European Review for Medical and Pharmacological Sciences 2020; 24: 2539-2547 Upregulation of lncRNA DANCR functions as an oncogenic role in non-small by regulating miR-214-5p/CIZ1 axis

Y.-R. CHEN1, Y.-S. WU2, W.-S. WANG3, J.-S. ZHANG4, Q.-G. WU5

1Shanghai Top Medicine & Technology Co. Ltd, Shanghai, China 2Department of Critical Care Medicine, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China 3Department of Respiratory Medicine, The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China 4Department of Respiratory Medicine, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China 5Department of Respirology, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China

Abstract. – OBJECTIVE: Lung cancer has Introduction an unfavorable prognosis due to the lack of ef- ficient diagnostic and therapeutic strategies. In the past 50 years, the incidence and mortality of Therefore, this study sought to figure out the ef- lung cancer have increased year by year, surpassing fect of long non-coding RNA (lncRNA) DANCR other types of tumors. According to population stati- on lung cancer progression. PATIENTS AND METHODS: LncRNA DAN- stics, the mortality of lung cancer in male population CR and miR-214-5p expressions in non-small comes to the first place among all malignant tumors cell lung cancer (NSCLC) were detected by Re- and which in the female population occupies the se- al Time-quantitative Polymerase Chain Reac- cond place1,2. Non-small cell lung cancer (NSCLC) tion (RT-qPCR). Function assays, including Cell is one of the most common subtypes of lung cancer. Counting Kit-8 (CCK-8) and flow cytometric anal- Finding effective therapeutic targets and providing ysis were conducted to clarify the role of DAN- CR and miR-214-5p in the progression of NS- new treatment for NSCLC are still emerging. CLC. Western blot, Dual-Luciferase reporter as- LncRNAs have been reported to exert diffe- say, and RNA immunoprecipitation assay (RIP) rent functions in diverse cancer types. LncRNA were performed to elucidate the underlying HANR is reported to function as an oncogene and mechanism. strengthen chemoresistance in hepatocellular car- RESULTS: LncRNA DANCR was upregulated cinoma3. LncRNA PEG10 has biological functions in NSCLC. The knockdown of lncRNA DANCR inhibited cell proliferation and accelerated cell in cell proliferation and invasion in esophageal can- 4 in NSCLC. LncRNA DANCR interact- cer . LncRNA AWPPH serves as an oncogenic role ed with miR-214-5p. MiR-214-5p over-expression in hepatocellular carcinoma by regulating YBX15. partially reversed the regulatory effects of DAN- In the current study, we determined that DAN- CR on proliferation and apoptosis in NSCLC. In CR was overexpressed in NSCLC. MiR-214-5p addition, CIZ1 was the downstream bind- ing miR-214-5p. LncRNA DANCR could regulate was downregulated in NSCLC. Besides, DANCR the miR-214-5p/CIZ1 axis. promoted cell proliferation and inhibited cell CONCLUSIONS: Downregulation of lncRNA apoptosis by targeting the miR-214-5p/CIZ1 axis. DANCR inhibited cell proliferation and induced cell apoptosis in NSCLC by regulating the miR- 214-5p/CIZ1 axis. LncRNA DANCR may act as Patients and Methods an oncogene and promote the progression of NSCLC. Clinical Specimens and Cell Lines Key Words: A total of 100 NSCLC specimens were obtai- LncRNA, DANCR, MiR-214-5p, CIZ1, NSCLC. ned from the patients who received surgery in

Corresponding Author: Qingguo Wu, MM; e-mail: [email protected] 2539 Y.-R. Chen, Y.-S. Wu, W.-S. Wang, J.-S. Zhang, Q.-G. Wu

Shanghai Public Health Clinical Center, Fudan Cell-Counting Kit-8 Assay (CCK-8) University from 2016 to 2018. The written in- The Cell Counting Kit-8 assay was involved in formed consent was achieved, and this study examining the cell proliferation in NSCLC. The was supported by the Ethics Committee of transfected cells were planted into 96-wells plates Shanghai Public Health Clinical Center, Fudan (6 × 103/well) and then, CCK-8 solution (Beyo- University. time, Shanghai, China) (10 µl/well) was used to Human healthy bronchial epithelial cell li- stain cells for 2 hours at 37°C. The optical density ne (16HBE) was obtained from Cell Bioscience (OD) value (450 nm) was then evaluated. Inc. (Shanghai, China). NSCLC cell lines (A549, SPCA1, H1299, and H358) were obtained from the Chinese Academy of Science Cell Bank (Shan- Flow Cytometric Analysis ghai, China). The cells were cultured in Roswell To detect the apoptotic cells, the Annexin Park Memorial Institute-1640 (RPMI-1640) me- V-FITC/PI Apoptosis Detection Kit (Vazyme, dium (Thermo Fisher Scientific, Waltham, MA, Nanjing, China) was performed according to the USA) containing 10% fetal bovine serum (FBS; instruction. The flow cytometric analysis was ta- Life Technologies, Gaithersburg, MD, USA) and ken place at BD FACSCanto II (BD Biosciences, 1% penicillin in a humidified incubator that con- Franklin Lakes, NJ, USA) flow cytometry. taining 5% CO2 at 37°C. Cell Transfection Western Blot The lentiviral targeting lncRNA DANCR was The was extracted from cells by Re- cloned into the vector, which upregulated ln- agent radioimmunoprecipitation assay (RIPA; cRNA DANCR (Biosettia Inc., San Diego, CA, Beyotime, Shanghai, China). After SDS-PAGE, USA). For knockdown DANCR, the selected cells the protein samples were transferred on poly- were transfected with DANCR siRNA following vinylidene difluoride (PVDF) membranes (Ro- the standard protocol. The cells were transfected che, Basel, Switzerland). Cell Signaling Tech- by miRNA mimics or inhibitor which were pur- nology (CST, Danvers, MA, USA) provided us chased from GenePharma Co., Ltd. (Shanghai, rabbit anti-GAPDH and rabbit anti-CIZ1, as well China). as goat anti-rabbit secondary antibody. The mem- branes were blocked in 5% skim milk and incu- RNA Extraction and Real bated with primary and secondary antibodies. Time-quantitative Polymerase The grey values of the exposed bands were de- Chain Reaction (RT-qPCR) tected by the chemiluminescent film with Image The total RNA was separated by TRIzol rea- J software (NIH, Bethesda, MD, USA). gent (Invitrogen, Carlsbad, CA, USA), and was reversely transcribed to complementary deoxyri- Dual-Luciferase Reporter Assay bose nucleic acids (cDNAs) by Transcription Kit Binding sequences in 3ʹ-untranslated region (TaKaRa Biotechnology, Dalian, China). QRT- (3ʹ-UTR) were amplified and cloned in the pGL3 PCR reaction conditions were as follows: 94°C vector (Promega, Madison, WI, USA), namely for 30 s, 55°C for 30 s and 72°C for 90 s, for a total WT 3ʹ-UTR. Quick-change site-directed muta- of 40 cycles. The relative expression level of the genesis kit (Stratagene, La Jolla, CA, USA) was target gene was expressed by 2-ΔΔCt. The primers used for site-directed mutagenesis in binding se- were as follows: DANCR-forward: 5’-CTGCAT- quences, that were MUT 3ʹ-UTR. The cells were TCCTGAACCGTTATCT-3’, DANCR-reverse: co-transfected with WT-3ʹ-UTR/MUT-3ʹ-UTR 5’-GGGTGTAATCCACGTTTCTCAT-3’. MiR- and NC/miR-214-5p mimics for 48 h, followed 214-5p-forward: 5’-TCTCTTGCTATAGAAGCA- by determination of Luciferase activity CAAC-3’, miR-214-5p-reverse: 5’-TCCTCCACA- ATCATGCTGTGT-3’; U6-forward: 5’-GCTTCG- GCAGCACATATACTAAAAT-3’, U6-reverse: RNA Immunoprecipitation Assay (RIP) 5’-CGCTTCAGAATTTGCGTGTCAT-3’; glyce- By using Magna RIP RNA-Binding Protein raldehyde 3-phosphate dehydrogenase (GAPDH)- Immunoprecipitation Kit (Millipore, Billerica, forward: 5’-CGCTCTCTGCTCCTCCTGTTC-3’, MA, USA), RIP assay was conducted according GAPDH-reverse: 5’-ATCCGTTGACTCCGAC- to the protocol. Co-precipitated RNAs were as- CTTCAC-3’. sessed by RT-qPCR assay.

2540 RNA-DANCR in NSCLC

Statistical Analysis remarkably enriched in the lncRNA DANCR The data were presented as mean ± SD (stan- group, revealing that lncRNA DANCR might dard deviation). Statistical Product and Service function as a miR-214-5p sponge (Figure 2D). Solutions (SPSS) 17.0 (SPSS Inc., Chicago, IL, USA) was used for statistically analysis. Chi-squa- MiR-214-5p Overexpression re test, analysis of variance (ANOVA) test, and Repressed Cell Proliferation and the Kaplan-Meier were performed. *p<0.05 was Induced Cell Apoptosis considered statistically significant. MiR-214-4p was lowly expressed in NSCLC tissues and cells (Figures 3A, 3B). The tran- sfection efficacy of miR-214-4p mimics was Results firstly verified (Figure 3C). Later, CCK-8 and flow cytometric assay were conducted to inve- DANCR Was Over-Expressed in stigate the effect of miR-214-5p on cell prolife- NSCLC Tissues and Cell Lines ration and apoptosis. These assays showed that For determination of the expression level of upregulated miR-214-5p repressed cell prolife- DANCR, the qRT-PCR assay was involved. ration (Figure 3D) and induced cell apoptosis DANCR was significantly upregulated in tumor (Figure 3E). specimens (Figure 1A). Similarly, we examined the expression of DANCR in NSCLC cell lines. It CDKN1A Interacting Zinc Finger turned out that NSCLC cell lines had a relatively Protein 1 (CIZ1) Was a Downstream higher expression level of DANCR in comparison Target of miR-214-5p with the human normal bronchial epithelial cell Three publicly available databases Target- line (Figure 1B). qRT-PCR was used to detecting Scan, miRWalk, and MiRanda were utilized to the transfection efficiency of si-DANCR, and the predict downstream target of miR-214-5p. We transfection of si-DANCR effectively reduced the considered that CIZ1 was a potential target of DANCR level (Figure 1C). miR-214-5p. Using a similar way, CIZ1 was verified to be the downstream gene binding miR-214-5p (Figure 4A). Furthermore, RT-PCR Knockdown of DANCR Suppressed results showed that CIZ1 expression in the cells Cell Proliferation and Induced Cell transfected with miR-214-5p mimics was redu- Apoptosis In Vitro ced (Figure 4B). Meanwhile, through the We- Subsequently, we conducted CCK-8 assay stern blot assay, it was found that the protein to examine cell proliferation. As Figure 1D level of CIZ1 could be inhibited by upregulating showed, DANCR downregulated group had a miR-214-5p (Figure 4C). relatively lower OD value compared with the control group. We examined the apoptotic cells through flow cytometric analysis, and it revea- DANCR Exerted its Function Via led that downregulated DANCR promoted cell Regulating miR-214-5p/CIZ1 Axis apoptosis in vitro (Figure 1E). Taken together, Additionally, the reduction of the Luciferase we considered that the knockdown of DANCR activity caused by the co-transfection of CIZ1- led to suppression in cell proliferation and pro- wild type and miR-214-5p mimics could be partly motion in cell apoptosis. cancelled via over-expressing lncRNA DANCR (Figure 5A). To investigate the interaction betwe- LncRNA DANCR Interacted en lncRNA DANCR and CIZ1, we also found With miR-214-5p that CIZ1 expression in cells transfected with By online publicly available databases, we si-DANCR was lower than that of the control found that DANCR may have underlying inte- cells (Figures 5B, 5C). raction with miR-214-5p (Figure 2A). Besides, miR-214-5p was upregulated in cells transfected with si-DANCR (Figure 2B). Dual-Luciferase Discussion reporter assay showed the decreased activity of after co-transfection of DANCR-wild type and LncRNAs have been proven to have multiple miR-214-5p mimics (Figure 2C). The result of functions in tumor progression. LncRNAs have the RIP assay showed that miR-214-5p could be been used for novel biomarkers and therapeutic

2541 Y.-R. Chen, Y.-S. Wu, W.-S. Wang, J.-S. Zhang, Q.-G. Wu

Figure 1. LncRNA DANCR was upregulated in NSCLC tissues and cell lines and the knockdown of DANCR suppressed cell proliferation and induced cell apoptosis in vitro. A, RT-qPCR analysis of lncRNA DANCR expression in NSCLC tissues and the adjacent tissues. B, RT-qPCR analysis of lncRNA DANCR expression in NSCLC cell lines. C, LncRNA DANCR expression was downregulated by si-DANCR transfection. D, CCK-8 analysis of the effect of lncRNA DANCR on cell proliferation. E, Flow cytometric analysis of the effect of lncRNA DANCR on cell apoptosis. *p<0.05, **p<0.01, ***p<0.001.

2542 RNA-DANCR in NSCLC

Figure 2. LncRNA DANCR was interacted with miR-214-5p. A, By analyzing online databases, we found that there was a potential functional relationship between miR-214-5p and lncRNA DANCR. B, Increased miR-214-5p was exhibited in cells transfected with si- DANCR. C, Dual-Luciferase reporter showed the reduction activity of the cells co-transfected with lncRNA DANCR-wild type and miR-214-5p mimics. D, The result of RIP assay showed that miR-214-5p could be remarkably enriched in the lncRNA DANCR group. **p<0.01. targets for many cancers6. DANCR plays a vi- Many investigations have proven the inte- tal role in regulating the progression process of raction between lncRNAs and miRNAs in ma- diverse cancer types7. In particular, DANCR is lignant behaviors of cancer. LncRNA TUG1 of significance during the progression of lung affects cell proliferation and migration in pa- cancer8. As a ceRNA, DANCR exerts a functio- pillary thyroid cancer by negatively regulating nal effect on osteosarcoma progression9. In this miR-14510. LINC00319 is reported to play an study, we found that DANCR was upregulated in oncogenic role in ovarian cancer via regulating NSCLC. The knockdown of DANCR inhibited miR-423-5p/NACC1 axis11. Herein, in the current cell proliferation and induced cell apoptosis in study, miR-214-5p was predicted to be a potential NSCLC. target of DANCR. Through a series of functional

2543 Y.-R. Chen, Y.-S. Wu, W.-S. Wang, J.-S. Zhang, Q.-G. Wu

Figure 3. Overexpression of miR-214-5p repressed cell proliferation and induced cell apoptosis. A, B, MiR-214-5p was significantly downregulated in tumor tissues and cell lines.C, MiR-214-5p expression was upregulated after transfection with miR-214-5p mimics. D, CCK-8 analysis of the effect of miR-214-5p on cell proliferation. E, Flow cytometric analysis of the effect of miR-214-5p on cell apoptosis. *p<0.05, **p<0.01, ***p<0.001.

2544 RNA-DANCR in NSCLC

Figure 4. CDKN1A interacting zinc finger protein 1 (CIZ1) was a direct downstream target of miR-214-5p. A, The databases online (starBase and miRDB) predicted that CIZ1 might be a downstream target of miR-214-5p and the Luciferase reporter assay uncovered that co-transfection of CIZ1-wild type and miR-214-5p mimics decreased the Luciferase activity. B, RT-qPCR results showed that CIZ1 expression in the cells transfected with miR-214-5p mimics was reduced. C, Western blot analysis of the expression levels of CIZ1 expression in cells transfected with miR-214- 5p mimics. **p<0.01. experiments, we indicated that miR-214-5p was Similarly, the potential target of miR-214-5p a target of DANCR. MiR-214-5p over-expression was predicted, and CIZ1 was screened out. CIZ1 suppressed cell proliferation and accelerated cell is vital in the regulation of cell proliferation apoptosis in NSCLC. and progression via interacting with

2545 Y.-R. Chen, Y.-S. Wu, W.-S. Wang, J.-S. Zhang, Q.-G. Wu

Figure 5. DANCR exerted its function via regulating miR-214-5p/CIZ1 axis. A, The reduction of the Luciferase activity caused by co-transfection of CIZ1-WT and miR-214-5p mimics could be partly reversed via upregulating lncRNA DANCR. B, RT-qPCR analysis of the expression of CIZ1 responding to lncRNA DANCR downregulation. C, Western blot analysis of the expression of CIZ1 responding to lncRNA DANCR downregulation. *p<0.05, **p<0.01. regulatory proteins12. Hence, CIZ1 is regarded and induced cell apoptosis in vitro. MiR-214-5p as a driver of tumor growth. CIZ1 is reported to was validated to interact with DANCR, and its function as an oncogene in gallbladder cancer13. overexpression repressed cell proliferation and In hepatocellular carcinoma, CIZ1 regulates cell accelerated cell apoptosis. Besides, CIZ1 was proliferation and cell migration14. Our findings determined to be a downstream target of miR- illustrated that CIZ1 was a downstream gene bin- 214-5p. In sum, our research showed that lncRNA ding miR-214-5p. DANCR served as an oncogenic role in NSCLC via regulating the miR-214-5p/CIZ1 axis. Conclusions

In this study, we first detected that lncRNA DANCR was upregulated in NSCLC. The knock- Conflict of Interest down of DANCR inhibited cell proliferation The Authors declare that they have no conflict of interests.

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