supplementary Table S2. Cell proliferation was detected using the CCK-8 assay (Beyotime, Shanghai) based on the product description. Luciferase reporter assays SiRNAs or plasmids were used to transfect cells, which had been incubated for 24 h in 96-well plates. A microplate reader TCF (T cell factor) transcription factor activity was used to (Bio-Rad) was used to measure the absorbance in each well at measure canonical Wnt signaling pathway activity. TOP or FOP 0, 24, 48 and 72 h after transfection. flash and Renilla-luciferase plasmids were used to transfect renal cells. The luciferase activity was analyzed using a DLR assay sys- The 5-ethyl-2′-deoxyuridine (EdU) tem (Promega, Madison, WI, USA). PmirGLO Dual-luciferase incorporation assay vectors (Fubio, Shanghai, China) were used to clone the binding and mutant sequences. HIF1A-AS2 or SOX4 wild type (WT) An EdU Apollo DNA in vitro kit (Ribobio, Guangzhou, China) or mutant type (MUT) was constructed and co-transfected used the EdU incorporation assay based on the product along with miR-30a-5p mimics or normal control (NC), then descriptions. Briefly, cells transfected with siRNA or plasmid transfected with Lipofectamine 3000 and incubated for 48 h. A were incubated for 2 h at 37 °C, then treated with 100 μL of microplate reader was used to measure the luciferase activities. 50 μM EdU per well, followed by fluorescence microscopy to visualize the cells. Animal experiments

Cell migration assay The 5-week-old male BALB/c nude mice were divided into 2 groups, with each group comprised of 6 mice. LV-NC and The cells were transferred to 6-well plates, and were cultured LV-HIF1A-AS2 were made by Gene Pharma (Shanghai). A in an incubator until 90%–100% confluent, followed by siRNA total of 2 × 106 OS-RC-2 cells were injected into the mouse or plasma transfection of the cells. A 200 μL sterilized pipet dorsal flank regions, and tumor growth was measured every 590 Chen et al. HIF1A-AS2 accelerates RCC via sponging miR-30a-5p

5 days. The formula, a × b2/2 (a: long diameter; b: short dia- P < 0.01), ACHN cells (approximately 5.184-fold, P < 0.01), meter) was used to calculate tumor volume. Finally, mice were and OS-RC-2 cells (approximately 1.998-fold, P < 0.001) sacrificed after 30 days, and each subcutaneous tumor was (Figure 1E–1G). The relative expressions of miR-30a-5p were weighed. The animal experimental protocol was approved by decreased in renal carcinoma cells, with 786-O approximately the Ethics Committee of Soochow University (Approval No. 62.79% (P < 0.01), OS-RC-2 approximately 78.83% (P < 0.01), ECSU-20190002018). and ACHN approximately 55.70% (P < 0.01) (Figure 1H). High expression of HIF1A-AS2 was closely associated with RNA fluorescence in situ hybridization (FISH) tumor size (P < 0.05), differentiation (P < 0.01), and TNM stages (P < 0.01) (Table 1). However, there was no correlation For FISH analyses, renal cells were immobilized in 4% for- between HIF1A-AS2 and sex, age, and lymph node metastasis. maldehyde, treated with pepsin, and dehydrated with ethanol. Follow-up animal studies indicated that HIF1A-AS2 promoted The 786-O, ACHN, OS-RC-2, and 293T cells were incubated tumorigenicity in vivo (Figure 7A–7F). These results showed in hybridization buffer with FISH probes using HIF1A-AS2 that HIF1A-AS2 acted as a tumor enhancer, and that miR- (Robbio, Guangzhou, China) for 24 h. After hybridization, 30a-5p acted as an anti-oncogene in the kidney carcinomas. the slides were rinsed and dehydrated. The slides were then mounted using Prolong® Gold Antifade Reagent (Thermo LncRNAHIF1A-AS2 was mainly distributed in Fisher Scientific) using 4′,6-diamidino-2-phenylindole for the cytoplasm detection, followed by confocal laser scanning microscopy (Zeiss, Jena, Germany). The cellular localizations of lncRNAs were used to assess pos- sible functions and then revealed their potential mechanisms Statistical analysis of action, including chromatin remodeling and translational regulation. Using the fractionation indicators of 18S RNA Every experimental assay was conducted at least in triplicate. and U6, FISH was used to show that HIF1A-AS2 was mainly The data are presented as the mean ± standard deviation (SD). ­localized to the cytoplasm of kidney cell lines [Figure 1I (a–d)]. Statistical analyses were conducted using SPSS statistical soft- ware for Windows, version 20.0 (SPSS, Chicago, IL, USA). The Knockdown of HIF1A-AS2 suppressed the relative expression analyses of HIF1A-AS2 were conducted cell proliferation of kidney cell lines, and the using the paired sample t-test. Analysis of variance was used upregulation of HIF1A-AS2 promoted cell to analyze the CCK-8 assay data. Other data analyses used the proliferation of renal cell lines independent samples t-test. P < 0.05 was regarded as statisti- cally significant. The relative expression of HIF1A-AS2 was detected by qRT- PCR at 48 h after transfection of siRNAs in 786-O, ACHN, Results and OS-RC-2 cells, and after transfection of pcDNA3.1-HI- F1A-AS2 into 293T cells. The relative expression levels of Upregulated expression of HIF1A-AS2 and HIF1A-AS2 were downregulated by 83.04% in 786-O (P downregulated expression of miR-30a-5p in < 0.001), downregulated by 60.30% in ACHN (P < 0.01), kidney carcinomas and decreased by 80.17% in OS-RC-2 cells (P < 0.001). The relative expression levels of HIF1A-AS2 were significantly Compared to para-carcinoma specimens, the relative expres- decreased by si-HIF1A-AS2 after 48 h of transfection (Figure sion of HIF1A-AS2 was increased in 69.88% (58 of 83) car- 2A), and the relative expression levels of HIF1A-AS2 were cinoma specimens (P < 0.001) by approximately 3.472-fold prominently increased by 14.43-fold in 293T cells (P < (Figure 1A and 1B), with the relative expression of miR-30a-5p 0.001) at 48 h after transfection of pcDNA3.1-HIF1A-AS2 decreasing in 71.08% (59 of 83) of carcinoma samples (P < (Figure 2B). 0.01) (Figure 1C and 1D). Compared to 293T cells, the rel- CCK-8 assays detected whether si-HIF1A-AS2 restrained ative expression of HIF1A-AS2 was significantly upregulated cell proliferation in 786-O, ACHN and OS-RC-2 cell lines and in kidney carcinoma cells, 786-O (approximately 4.200-fold, whether pcDNA3.1-HIF1A-AS2 promoted the proliferation Cancer Biol Med Vol 18, No 2 May 2021 591

A E 8 High expression 6 I 293T OSRC-2 Low expression ** Cy3 DAPI Merge Cy3 DAPI Merge 6 5 a c 4 4 2 3 U6 0 5101520 25 30 35 40 45 50 55 60 65 70 75 80 85 2 –2 level (mean ∆∆ CT ) 1 –4 Relative HIF1A-AS2 expression Relative HIF1A-AS2 expression 0 –6 786-O 293T 8 F 8

B 18S 7 ** ** 6 6 5 4 4 3

2 2 1 Probe Relative HIF1A-AS2 expression Relative HIF1A-AS2 expression 0 0 ACHN 293T HIF 1A-AS2 Tumor Normal

3.5 High expression 8 C 3.0 Low expression G 786-O ACHN 2.5 7 ** b d

2 2.0 1.5 6 1.0 5 0.5 0.0 4 51015202530354045505560 65 70 75 80 85 –0.5 3 U6 –1.0 –1.5 2 Relative HIF1A-AS –2.0 –2.5 1

expression level (mean ∆∆ CT ) –3.0

Relative HIF1A-AS2 expression 0 –3.5 OSRC-2 293T

1.4 D 2.0 18 S H ** ** 1.2 ** 1.5 1.0 0.8 ** 1.0 0.6 0.4 0.5 0.2 Probe 0.0 Relative miR-30a-5p expression Relative miR-30a-5p expression 0.0 786-O 293T OS-RC-2 ACHN 293T HIF 1A-AS2 Tumor Normal

Figure 1 The relative expressions of HIF1A-AS2 and miR-30a-5p in renal carcinoma patients’ samples and cells. Samples were detected, and the distribution of HIF1A-AS2 was mainly in the cytoplasm. GAPDH was the internal control gene. The relative expression patterns of HIF1A-AS2 (A and B) and miR-30a-5p (C and D) are shown in paired kidney carcinoma tissues and normal tissues. We also compared their expression levels in renal carcinoma cells (786-O, ACHN, and OS-RC-2 cells) and 293T cells (F-H). The distribution of HIF1A-AS2 was analyzed by fluorescence in situ hybridization in 786-O, ACHN and OS-RC-2, and 293T cells [I (a-d)]. The18S RNA and U6 indicated cytoplasm and nucleus, respectively. (*P < 0.05; **P < 0.01). in 293T renal cells. The data showed that knockdown of Together, the results showed that downregulation of HIF1A-AS2 [Figure 2C (a–c)] significantly restrained cell HIF1A-AS2 decreased cell proliferation in renal cell lines, and proliferation in kidney carcinoma cells (P < 0.01 in renal cell upregulation of HIF1A-AS2 facilitated cell proliferation in lines). PcDNA3.1-HIF1A-AS2 [Figure 2C (d)] in 293T cells renal cell lines. remarkably accelerated cell proliferation (P < 0.01). Cell proliferation was also detected using EdU assays. As Downregulation of HIF1A-AS2 decreased shown [Figure 2D (a–d)], compared to the control group, cell migration of kidney cell lines, and EdU positive 786-O, ACHN, and OS-RC-2 cells in the overexpression of HIF1A-AS2 promoted knockdown of HIF1A-AS2 groups were decreased, while the kidney cell line migration ­pcDNA3.1-HIF1A-AS2 groups showed the reverse results. The EdU assay showed that the EdU positive cell quanti- Cells were transfected with siRNA and plasmids in 6-well ties in the si-HIF1A-AS2 group were decreased by 44.15% in plates, and cell scratch assays were used to detect the roles 786-O cells (P < 0.05) [Figure 2D (a)], reduced by 64.41% of siRNA and plasmids in cell migration. The scratch assay in ACHN cells (P < 0.001) [Figure 2D (b)], and reduced by showed that the ratio of the relative migration of the si-HI- 66.81% in OS-RC-2 cells (P < 0.001) [Figure 2D (c)]. The Edu F1A-AS2 group was decreased by 48.38% in 786-O cells (P positive cell quantities were increased by 4.110-fold in 293T < 0.01) [Figure 2E (a)], 43.57% in ACHN cells (P < 0.01) cells (P < 0.05) [Figure 2D (d)] in the pcDNA3.1-HIF1A-AS2 [Figure 2E (b)], and 54.80% in OS-RC-2 cells (P < 0.01) group. [Figure 2E (c)]. The ratio of the relative migration of the 592 Chen et al. HIF1A-AS2 accelerates RCC via sponging miR-30a-5p

Table 1 Correlation between HIF1A-AS2 expression and clinicopathological characteristics of renal cell cancer patients (clear cell renal cell carcinomas) Characteristics Total Expression of HIF1A-AS2 P Higher, n = 58 Lower, n = 25 Gender 0.326

Male 51 38 (74.5%) 13 (25.5%)

Female 32 20 (62.5%) 12 (37.5%)

Age (years) 0.456

≤ 50 28 18 (64.3%) 10 (35.7%)

> 50 55 40 (72.7%) 15 (27.3%)

Tumor size (cm) 0.027*

≤ 7 31 17 (54.8%) 14 (45.2%)

> 7 52 41 (78.8%) 11 (21.2%)

Differentiation 0.007**

Moderate/poor 49 40 (81.6%) 9 (18.4%)

Well 34 18 (52.9%) 16 (47.1%)

TNM stage 0.007**

T0-1 31 16 (51.6%) 15 (48.4%)

T2-4 52 42 (80.8%) 10 (19.2%)

Lymph node metastasis 0.548

N0 67 48 (71.6%) 19 (28.4%)

N1 or above 16 10 (62.50%) 6 (37.5%)

*P < 0.05; **P < 0.01. Tumor node metastasis according to staging TNM of the American Joint Committee on Cancer in 2010.

pcDNA3.1-HIF1A-AS2 group was upregulated 2.118-fold 786-O cells (P < 0.001), 3.053-fold in ACHN cells (P < 0.01), in 293T cells (P < 0.01) [Figure 2E (d)]. The results demon- and 3.502-fold in OS-RC-2 cells (P < 0.001) [Figure 2F (a–c)] strated that downregulation of HIF1A-AS2 decreased renal after transfection with HIF1A-AS2 knockdown. cell migration, while upregulation of HIF1A-AS2 promoted Compared to the negative control groups, the ratios of renal cell migration. apoptosis were downregulated by 66.22% in 293T cells (P < 0.001) [Figure 2F (d)] after transfection with pcDNA3.1-HI- Silencing HIF1A-AS2 enhanced renal cell F1A-AS2. Overall, knockdown of HIF1A-AS2 accelerated renal apoptosis, and upregulation of HIF1A-AS2 cell apoptosis, and upregulation of HIF1A-AS2 suppressed suppressed renal cell apoptosis renal cell apoptosis.

Knockdown or overexpression of HIF1A-AS2 may regulate Upregulated miR-30a-5p inhibited renal renal cell apoptosis after transfection of plasmids and siRNA. carcinoma cell proliferation and migration, Flow cytometry was used to detect cell apoptosis. The apop- and promoted renal carcinoma cell apoptosis totic renal cells were observably upregulated after transfection with si-HIF1A-AS2. Compared to the si-NC groups, the ratios The relative expression levels of miR-30a-5p were signifi- of apoptosis were significantly upregulated by 3.859-fold in cantly reduced by 54.28% in 786-O cells (P < 0.01), 72.66% Cancer Biol Med Vol 18, No 2 May 2021 593

20 pcDNA3.1-HIF1A-AS2 Negative control si-NC si-HIF1A-AS2 AB EFT = 0 h 786-O T = 24 h si-NC si-HIF1A-AS2 6 6 2.0 ession 10 Q1 Q2 10 Q1 Q2 15 ** a si-HIF1A-AS2 450 si-HIF1A-AS2 si-NC a 0.030% 0.113% 0.026% 0.316% **

) 120 si-NC 5 400 1.6 10 5 2 10 ) 350 10 si-NC 100 300 1.2 104 4 PI 250 80 PI 10 ssion 786- O 200 5 60 ** 150 0.8 103 3

10 Apoptosis (% exp re ** 100 lative HIF1A-AS2 expr lative HIF1A-AS 40 Q3 50

Re Q4 Re 0.4 Q4 Q3 ** si-HIF1A- 0 ** 0 97.2% 2.63% 0 0 lative cell migration (% 20 86.9% 12.7% 786-O 293T AS2 Re 3 4 5 6 0.0 0 0 10 10 10 10 0 103 104 105 106 786-O ACHINOS-RC-2 786-O Annexin V-FITC Annexin V-FITC si-HIF1A-AS2 si-NC 3.0 786-O 6 C T = 0 h ACHN T = 24 h b 106 Q1 Q2 10 Q1 Q2 si-HIF1A-AS2 si-NC 786-O D Merge si-NC 400 2.5 Hoechst EdU si-NC b 3.38% 5.84% a si-HIF1A-AS2 si-HIF1A-AS2 0.737% 0.162% **

) 120 350 a 1.2 105 5 2.0 10 ) EdU 1.0 300

of 100 1.5 si-NC 250 OD si-NC 0.8 104 4 PI 10 * 80 HN 200 1.0 0.6 PI

** AC 150 0.4 60 0.5 positive cell 3 3 Apoptosis (% lative fold 100 0.2 10 10 si-HIF1A-AS2 Re 40 50 0.0 0.0 si-HIF1A- Q4 Q3 Q4 Q3 02448 72 786-O 0 0 0 Time (h) AS2 lative cell migration (% 20 92.1% 3.83% 84.0% 9.98% ACHN 2.0 si-HIF1A-AS2 Re b ACHN si-NC 0 0 103 104 105 106 0 103 104 105 106 si-NC ACHN b 1.2 si-HIF1A-AS2 ACHN 1.6 Annexin V-FITC Annexin V-FITC 1.0 EdU

1.2 of 0.8 T = 0 h OS-RC-2 T = 24 h si-NC si-NC si-NC 6 Q1 Q2 6 Q1

OD 10 Q2 si-HIF1A-AS2 c c 10 400 0.8 0.6 si-HIF1A-AS2 0.045% 0.196% 0.132% 3.30% ** ) 120 5 350 0.4 ** 105 10 ) 0.4 positive cell 100 300 lative fold 0.2 si-NC

si-HIF1A-AS2 Re 250 0.0 0.0 80 C- 2 104 104 PI 0 24 48 72 ACHN PI 200

Time (h) OS-R 60 ** 150 3 103 c 2.5 si-HIF1A-AS2 OS-RC-2 si-NC 10 Apoptosis (% 100 si-NC 40 OS-RC-2 c 1.2 si-HIF1A-AS2 si-HIF1A- 50 2.0 Q4 Q3 Q4 Q3 AS2 lative cell migration (% 0 l 20 0 0 EdU 1.0 9.08% Re 96.6% 3.18% 87.5% OS-RC-2 1.5 si-NC of 0.8 0 3 4 5 6 3 4 5 6 OD 0 10 10 10 10 0 10 10 10 10 1.0 0.6 OS-RC-2 Annexin-FITC Annexin-FITC 0.4 ** 0.5 positive cel Negative control pcDNA3.1-HIF1A-AS2 lative fold 0.2 pcDNA3.1-HIF1A-AS2 si-HIF1A-AS2 6 6 Re Q2 0.0 0.0 T = 0 h 293T T = 24 h pcDNA3.1-HIF1A-AS2 d 10 Q1 10 Q1 Q2 120 Negative control 0 24 48 72 OS-RC-2 d 0.172% Negative control 0.019% 0.000% 0.058% Time (h) ) 250 5 5 Negative control 10 10 100 293T pcDNA3.1-HIF1A-AS2 Negative ** ) 3.0 pcDNA3.1-HIF1A-AS2 6.0 * d 293T d 5.5 control 200 80 2.5 Negative control Negative U 104 104 5.0 60 PI Ed 4.5 PI 2.0 150 293T Control of 4.0 40 ** 1.5 3 3 Apoptosis (% OD 3.5 100 10 10 1.0 3.0 20 2.5 pcDNA3.- Q4 Q3 Q4 Q3 0.5 pcDNA3.1 positive cell 2.0 50 0 0 0 lative cell migration (% lative fold HIF1A-AS2 3.63% 1.5 90.4% 9.44% 96.3% 293T

0.0 Re Re 1.0 0 24 48 72 -HIF1A-AS2 0 3 4 5 6 3 4 5 6 Time (h) 0.5 293T 0110 10 10 10 0 0 10 10 10 0.0 Annexin V-FITC Annexin V-FITC 293T

Figure 2 Knockdown or overexpression of HIF1A-AS2 suppressed or promoted cell progression. The relative expression level of HIF1A-AS2 was significantly downregulated by si-HIF1A-AS2 (A) and pcDNA3.1-HIF1A-AS2 (B). Analysis of variance was used for the comparison of curves of cell proliferation. Cell proliferation was detected in both renal carcinoma cells after transfection of si-RNA [C (a-c)] and pcDNA3.1-HI- F1A-AS2 [C (d)]. Representative images of EdU assays and the relative fold changes of EdU positive cells were detected by si-RNA [D (a–c)] and pcDNA3.1-HIF1A-AS2 [D (d)]. The relative cell migration was suppressed after transfection of si-RNA, and the representative images were as follow [E (a–c)]. The relative cell migration was promoted after transfection of pcDNA3.1-HIF1A-AS2, and the representative images are shown in [E (d)]. Apoptotic cells were measured after transfection of si-RNA [F (a–c)] and pcDNA3.1 [F (d)] by flow cytometry analyses. *( P < 0.05; **P < 0.01). in OS-RC-2 cells (P < 0.01), and 47.42% in ACHN cells (P < proliferation and suppression of miR-30a-5p accelerated renal 0.01) at 48 h after transfection with an miR-30a-5p inhibitor carcinoma cell proliferation. (Figure 3A). The relative expression levels of miR-30a-5p were The ratios of relative migrations were downregulated increased approximately 2.916-fold in 786-O cells (P < 0.001), 49.83% in 786-O cells (P < 0.01), 47.68% in OS-RC-2 cells 4.265-fold in OS-RC-2 cells (P < 0.001), and 2.084 times in (P < 0.001), and 78.98% in ACHN cells (P < 0.05) [Figure 3D 786-O cells (P < 0.001) at 48 h after transfection with miR- (a–c)] after transfection with miR-30a-5p mimics. The ratio 30a-5p mimics (Figure 3A). of the relative migration was upregulated 2.108-fold in 786-O The CCK-8 data showed that miR-30a-5p mimics decreased cells (P < 0.001), 2.212-fold in OS-RC-2 cells (P < 0.001), renal carcinoma cell proliferation, while the miR-30a-5p inhib- and 2.053-fold in ACHN cells (P < 0.01) [Figure 3D (a–c)] itor accelerated renal carcinoma cell proliferation [Figure 3B after transfection with the miR-30a-5p inhibitor. These results (a–c)] (P < 0.01). showed that higher miR-30a-5p expression suppressed renal The quantity of Edu-positive cells in the miR-30a-5p mimic carcinoma cell migration and decreased miR-30a-5p acceler- group was decreased by 51.82% in 786-O cells (P < 0.001), ated renal carcinoma cell migration. 51.10% in OS-RC-2 cells (P < 0.01), and 74.13% in ACHN Compared with the NC groups, the ratios of apoptosis were cells (P < 0.01) [Figure 3C (a–c)]. The level of Edu positive increased by 1.786-fold in 786-O cells (P < 0.05), 3.246-fold cells in the miR-30a-5p inhibitor group was upregulated in OS-RC-2 cells (P < 0.01), and 2.022-fold in ACHN cells (P approximately 2.421-fold in 786-O cells (P < 0.001), 2.032- < 0.001) [Figure 3E (a–c)] after transfection with miR-30a-5p fold in OS-RC-2 cells (P < 0.001), and 1.926-fold (P < 0.05) mimics. Compared to the NC groups, the ratios of apoptosis in ACHN cells [Figure 3C (a–c)]. Our study showed that were decreased by 72.91% in 786-O cells (P < 0.01), 81.07% upregulation of miR-30a-5p decreased renal carcinoma cell in OS-RC-2 cells (P < 0.001), and 60.73% in ACHN cells (P < 594 Chen et al. HIF1A-AS2 accelerates RCC via sponging miR-30a-5p

b c a 786-O OS-RC-2 ACHN C Hoechst EdU MergeHoechst EdU MergeHoechst EdU Merge A miR-30a-5p inhibitor D NC T = 0 h 786-O T = 24 h miR-30a-5p miR-30a-5p mimics a 4.5 ** mimics miR-30a-5p mimics 4.0 miR-30a-5p NC inhibition 3.5 miR-30a-5p inhibition NC 250 ession ** 3.0 ** ) 200 2.5 ** miR-30a-5p 2.0 inhibition NC 150 1.5

1.0 100 lative miR-30a-5p expr ** ** Re 0.5 ** ** lative cell migration (% 50 Re 0.0 miR-30a-5p mimics miR-30a-5p mimics miR-30a-5p mimics miR-30a- 786-O OS-RC-2 ACHN miR-30a-5p inhibition miR-30a-5p inhibition 0 l l l 2.5 miR-30a-5p inhibition 2.4 5p mimics NC ** NC * 786-O 2.6 NC ** 2.2 2.4 2.0 2.0 2.2 1.8 2.0 B 1.6 miR-30a-5p inhibition 1.8 1.4 1.5 1.4 786-O 1.6 EdU positive cel NC EdU positive cel a 1.2 EdU positive cel 1.4 1.2 of of miR-30a-5p mimics of 1.2 1.0 1.0 1.0 1.0 0.8 T = 0 hOS-RC-2T = 24 h 0.8 0.6 0.8 0.6 ** 0.5 ** ** b 0.4 0.4 OD lative fold lative fold 0.6 lative fold 0.2 0.2 Re Re miR-30a-5p mimics Re 0.0 0.0 0.0 miR-30a-5p 0.4 786-O OS-RC-2 ACHN inhibition miR-30a-5p inhibition 0.2 250 NC ** miR-30a-5p inhibition NC miR-30a-5p mimics miR-30a-5p inhibition 0.0 E ) 6 Q2 6 6 miR-30a-5p mimics 02448 72 10 Q1 10 Q1 Q2 10 Q1 Q2 200 a 0.274% 0.254% 0.294% 1.00% 0.094% 1.37% 240 NC Time (h) 105 105 105 * 200 NC 150 ) 104 104 104 160

miR-30a-5p inhibition PI 100 PI

b PI 1.2 120 NC OS-RC-2 3 ** 786- O 3 3 10 10 10 lative cell migration (% 50 1.0 miR-30a-5p mimics 80 Re Q4 Apoptosis (% Q4 Q3 Q4 Q3 Q3 0 0 0 88.1% 0.8 98.7% 0.761% 93.4% 5.33% 10.4% 40 ** miR-30a- 0 5p mimics 3 4 5 6 3 4 5 6 3 4 5 6 OS-RC-2 OD 0.6 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 Annexin V-FITC Annexin V-FITC Annexin V-FITC 786-O 0.4 6 miR-30a-5p inhibition 6 10 6 b 10 Q1 Q2 Q1 Q2 10 Q1 Q2 NC 0.2 0.168% 0.722% 0.143% 0.924% miR-30a-5p mimics 0.430% 0.468% 450 5 5 10 105 ** 0.0 10 400 02448 350

72 ) 4 4 4 C- 2 10 10 10 300 T = 0 hACHNT = 24 h PI PI Time (h) PI 250 c OS-R 3 3 200 miR-30a-5p mimics 103 10 10 1.4 miR-30a-5p inhibition 150 NC Apoptosis (% miR-30a-5p Q4 Q3 Q4 Q3 Q4 Q3 c ACHN 0 0 100 inhibition 1.2 NC 0 99.0% 0.410% 94.7% 4.15% 87.4% 11.5% miR-30a-5p inhibition 50 240 ** miR-30a-5p mimics 3 4 5 6 ** 1.0 0 103 104 105 106 0 103 104 105 106 0 10 10 10 10 0 ) Annexin V-FITC Annexin V-FITC Annexin V-FITC OS-RC-2 200 0.8 miR-30a-5p inhibition 6 6 OD 106 Q1 Q2 10 Q1 Q2 10 Q1 Q2 miR-30a-5p mimics 0.6 c 160 0.074% 1.21% 0.263% 1.51% 0.353% 2.62% 220 NC ** 0.4 5 105 105 200 10 NC 120 0.2 180 4 104 104 ) 160

10 PI HN PI 0.0 PI 140 80 AC 02448 72 120 lative cell migration (% * 3 3 103 Re 40 Time (h) 10 10 100 80

Q3 Q4 Q3 Apoptosis (% Q4 Q3 Q4 60 miR-30a- 0 0 0 5.58% 0 85.2% 11.8% ** 96.0% 2.66% 92.6% 40 5p mimics ACHN 0 103 104 105 106 0 103 104 105 106 0 103 104 105 106 20 0 Annexin V-FITC Annexin V-FITC Annexin V-FITC ACHN

Figure 3 The miR-30a-5p acted as an anti-oncogene. The relative expression level of miR-30a-5p was significantly downregulated by the miR-30a-5p inhibitor and increased by miR-30a-5p mimics (A). We detected cell proliferation after miR-30a-5p inhibitor and miR-30a-5p mimic transfection [B (a-c)] in renal carcinomas cells. Representative images of EdU assays and the relative fold changes of EdU positive cells were detected by the miR-30a-5p inhibitor and miR-30a-5p mimics [C (a–c)]. The relative cell migration was inhibited or accelerated after miR-30a-5p mimics or inhibitor transfection in renal cancer [D (a–c)] cell lines. Flow cytometry analysis was used to measured apoptotic cells after miR-30a-5p mimics or inhibitor transfection in the renal cancer [E (a–c)] cell lines.(*P < 0.05; **P < 0.01).

0.01) [Figure 3E (a–c)] after transfection with the miR-30a-5p pmirGLO-HIF1A-AS2-Wt, the luciferase activity was reduced inhibitor. Overall, increased miR-30a-5p expression increased 51.44% in 786-O cells (P < 0.001) and 49.42% in OS-RC-2 renal carcinoma cell apoptosis and reduced miR-30a-5p cells (P < 0.001) in the co-transfections miR-30a-5p mimics + decreased renal carcinoma cell apoptosis. pmirGLO-HIF1A-AS2-Wt groups, however, miR-30a-5p did not suppress the HIF1A-AS2 mutant reporter vector luciferase HIF1A-AS2 sponged miR-30a-5p activity (Figure 4B). Overall, the luciferase reporter assays ver- ified that HIF1A-AS2 sponged miR-30a-5p. Compared to the NC groups, the relative expressions of miR-30a-5p were increased 2.727-fold in 786-O cells (P < HIF1A-AS2 sponging miR-30a-5p mediated 0.01) and 4.427-fold in OS-RC-2 cells (P < 0.001) (Figure renal carcinoma cell progression 4A) in the si-HIF1A-AS2 groups. Figure 4B shows that bio- informatics databases were used to predict binding sites of First, we verified that miR-30a-5p co-regulated progression HIF1A-AS2 and miR-30a-5p. The predictions were confirmed induced by HIF1A-AS2 in renal carcinomas cells. The si-HI- using the luciferase reporter assay. In addition, miR-30a-5p F1A-AS2 co-transfected miR-30a-5p mimics better suppressed mimics suppressed HIF1A-AS2 wild-type reporter luciferase renal carcinoma cell proliferation (Figure 4C–4F) and migra- activity; when compared to the co-transfections using NC+ tion (Figure 4G and 4H) than the si-NC co-transfection with Cancer Biol Med Vol 18, No 2 May 2021 595

786-O OS-RC-2 5.0 si-NC si-NC + NC T = 0 h 786-O T = 24 h si-HIF1A-AS2 ** 1.5 si-NC + NC 786-O 1.6 si-HIF1A-AS2+miR-30a-5p inhibition OS-RC-2 Hoechst EdU MergeHoechst EdU Merge AC4.5 si-HIF1A-AS2+miR-30a-5p inhibition DEsi-HIF1A-AS2+NC FG si-HIF1A-AS2+NC si-HIF1A-AS2+miR-30a-5p mimic si-HIF1A-AS2+miR-30a-5p mimic

ession 1.2 4.0 1.2 si-NC 3.5 ** 0.9 +NC si-NC+NC 3.0 0.8 OD 0.6 OD 2.5 0.4 2.0 0.3 si-HIF1A-AS2 1.5 +miR-30a-5p 0.0 0.0 +inhibition 1.0 0 24 48 72 02448 72 si-HIF1A-AS2 0.5 Time (h) Time (h) lative miR-30a-5p expr +miR-30a-5p

Re 0.0 si-HIF1A-AS2 inhibition 786-O OS-RC-2 +NC

si-HIF1A-AS2 +miR-30a-5p +mimics si-HIF1A-AS2 HIF1A-AS2-Wt5′A UUUUUUUUUUCUUGUUUACA3′ B +NC

miR-30a-5p 3′GAAGGUCAGCUCCUACAAAUGU5′

HIF1A-AS2-Mut 5′A UUUUUUUUUUCUACAAAUGU 3′ miR-30a-5p mimics+si-HIF1A-AS2 miR-30a-5p mimics+si-HIF1A-AS2 NC+si-HIF1A-AS2 NC+si-HIF1A-AS2 RNAhybrid 1.2 1.2 NC 1.2 miR-30a-5p inhibition+si-HIF1A-AS2 miR-30a-5p inhibition+si-HIF1A-AS2 si-HIF1A-AS2 miR-30a-5p mimics https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid NC+si-NC NC+si-NC +miR-30a-5p 1.0 miRDB 1.0 1.0 http://mirdb.org/ miR-30a-5p mimics 0.8 DIANA tools 0.8 0.8 - LncBase experimental v2 0.6 http://carolina.imis.athena-innovation.gr/diana_tools/ ** web/index.php?r=Incbasev2%2Findex-experimental NC+si-NC ** EdU positive cell 0.6 0.6 miR-30a-5p inhibition+si-HIF1A-AS2 EdU positive cell ** NC+si-HIF1A-AS2 )

0.4 of miR-30a-5p mimics+si-HIF1A-AS2 ** of 120 0.2 0.4 0.4

lative luciferase activity 100

Re 0.0 0.2 ** 0.2 ** 80 lative fold lative fold C-2 Wt 0.0

C-2 Mut Re 0.0 60

786-O Wt Re 786-O Mut ** OS-R 786-O OS-R OS-RC-2 40 ** 20 si-NC+NC si-HIF1A-AS2+miR-30a-5p inhibition si-HIF1A-AS2+NC si-HIF1A-AS2+miR-30a-5p mimics lative cell migration (% miR-30a-5p mimics+si-HIF1A-AS2 Re 0 NC+si-HIF1A-AS2 786-O 106 106 106 106 miR-30a-5p inhibition+si-HIF1A-AS2 T = 0 h T = 24 h NC+si-NC OS-RC-2 I 1400 ** H 5 5 5 5 10 10 10 10 1200 ) 1000 si-NC+NC 104 104 104 104 PI PI PI PI **

786- O 800

103 103 103 103 600 Apoptosis (% 400 0 0 0 0 si-HIF1A-AS2 200 NC+si-NC +miR-30a-5p miR-30a-5p inhibition+si-HIF1A-AS2 ) NC+si-HIF1A-AS2 120 miR-30a-5p mimics+si-HIF1A-AS2 01103 04 105 106 01103 04 105 106 01103 04 105 106 01103 04 105 106 0 inhibition 789-O 100 Annexin V-FITC Annexin V-FITC Annexin V-FITC Annexin V-FITC 80 miR-30a-5p mimics+si-HIF1A-AS2 106 106 106 106 NC+si-HIF1A-AS2 miR-30a-5p inhibition+si-HIF1A-AS2 60 ** J 900 NC+si-NC ** 105 105 105 105 800 si-HIF1A-AS2 40 ** 700 +NC 20 ) lative cell migration (% 4 4 4 4 ** 10 10 10 10 600 Re PI PI PI 0 PI C- 2 500 OS-RC-2

3 3 3 3 400 OS-R 10 10 10 10 si-HIF1A-AS2 Apoptosis (% 300 +miR-30a-5p 200 0 0 0 0 mimics 100 01103 04 105 106 01103 04 105 106 01103 04 105 106 01103 04 105 106 0 Annexin V-FITC Annexin V-FITC Annexin V-FITC Annexin V-FITC OS-RC-2

Figure 4 HIF1A-AS2 was a target of miR-30a-5p. The relative expression of miR-30a-5p was increased by si-HIF1A-AS2 (A). Dual-luciferase reporter assays were performed in 786-O and OS-RC-2 cells co-transfected with HIF1A-AS2-Wt or HIF1A-AS2-Mut and miR-30a-5p mimics or normal control (NC) (B). We detected cell proliferation after co-transfection with si-NC+NC in both renal carcinoma cell lines, si-HIF1A- AS2+miR-30a-5p inhibitor or si-HIF1A-AS2+miR-30a-5p mimics by CCK-8 and Edu assays (C-F). The relative cell migration after co-trans- fection with si-NC+NC, si-HIF1A-AS2+miR-30a-5p inhibitor or si-HIF1A-AS2+miR-30a-5p mimics, and the representative images are shown (G and H). The apoptotic cells were measured by flow cytometry analysis after co-transfection with si-NC+NC, si-HIF1A-AS2+miR-30a-5p inhibitor or si-HIF1A-AS2+miR-30a-5p mimics (I and J). (*P < 0.05; **P < 0.01).

NC (si-NC+NC), and compared with the si-NC+NC group, and increased it by 20.68% in 786-O cells and 32.87% in apoptosis was increased in the si-HIF1A-AS2 co-transfection OS-RC-2 cells (Figure 4E and 4F). miR-30a-5p mimics (si-HIF1A-AS2+miR-30a-5p) group Compared to si-NC+NC, si-HIF1A-AS2 co-transfected (Figure 4I and 4J). In contrast, the miR-30a-5p inhibitor par- miR-30a-5p mimics decreased the ratio of the relative migra- tially reversed the inhibition effects on renal carcinoma cell tions by 74.80% in 786-O cells (P < 0.001) and 73.91% in progression induced by si-HIF1A-AS2. OS-RC-2 cells (P < 0.001). Moreover, the miR-30a-5p inhib- The CCK-8 assays showed that si-HIF1A-AS2 co-trans- itor partially reversed the inhibitory effects on renal carci- fected miR-30a-5p mimics (Figure 4C and 4D) decreased noma cell migration induced by si-HIF1A-AS2, and increased renal carcinoma cell proliferation in both 786-O and OS-RC-2 it by 37.20% in 786-O cells and 17.78% in OS-RC-2 cells cells (all, P < 0.01). In addition, the miR-30a-5p inhibitor par- (Figure 4G and 4H). tially reversed the decreased effects on renal carcinoma cell Compared to si-NC+NC, si-HIF1A-AS2 co-transfected proliferation induced by si-HIF1A-AS2 (Figure 4C and 4D). miR-30a-5p mimics increased the ratio of relative apopto- Compared to si-NC+NC, si-HIF1A-AS2 co-transfected sis by 13.08-fold in 786-O cells (P < 0.001) and 8.292-fold miR-30a-5p mimics decreased the quantity of Edu positive in OS-RC-2 cells (P < 0.001). Furthermore, the miR-30a-5p cells by 85.99% in 786-O cells (P < 0.001) and 82.23% in inhibitor partially reversed the increased apoptosis in renal OS-RC-2 cells (P < 0.001). In addition, the miR-30a-5p inhib- carcinoma cells induced by si-HIF1A-AS2 and reduced itor partially reversed the inhibitory effects on renal carcinoma it by 315.6% in 786-O cells and 192.4% in OS-RC-2 cells cell quantity of Edu positive cells induced by si-HIF1A-AS2, (Figure 4I and 4J). 596 Chen et al. HIF1A-AS2 accelerates RCC via sponging miR-30a-5p

HIF1A-AS2 sponging miR-30a-5p closely dramatically decreased SOX4 wild-type reporter luciferase regulated SOX4 activity, which was decreased to 50.43% in 786-O cells (P < 0.001) and 48.45% in OS-RC-2 cells (P < 0.001) in the We used bioinformatics databases to predict mutual binding co-transfection with miR-30a-5p mimics + SOX4-3’-UTR-Wt. sites of SOX4 and miR-30a-5p, which is shown in Figure 5A. In contrast, miR-30a-5p could not decrease the SOX4 mutant The predicted binding sites and binding effects were deter- binding site reporter vector luciferase activity (Figure 5A). mined using the luciferase reporter assay. We confirmed whether HIF1A-AS2 regulated SOX4 expres- Compared to co-transfections with NC + pmirGLO-HI- sion in a miR-30a-5p-dependent manner in renal carcinoma F1A-AS2-Wt, our results confirmed that miR-30a-5p mimics cells (Figure 5B–5D). Compared to the si-NC groups, the

SOX4-Wt5′ C CCCUAAUUUCUCCAUGUUUACA 3′ 293T Lv-HIF1A-AS2 A D E F 786-O OS-RC-2 1.2 Lv-NC 786-O miR-30a-5p 3′ GAAGGUCAGCUCCUACAAAUGU 5′ pcDNA3.1-HIF1A-AS2 – + l 786-O OS-RC-2 Negative control 1.0 miR-30a-5p inhibition + – – + –– + – LV-HIF1A-AS2 ––++ SOX4-Mut 5′ CCCCUAAUUUCUCCAACAAAUGA 3′ miR-30a-5p mimics – + – – + – SOX4 NC 0.8 1.2 miR-30a-5p mimics ++ β ++––ession leve NC –– –– -catenin LV-NC * ** ** ** 1.0 SOX4 Met 0.6 ** β-actin SOX4 ** 0.8 C- ** β-catenin otein expr 0.4 0.6 ** ** CyclinD1 0.4 Fra-1 Met 0.2 lative pr

0.2 VEGF Re

lative luciferase activity 0.0 β-actin C-myc Re 0.0 miR-30a-5p mimics X4 Met NC CyclinD1 SO Fra-1 VEGF 3.0 miR-30a-5p inhibition c-Myc -Catenin CyclinD1 C-2 Wt * β 786-O Wt C-2 Mut l 786-O Mut OS-R Fra-1 OS-R 2.5 ** l

1.4 si-HIF1A-AS2 l VEGF 1.2 Lv-HIF1A-AS2 si-NC miR-30a-5p mimics Lv-NC OS-RC-2 NC 2.0 B 1.2 C 8 miR-30a-5p inhibition ession leve Negative control β-actin 1.0 ession pcDNA3.1-HIF1A-AS2 * 4.5 * ession 1.0 7 1.5 4.0 293T ession leve 0.8 ession leve 3.5 ** 6 ** 0.6 ** ** ** 0.8 otein expr 3.0 ** ** 5 1.0 2.5 ** * ** 0.4 ** 0.6 4 2.0 ** ** ** otein expr X4 mRNA expr ** otein expr

* lative pr 1.5 0.2

X4 mRNA expr 0.5 0.4 3 **

Re 1.0

** 2 0.5 lative pr 0.0 n 0.2 lative pr 0.0 Re lative SO X4 1 0.0 Met Re

lative SO Fra-1 VEGF

Re SO ** 786-O OS-RC-2 n c c-Myc 0.0 0 X4 -Cateni Re Met CyclinD1 SO Fra-1 VEGF β 786-O OS-RC-2 786-O OS-RC-2 c-My β-Cateni CyclinD1

Negative control+DMSO Negative control+IWR-1 pcDNA3.1-HIF1A-AS2+IWR-1 pcDNA3.1-HIF1A-AS2+DMSO Negative control+DMSO pcDNA3.1-HIF1A-AS2+DMSO T = 0 h 786-O T = 24 h pcDNA3.1-HIF1A-AS2+IWR-1 6 6 6 6 Negative control+IWR-1 G I J 10 10 10 10 350 ** ** 5 5 5 5 300 1.2 si-HIF1A-AS2 10 10 10 10 si-NC ) 250 1.0 Negative control 104 104 104 104 PI PI PI +IWR-1 PI 200 0.8 103 103 103 103 150

lative fold ** 0.6 **

** Apoptosis (% 100 0 0 0 0 0.4 50 P/FOP re 0 103 104 105 106 0 103 104 105 106 0 103 104 105 106 0 103 104 105 106 0

TO 0.2 pcDNA3.1-HIF1A-AS2 Annxin-FITC Annxin-FITC Annxin-FITC Annxin-FITC 786-O 0.0 +IWR-1 786-O OS-RC-2 Negative control+IWR-1 pcDNA3.1-HIF1A-AS2+IWR-1 pcDNA3.1-HIF1A-AS2+DMSO Negative control+DMSO 200 pcDNA3.1-HIF1A-AS2+IWR-1 3.0 pcDNA3.1-HIF1A-AS2+IWR-1 ** Negative control+IWR-1 pcDNA3.1-HIF1A-AS2+DMSO 786-O pcDNA3.1-HIF1A-AS2+DMSO ) 786-O H ** Negative control+DMSO IWR-1+negative control pcDNA3.1-HIF1A-AS2 2.5 DMSO+negative control K ** ** ** +DMSO 150 – +–+ 2.2

pcDNA3.1-HIF1A-AS2 ession 2.0 2.0 Empty vector + – – + 1.8 1.5 100 1.6 OD ** 1.4 IWR-1 – – ++ 1.0 1.2 1.0 Negative control 50

0.5 lative cell migration (% DMSO + + – – 0.8 +DMSO

Re 0.6 0.0 β -catenin mRNA expr β-catenin 0.4 0 24 48 72 0 0.2 lative Time (h) 786-O 0.0 β-actin Re 786-O

Negative control+si-NC si-NC 786-O OS-RC-2 pcDNA3.1-HIF1A-AS2+si-SOX4 786-O OS-RC-2 Negative control+si-SOX4 si-SOX4 M N L 1.2 si-SOX4 – + – + SOX4 3.0 pcDNA3.1-HIF1A-AS2+si-NC l

l β-actin si-NC + ––+ l 1.0 si-SOX4 si-SOX4 2.5 * si-NC 786-O 1.2 si-NC OS-RC-2 SOX4 1.2 Negative control – + – + – + – + ** 1.0 ession 0.8 β-catenin 1.0 ession leve 2.0 ession leve

ession leve pcDNA3.1-HIF1A-AS2 + ––+ + ––+ 0.8 0.8

Met X4 expr 0.6 ** ** ** ** si-NC 1.5 0.6 ** ** 0.6 ++– – ++– – ** C-myc ** ** ** otein expr otein expr 0.4 0.4 ** – + + – – + + – 0.4 otein expr ** ** si-SOX4 1.0 CyclinD1 ** lative SO 0.2 ** 0.2 Fra-1 ** Re lative pr ** 0.2 lative pr 0.5 ** lative pr VEGF 0.0 0.0 Re Re n c X4 Re Met X4 β-actin SO Fra-1 VEGF SO Met 0.0 0.0 c-My c-Myc Fra-1 VEGF -Cateni CyclinD1 β β-Catenin CyclinD1 786-O OS-RC-2 786-O OS-RC-2

Figure 5 HIF1A-AS2 positively regulates SOX4 expression via sponging miR-30a-5p. SOX4-Wt and miR-30a-5p mimics co-transfection inhibited luciferase activity (A) as shown using the dual-luciferase reporter assay. The relative expression level of SOX4 was decreased by si-HIF1A-AS2 (B). The relative expression level of SOX4 was regulated by miR-30a-5p mimics and inhibition (C). Overexpressing miR-30a-5p downregulated SOX4 expression and knockdown of miR-30a-5p upregulated SOX4 expression in renal carcinomas cells (D). Overexpression of HIF1A-AS2 upregulated SOX4 expression in 293T cells (E). Knockdown of HIF1A-AS2 downregulated SOX4 expression in renal carcinomas cells (F). Knockdown of HIF1A-AS2 downregulated transcriptional activity of the Wnt/β-catenin pathway as determined by the TOP flash/FOP flash reporter luciferase activity assay (G). CCK-8 assay (H), migration (I), flow cytometric analysis (J), Western blot and qPCR (K) showed that IWR-1 inhibited and even reversed the effect of pcDNA3.1-HIF1A-AS2 on cell progression in 786-O cells. Knockdown of SOX4 downregulated β-catenin expression in renal carcinomas cells (M). Knockdown of SOX4 reversed malignant phenotypic promotion of renal carcinomas cells induced by HIF1A-AS2. Si-SOX4 reversed SOX4 expression promotion induced by overexpression of HIF1A-AS2 in renal carcinomas cells (N). The relative expression level of SOX4 was decreased by si-SOX4 (L). (*P < 0.05; **P < 0.01). Cancer Biol Med Vol 18, No 2 May 2021 597

SOX4 expressions were decreased by 70.92% in 786-O cells co-transfected IWR-1 group compared with the pcDNA3.1- (P < 0.001) and 60.93% in OS-RC-2 cells (P < 0.05) (Figure HIF1-AS2+DMSO group in 786-O cells. In contrast, the 5B) in the si-HIF1A-AS2 groups. Our study showed that the IWR-1 inhibitor partially reversed the tumorigenic effects relative expression of HIF1A-AS2 was closely related to SOX4 induced by HIF1A-AS2. Our further experiments showed expression, and that downregulated HIF1A-AS2 reduced that IWR-1 dramatically reversed the promotion of β-catenin SOX4 expression in renal carcinomas cells. Furthermore, expression induced by overexpressing HIF1A-AS2 in renal car- compared to the si-NC groups, the relative expression levels cinomas cells (Figure 5K). of SOX4 were decreased by 63.09% in 786-O cells (P < 0.01) and 76.90% in OS-RC-2 cells (P < 0.001) (Figure 5C) in the Silencing of SOX4 reversed the malignant miR-30a-5p mimic groups, and the relative expression levels renal carcinoma cell phenotype promotion of of SOX4 were increased 5.150-fold in 786-O cells (P < 0.01) HIF1A-AS2 overexpression and 5.821-fold in OS-RC-2 cells (P < 0.05) (Figure 5C) in the miR-30a-5p inhibition groups. Our studies suggested that We wished to confirm whether HIF1A-AS2 regulated malig- overexpression of miR-30a-5p reduced SOX4 expression and nant phenotypes in a SOX4-dependent manner in renal car- miR-30a-5p inhibition upregulated SOX4 expression in renal cinomas cells. carcinomas cells (Figure 5D). Together, our results showed Further experiments confirmed that decreasing SOX4 that HIF1A-AS2 closely regulated SOX4 expression via spong- could alter WNT signaling in renal carcinomas cells. ing miR-30a-5p in renal carcinoma cells. Knockdown of SOX4 decreased MET, C-myc, cyclinD1, Fra- Our further experiments confirmed that decreased 1, VEGF, and β-catenin expressions in renal carcinomas cells HIF1A-AS2 could alter WNT signaling in renal carcinomas (Figure 5M). Further experiments showed that the knock- cells (Figure 5E and 5F). Western blot was used to detect the down of SOX4 dramatically reversed the promotion of SOX4 expression of WNT signaling-associated downstream . expression induced by overexpressing HIF1A-AS2 in renal Overexpression of HIF1A-AS2 increased SOX4, MET, C-myc, carcinoma cells (Figure 5N). The relative expression levels of cyclinD1, Fra-1, VEGF, and β-catenin expressions in renal SOX4 were reduced by 60.53% in 786-O cells (P < 0.01) and cells (Figure 5E); knockdown of HIF1A-AS2 decreased SOX4, 59.09% in OS-RC-2 cells (P < 0.01) at 48 h after transfec- MET, C-myc, cyclinD1, Fra-1, VEGF, and β-catenin expression tion of si-SOX4 (Figure 5L). Moreover, knockdown of SOX4 in renal carcinomas cells (Figure 5F). reversed the promotion of renal carcinoma cell proliferation TOP/FOP flash is a method for the determination of (Figure 6A–6D) induced by overexpression of HIF1A-AS2. intracellular beta-catenin-mediated transcription activity. Furthermore, SOX4 knockdown reversed renal carcinoma TOP/FOP flash reporter assays were used to detect whether cell migration (Figure 6E and 6F) induced by overexpression HIF1A-AS2 functioned via the WNT/β-catenin signaling of HIF1A-AS2. In addition, SOX4 knockdown reversed renal pathway. Data illustrated that decreasing HIF1A-AS2 expres- carcinoma cell apoptosis suppression (Figure 6G and 6H) sion downregulated Wnt-activity in the 786-O and OS-RC-2 induced by overexpression of HIF1A-AS2. cells (Figure 5G). Compared to negative control+si-NC, pcDNA3.1-HI-

IWR-1 (a β-catenin inhibitor, IC50 = 180 nM, F1A-AS2 co-transfected si-NC increased the quantity of Edu sc-295215, Santa Cruz), which is a tyrosine kinase inhibitor, positive cells by 1.892-fold in 786-O cells (P < 0.01) and 2.09- suppressed the Wnt/β-catenin signaling pathway. To further fold in OS-RC-2 cells (P < 0.001), and si-SOX4 significantly verify whether HIF1A-AS2 was involved in the Wnt signaling decreased the quantity of Edu positive cells by 65.33% in pathway we used IWR-1. IWR-1 was involved in the inhibitory 786-O cells (P < 0.05) and 56.34% in OS-RC-2 cells (P < 0.01). effects of si-HIF1A-AS2 in kidney carcinoma cells. Cells that Moreover, si-SOX4 partially reversed its stimulatory effects on were continuously transfected with pcDNA3.1-HIF1A-AS2 renal carcinoma cells, and the quantity of Edu positive cells and co-incubated with IWR-1 showed stronger inhibi- induced by pcDNA3.1-HIF1A-AS2 decreased by 104.5% in tory effects on cell proliferation (Figure 5H) and migration 786-O cells and 78.93% in OS-RC-2 cells (Figure 6A and 6B). (Figure 5I) of kidney carcinoma cells, with comparison to The CCK-8 assay results showed that pcDNA3.1-HIF1A-AS2 the pcDNA3.1-HIF1-AS2+DMSO group. Apoptosis (Figure co-transfected si-NC accelerated renal carcinoma cell prolif- 5J) was remarkably increased in the pcDNA3.1-HIF1AS2 eration, and si-SOX4 co-transfected si-NC was reversed (P < 598 Chen et al. HIF1A-AS2 accelerates RCC via sponging miR-30a-5p

T = 0 hTOS-RC-2 = 24 h A 786-O BEOS-RC-2 T = 0 hT786-O = 24 h F Hoechst EdU MergeMHoechst EdU erge pcDNA3.1- pcDNA3.1- pcDNA3.1- HIF1A-AS2 HIF1A-AS2 HIF1A-AS2 +si-NC +si-NC +si-NC

Negative control Negative control Negative control +si-NC +si-NC +si-NC

pcDNA3.1- pcDNA3.1- pcDNA3.1- HIF1A-AS2 HIF1A-AS2 HIF1A-AS2 +si-SOX4 +si-SOX4 +si-SOX4

Negative control Negative control Negative control +si-SOX4 +si-SOX4 +si-SOX4

Negative control+si-SOX4 Negative control+si-SOX4 pcDNA3.1-HIF1A-AS2+si-NC pcDNA3.1-HIF1A-AS2+si-SOX4 pcDNA3.1-HIF1A-AS2+si-SOX4 pcDNA3.1-HIF1A-AS2+si-NC Negative control+si-NC Negative control+si-NC Negative control+si-NC Negative control+si-NC pcDNA3.1-HIF1A-AS2+si-NC pcDNA3.1-HIF1A-AS2+si-NC pcDNA3.1-HIF1A-AS2+si-SOX4 pcDNA3.1-HIF1A-AS2+si-SOX4 2.2 Negative control+si-SOX4 Negative control+si-SOX4 250 220 **

2.2 ) ** ) 200 2.0 ** 2.0 ** 180 1.8 1.8 200 160 1.6 1.6 140 1.4 1.4 150 1.2 1.2 120 EdU positive cell 1.0 EdU positive cell 1.0 100 of of 100 0.8 0.8 80 0.6 ** 0.6 ** 60 **

lative cell migration (% 50 0.4 0.4 ** lative cell migration (% 40 Re

0.2 0.2 Re 20 lative fold 0.0 lative fold 0.0 00 Re 786-O Re OS-RC-2 786-O OS-RC-2

1.5 pcDNA3.1-HIF1A-AS2+si-NC CGNegative control+si-NC 786-O pcDNA3.1-HIF1A-AS2+si-NC pcDNA3.1-HIF1A-AS2+si-SOX4 Negative control+si-NCNegative control+si-SOX4 pcDNA3.1-HIF1A-AS2+si-SOX4 Negative control+si-SOX4 6 106 106 106 300 1.2 Negative control+si-SOX4 10 Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 pcDNA3.1-HIF1A-AS2+si-SOX4 0.234% 0.247% 0.301% 1.23% 0.060% 1.27% Negative control+si-NC 0.915% 0.267% pcDNA3.1-HIF1A-AS2+si-NC

5 5 5 250 ** 0.9 105 10 10 10 )

OD 200 104 104 104 104

0.6 PI PI PI PI

786- O 150

0.3 3 3 3 3 10 10 10 10 100 Apoptosis (% Q3 0.0 0 Q4 Q3 0 Q4 Q3 0 Q4 Q3 0 Q4 50 0 24 48 72 98.7% 0.821% 93.8% 4.97% 91.2% 7.29% 86.6% 12.1% ** Time (h) 0 103 104 105 106 0103 104 105 106 0103 104 105 106 0103 104 105 106 0 Annexin V-FITCAnnexin V-FITC Annexin V-FITC Annexin V-FITC 786-O

pcDNA3.1-HIF1A-AS2+si-NC Negative control+si-SOX4 1.4 OS-RC-2 Q1 Q1 Negative control+si-NC 106 Q2 106 Q2 106 Q1 Q2 106 Q1 Q2 Negative control+si-NC D pcDNA3.1-HIF1A-AS2+si-SOX4 H 0.415% 0.268% 0.166% 0.758% 0.167% 0.859% 0.235% 0.846% 300 pcDNA3.1-HIF1A-AS2+si-SOX4 pcDNA3.1-HIF1A-AS2+si-NC 1.2 Negative control+si-SOX4 105 105 105 105 1.0 250 ** )

4 4 4 4

0.8 C- 2 10 10 10 10 200 PI PI PI PI OD 0.6 150 OS-R 103 103 103 103 0.4 100 Apoptosis (% Q4 Q3 0.2 0 Q4 Q3 0 0 Q4 Q3 0 Q4 Q3 98.5% 0.844% 93.5% 5.53% 91.0% 7.96% 85.9% 13.0% 50 ** 0.0 0 103 104 105 106 0 103 104 105 106 0 103 104 105 106 0 103 104 105 106 0244872 0 Annexin V-FITC Annexin V-FITC Annexin V-FITC Annexin V-FITC OS-RC-2 Time (h)

Figure 6 HIF1A-AS2 positively regulates SOX4 expression via sponging miR-30a-5p. Knockdown SOX4 reversed cell proliferation promotion induced by overexpression of HIF1A-AS2 (EdU, A and B; CCK8, C and D). Knockdown of SOX4 reversed cell migration promotion induced by overexpression of HIF1A-AS2 (E and F). Knockdown SOX4 reversed cell apoptosis inhibition induced by overexpression of HIF1A-AS2 (G and H). (*P < 0.05; **P < 0.01).

0.01) in 786-O and OS-RC-2 cells. Si-SOX4 partially reversed OS-RC-2 cells (P < 0.01), and si-SOX4 co-transfected si-NC the stimulatory effects on renal carcinoma cell proliferation significantly increased the ratio of the relative apoptosis by induced by pcDNA3.1-HIF1A-AS2 (Figure 6C and 6D). 2.132-fold in 786-O cells (P < 0.01) and 2.133-fold in OS-RC-2 Compared to the negative control+si-NC, pcDNA3.1-HI- cells (P < 0.01). Furthermore, si-SOX4 partially reversed the F1A-AS2 co-transfected si-NC increased the ratio of the rela- repression of apoptosis in renal carcinoma cells induced by tive migration 2.053-fold in 786-O cells (P < 0.001) and 1.967- pcDNA3.1-HIF1A-AS2 and increased by 93.60% in 786-O fold in OS-RC-2 cells (P < 0.001), and si-SOX4 co-transfected cells and 99.59% in OS-RC-2 cells (Figure 6G and 6H). si-NC significantly decreased the ratio of the relative migration Together, our results indicated that HIF1A-AS2 acceler- to 67.34% in 786-O cells (P < 0.01) and 50.27% in OS-RC-2 ated malignant renal carcinoma cell phenotypes in a SOX4- cells (P < 0.01). Moreover, si-SOX4 partially reversed the stim- dependent manner. ulatory effects on renal carcinoma cell migration induced by pcDNA3.1-HIF1A-AS2 and decreased by 97.08% in 786-O Knockdown of HIF1A-AS2 decreased cells and 91.12% in OS-RC-2 cells (Figure 6E and 6F). tumorigenicity of kidney carcinoma cells Compared to the negative control+si-NC, pcDNA3.1-HI- F1A-AS2 co-transfected si-NC decreased the ratio of relative Xenograft models were further used to determine whether apoptosis 78.16% in 786-O cells (P < 0.01) and 74.04% in HIF1A-AS2 regulated tumorigenicity of kidney carcinoma Cancer Biol Med Vol 18, No 2 May 2021 599

LV-HIF1A-AS2 – + l A B 1.4 Lv-NC CD1.2 Lv-HIF1A-AS2 1.2 LV-HIF1A-AS2 Lv-HIF1A-AS2 LV-NC + – Lv-NC LV-NC ession 1.2 1.0 ) 1.0 β-catenin ssion leve

1.0 Met re 0.8 LV-NC ** ** 0.8 0.8 C-myc 0.6 ** ** 0.6 CyclinD1 0.4 0.6 otein exp ** ** ** ** LV-HIF1A-AS2 ** Fra-1 0.2 mor weight (g 0.4 0.4 VEGF Tu lative pr 0.0 0.2 n c 0.2 SOX4 Re X4 Met lative HIF1A-AS2 expr Fra-1 VEGF β-actin SO c-My 0.0

Re -Cateni 0.0 β CyclinD1

E FGRenal carcinomas specimen Normal peritumoral specimen 2200 LV-NC a LV-HIF1A-AS2 Normal cell ) 2000 miR-30a-5p miR-30a-5p 3 miR-30a-5p LnRNA HIF1A-AS2 3 1 oncogene

m 1800 5′ 3′ 1600 miR-30a-5p miR-30a-5p miR-30a-5p 4 2 miR-30a-5p miR-30a-5p 1400 miR-30a-5p +activation Inc-HIF1A-AS2 1200 Protein Cancer cell

X4 6 1000 5 SOX4-binding stability Translation of miR-30a-5p

800 SO LV-HIF1A-AS2 LV-NC HIF1A-AS2 oncogene 600 5′ activation 3′ mor volume (m 400 b Tu

200 Proliferation ↑ Invasion etc. ↑ 0 Apoplosis ↓ Migration ↑ 0510 15 20 25 30 Time (d)

Figure 7 The HIF1A-AS2 effect on renal carcinoma cell tumorigenicity. Tumors collected from mice are shown (A). The relative expression level of HIF1A-AS2 was decreased by LV-HIF1A-AS2 (B). Knockdown of HIF1A-AS2 downregulated SOX4. (C). The tumor weights of LV-HIF1A-AS2 or LV-NC treatment groups were measured and analyzed (D). We measured and analyzed the tumor volumes of the LV-HIF1A-AS2 or LV-NC treatment groups (E). Knockdown of HIF1A-AS2 downregulated SOX4 expression in renal cancer tissue and in vivo renal carcinoma cells [F(a and b)]. The schematic diagram of the oncogenic role of HIF1A-AS2 in renal carcinoma cells (G). HIF1A-AS2 functions as a miRNA sponge to positively regulate SOX4 expression by sponging miR-30a-5p and subsequently promoting malignant phenotypes of renal carcinoma cells, thus playing an oncogenic role in renal carcinomas pathogenesis.(*P < 0.05; **P < 0.01).

cells. We found that knockdown of HIF1A-AS2 decreased and subsequently accelerating malignant renal carcinoma the tumorigenicity of kidney carcinoma cells in vivo (Figure phenotypes. 7A–7F). Tumors collected from mice are shown and meas- ured (Figure 7A). Downregulation of HIF1A-AS2 expres- Discussion sion was significant when compared to the control group of kidney carcinoma cells in vivo (Figure 7B). We found that Studies have reported that lncRNAs are abnormally expressed LV-HIF1A-AS2 downregulated SOX4, β-catenin, Met, C-myc, RNAs with more than 200 nucleotides, which play special roles cyclinD1, Fra-1, VEGF, and the expression of kidney carci- in various diseases, especially malignant tumor formation12-28. noma cells in vivo (Figure 7C). Compared to the LV-NC treat- As key components in regulating and tumor ment group, the tumor weights were less in the LV-HIF1A-AS2 progression, studies of lncRNAs have expanded our under- group (Figure 7D). Tumor growth of the LV-NC treatment standing of their biological behavior during diseases, espe- group was faster than that in the LV-HIF1A-AS2 group cially during carcinoma18-28. In addition, reports have shown (Figure 7E). Immunohistochemistry assays showed that the that the lncRNAs are significant biomarkers and possible relative protein expression level of HIF1A-AS2 was upregu- treatment targets. lated in the renal cancer tissues, and that the knockdown of LncRNA HIF1A-AS2 is located on 14, HIF1A-AS2 downregulated SOX4 expression in vivo in renal NC_000014.9, and has been found to be overexpressed, to carcinoma cells [Figure 7F(a, b)]. These data showed that act as an oncogene in many tumor tissues, including gastric HIF1A-AS2 facilitated tumorigenicity of kidney carcinoma carcinoma29, triple-negative breast carcinoma30-33, bladder cells in vivo. carcinoma34,35, glioblastoma multiforme36 and osteosar- As shown in Figure 7G, HIF1A-AS2 was significantly coma38. HIF1A-AS2 is also involved in the progression of upregulated in renal carcinoma cells and HIF1A-AS2 sponged diseases in tissues, such as human umbilical vein endothelial miR-30a-5p to closely regulate SOX4 expression. Upregulated cells, colorectal cancer, adipose used to extract stem cells, cor- SOX4 protein facilitated transcription and translation of pro- onary artery disease, and preeclampsia37,39,40. In most cases, teins operating through abnormal protein signaling pathways, HIF1A-AS2 is involved in the progression and tumorigenesis 600 Chen et al. HIF1A-AS2 accelerates RCC via sponging miR-30a-5p of carcinomas and functions as an oncogene. Knowledge of the miR-214-5p axis in the osteosarcoma54. SOX4 also participates basic structures and interactions of lncRNAs with other cellu- in the epithelial-mesenchymal transition via lncRNA HCP5/ lar biomolecules can provide direction for further research to miR-140-5p sponging55. Our results further elucidated the reveal the mechanism of tumorigenesis and tumor progres- role of HIF1A-AS2 in renal carcinoma, and expanded the sion. However, the relationship between HIF1A-AS2 and kid- knowledge of the role of HIF1A-AS2 in diverse diseases. ney carcinoma has been rarely reported. A correlation with miR-30a-5p and HIF1A-AS2 in renal Studies have reported a mutual function between lncRNAs carcinomas remains unknown. Through bioinformatics anal- and miRNAs50-56, which was further confirmed by our report. yses, we identified a putative binding site between HIF1A-AS2 LncRNAs act as miRNAs sponges or baits to titrate miRNA and miR-30a-5p, and found that HIF1A-AS2 acted as the concentrations, thereby decreasing and preventing miRNAs sponge of miR-30a-5p, which directly bound to miR-30a-5p. from binding to specific mRNAs. Studies have also reported Furthermore, HIF1A-AS2 overexpression led to downregula- the roles of HIF1A-AS2 in tumors, such as HIF1A-AS2 tar- tion of miR-30a-5p, and HIF1A-AS2 knockdown upregulated geting miR-548c-3p in breast cancer31, sponging miR-129-5p the relative expression level of miR-30a-5p. The MiR-30a-5p in colorectal cancer and miR-33b-5p in osteosarcoma37,41, inhibitor partially reversed the effects, and miR-30a-5p mim- sponging miR-665 in osteogenic differentiation38, and spong- ics enhanced the effects induced by HIF1A-AS2 knockdown ing miR-153-3p in human umbilical vein cells56. Our findings on renal carcinoma cells. The miR-30a-5p directly targeted were consistent with these existing reports. SOX4 to reduce the relative protein expression levels of SOX4 The miRNAs are 18–25 nucleotides of non-coding RNAs, in renal carcinomas. Likewise, the relative protein expression which are involved in tumorigenesis and progression of var- level of SOX4 was upregulated during HIF1A-AS2 overexpres- ious diseases50-55. Based on our knowledge, miRNA plays an sion, to mediate the function of HIF1A-AS2. Overexpression indirect role in protein regulation, by binding to the 3′-UTR of HIF1A-AS2 reduced the relative expression of miR-30a-5p of specific mRNAs, to induce degradation or transcriptional and subsequently increased the relative protein expression of suppression of target genes. The miR-30a-5p was confirmed SOX4 at the post-transcriptional level in renal cancer cells. by bioinformatics analyses and was further verified by in the Silencing of SOX4 reversed the malignant renal carcinoma cell present study. MiR-30a-5p has been studied in numerous phenotype promoted by overexpressed HIF1A-AS2. diseases, such as cholangiocarcinoma42, osteoarthritis43, lung We elucidated the mutual function of HIF1A-AS2 and miR- cancer44-46, and colorectal cancer47,48. Furthermore, miR- 30a-5p in renal carcinomas, which could provide a novel bio- 30a-5p interacts with several ncRNAs, such as LINC0046143,44, marker and therapeutic target for the diagnosis and treatment NORAD45, DLEU246, LIN28B47, and FEZF1-AS148. Based on of renal carcinomas. We also demonstrated that the relative experimental results, miR-30a-5p as a negative regulatory expression of HIF1A-AS2 was significantly increased in renal agent participated in the progression of renal carcinoma. The carcinoma samples and cell lines. The relative expression of present study showed that miR-30a-5p inhibited the pro- HIF1A-AS2 was positively correlated with differentiation and gression and mediated the function of HIF1A-AS2 in renal the TNM stage in renal carcinomas. Downregulated expres- carcinomas. sion of HIF1A-AS2 inhibited renal carcinoma cell proliferation SOX4, the target gene of miR-30a-5p, was predicted by bio- or migration, and upregulated apoptosis. Overexpression of informatics analyses in addition to our experimental stud- HIF1A-AS2 showed the opposite effect. We have demonstrated ies. SOX4 belongs to the SOX transcription factor family, that HIF1A-AS2 enhanced the development and progression and binds to the A/TA/TCAAG motif to regulate target gene of renal cancers by promoting cell proliferation, migration, transcriptional activity via the high mobility group domain, and reducing apoptosis. Furthermore, HIF1A-AS2 sponged which regulates various biological functions, such as embry- miR-30a-5p in a ceRNA-dependent manner. Mechanistically, onic development and cell progression51-55. It regulates cell upregulation of HIF1A-AS2 decreased the relative expression differentiation, proliferation, and metastasis. SOX4 via the of miR-30a-5p and subsequently promoted the relative expres- lnc01694/miR-340-5p/Sox4 axis regulates gallbladder can- sion of SOX4 and WNT signaling at the posttranscriptional cer51. SOX4 is regulated by SNHGR-miR-489-3p competitive level. In summary, our study showed that HIF1A-AS2 acted as binding in acute myeloid leukemia52. Sox4 is targeted by lnc- a tumor promoter by miRNA sponging, and may therefore be NNT‑AS1/miR‑142‑5p axis in gastric cancer53, and lnc-FTX/ a potential therapeutic target in renal carcinomas. Cancer Biol Med Vol 18, No 2 May 2021 601

Conclusions 5. Clark DJ, Dhanasekaran SM, Petralia F, Pan J, Song X, Hu Y, et al. Integrated proteogenomic characterization of clear cell renal cell carcinoma. Cell. 2020; 180: 207. The results showed that HIF1A-AS2 sponged miR-30a-5p to 6. Zhu H, Wang S, Shen H, Zheng X, Xu X. SP1/AKT/FOXO3 closely regulate SOX4 expression, and subsequently acceler- signaling is involved in miR-362-3p-mediated inhibition of cell- ated the malignant phenotypes of renal carcinoma cells and cycle pathway and EMT progression in renal cell carcinoma. Front Cell Dev Biol. 2020; 8: 297. Wnt/β-catenin signaling, acting as an oncogene in the path- 7. Gargalionis AN, Sarlani E, Stofas A, Malakou LS, Adamopoulos C, ogenic mechanism of kidney carcinomas. Our results provide Bamias A, et al. Polycystin-1 induces activation of the PI3K/AKT/ useful pathways to further explore the pathogenesis of renal mTOR pathway and promotes angiogenesis in renal cell carcinoma. carcinoma progression and development. In conclusion, the Cancer Lett. 2020; 489: 135-43. results showed that the HIF1A-AS2-miR-30a-5p-SOX4 axis 8. Miao D, Margolis CA, Gao W, Voss MH, Li W, Martini DJ, et al. played significant roles in the progression and development Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma. Science. 2018; 359: 801-6. of renal carcinomas. HIF1A-AS2 and miR-30a-5p were the 9. He A, He S, Huang C, Chen Z, Wu Y, Gong Y, et al. MTDH novel and important tumor biomarkers, which could be used promotes metastasis of clear cell renal cell carcinoma by activating as diagnostic biomarkers and remedial targets for malignant SND1-mediated ERK signaling and epithelial-mesenchymal renal carcinomas. transition. Aging (Albany NY). 2020; 12: 1465-87. 10. Chow PM, Liu SH, Chang YW, Kuo KL, Lin WC, Huang KH. The Grant support covalent CDK7 inhibitor THZ1 enhances temsirolimus-induced cytotoxicity via autophagy suppression in human renal cell carcinoma. Cancer Lett. 2020; 471: 27-37. This work was supported by grants from the National Natural 11. Hamilton MJ, Young M, Jang K, Sauer S, Neang VE, King AT, et Science Foundations of China (Grant Nos. 81702511, 81472401, al. HOTAIRM1 lncRNA is downregulated in clear cell renal cell 81772708, and 2016YFA0201204), the Jiangsu Provincial Key carcinoma and inhibits the hypoxia pathway. Cancer Lett. 2020; Medical Discipline (Grant No. ZDXKA2016012), the Clinical 472: 50-8. Medicine Center of Suzhou (Grant No. SZZXJ201501), and 12. Wang C, Chen Y, Chen K, Zhang L. Long noncoding RNA LINC01134 promotes hepatocellular carcinoma metastasis via programs for Recruitment of Clinical Medical Top Team of activating AKT1S1 and NF-κB signaling. Front Cell Dev Biol. 2020; Suzhou. 8: 429. 13. Chen Y, Du H, Bao L. LncRNA PVT1 promotes ovarian cancer progression by silencing miR-214. Cancer Biol Med. 2018; 15: 238-50. Conflict of interest statement 14. Gong T, Li Y, Feng L, Fang M, Dai G, Huang X, et al. CASC21, a FOXP1 induced long non-coding RNA, promotes colorectal No potential conflicts of interest are disclosed. cancer growth by regulating CDK6. Aging (Albany NY). 2020; 12: 12086-106. 15. Cho SW, Xu J, Sun R, Mumbach MR, Carter AC, Chen YG, et al. References Promoter of lncRNA Gene HIF1A-AS2 is a tumor-suppressor DNA boundary element. Cell. 2018; 173: 1398-412.e22. 1. Barthel FP, Johnson KC, Varn FS, Moskalik AD, Tanner G, 16. Lü G, Li L, Wang B, Kuang L. LINC00623/miR-101/HRAS axis Kocakavuk E, et al. Longitudinal molecular trajectories of diffuse modulates IL-1 β-mediated ECM degradation, apoptosis and glioma in adults. Nature. 2019; 576: 112-20. senescence of osteoarthritis chondrocytes. Aging (Albany NY). 2. Moreno-Rubio J, Ponce S, Álvarez R, Olmedo ME, Falagan 2020; 12: 3218-37. S, Mielgo X, et al. Clinical-pathological and molecular 17. Chu Y, Liu Z, Liu J, Yu L, Zhang D, Pei F. Characterization of characterization of long-term survivors with advanced non-small lncRNA-perturbed TLR-signaling network identifies novel lncRNA cell lung cancer. Cancer Biol Med. 2020; 17: 444-57. prognostic biomarkers in colorectal cancer. Front Cell Dev Biol. 3. Yan C, Chang J, Song X, Yan F, Yu W, An Y, et al. Memory stem T 2020; 8: 503. cells generated by Wnt signaling from blood of human renal clear 18. Yu X, Ye X, Lin H, Feng N, Gao S, Zhang X, et al. Knockdown of cell carcinoma patients. Cancer Biol Med. 2019; 16: 109-24. long non-coding RNA inhibits autophagy in lung cancer. Cancer 4. Rose KM, Navaratnam AK, Faraj KS, Abdul-Muhsin HM, Syal Biol Med. 2018; 15: 228-37. A, Elias L, et al. Comparison of open and robot assisted radical 19. Jackson R, Kroehling L, Khitun A, Bailis W, Jarret A, York AG, et nephrectomy with level I and II inferior vena cava tumor al. The translation of non-canonical open reading frames controls thrombus: the mayo clinic experience. Urology. 2020; 136: 152-7. mucosal immunity. Nature. 2018; 564: 434-8. 602 Chen et al. HIF1A-AS2 accelerates RCC via sponging miR-30a-5p

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Supplementary materials

Table S1 The primers for real-time qPCR Gene Forward Primer sequence or Reverse HIF1A-AS2 Forward 5′-TCTGTGGCTCAGTTCCTTTTGT-3′

Reverse 5′-ATGTAGGAAGTGCCAGAGCC-3′

GAPDH Forward 5′-CGCTCTCTGCTCCTCCTGTTC-3′

Reverse 5′-ATCCGTTGACTCCGACCTTCAC-3 miR-30a-5p Forward 5′-AACGAGACGACGACAGAC-3′

Reverse 5′-GTAAACATCCTCGACTGGAAG-3′

SOX4 Forward 5′-AGCGACAAGATCCCTTTCATTC-3′

Reverse 5′-CGTTGCCGGACTTCACCTT-3′

Table S2 Antibodies used for Western blot Antibody Description Dilution Supplier Catalog Country SOX4 Mouse monoclonal 1:1000 Santa Cruz sc-130633 USA

Anti-VEGF Mouse monoclonal 1:1000 Santa Cruz sc-7269 USA

Anti-Met Mouse monoclonal 1:1000 Santa Cruz sc-8057 USA

Anti-β-catenin Mouse monoclonal 1:1000 Santa Cruz sc-7963 USA

Anti-c-myc Mouse monoclonal 1:500 Santa Cruz sc-40 USA

Anti-CyclinD1 Rabbit monoclonal 1:1000 Beyotime AF1183 China

Anti-Fra-1 Rabbit polyclonal 1:500 BBI D260339 China

Anti-β-Actin Mouse monoclonal 1:1000 Beyotime AF0003 China

VEGF, vascular endothelial growth factor; Met, MET proto-oncogene, receptor tyrosine kinase; β-catenin: catenin beta 1; Fra-1, FOS like 1, AP-1 transcription factor subunit; c-myc, MYC proto-oncogene, bHLH transcription factor. SOX4, SRY (sex determining region Y)-box 4.