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Am J Transl Res 2021;13(5):5702-5719 www.ajtr.org /ISSN:1943-8141/AJTR0126990 Original Article Knocking down LINC01116 can inhibit the regulation of TGF-β through miR-774-5p axis and inhibit the occurrence and development of glioma Jinfeng Lou1,2*, Pengfei Wang3*, Keliang Chang1, Genwei Wang1, Xiaoteng Geng1, Yang Wu2,4, Wanhong Zhang5, Guangming Niu1 1Department of Neurosurgery, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, Henan Province, China; 2Henan Provincial Engineering Research Center of Natural Drug Extraction and Medical Technology Application, Zhengzhou Railway Vocational & Technical College, Zhengzhou 451460, Henan Province, China; 3Microsurgery, The First Affiliated Hospital of Xinxiang Medical College, Weihui 453100, Xinxiang, Henan Province, China; 4Department of Neurosurgery, The People’s Hospital of Minquan County, Minquan 476800, Henan Province, China; 5Department of Neurosurgery, Kaifeng Central Hospital, Kaifeng 475000, Henan Province, China. *Co-first authors. Received November 25, 2020; Accepted February 21, 2021; Epub May 15, 2021; Published May 30, 2021 Abstract: Background: Many studies have shown that non-coding RNAs (ncRNAs), including long non-coding RNA (LncRNA) and micro RNA (miRNA), play a crucial regulatory role in glioma. LINC01116 is a newly discovered LncRNA, and the relationship between LncRNA and glioma is still under exploration. Method: LncRNAs with potential dif- ferences were screened through GEO database, and the expressions of LINC01116 and miR-744-5p/TGF-β1 in glioma tissues were tested using qRT-PCR. Changes in proliferation and migration/invasion of glioma were tested using CCK-8 and transwell assay. The expression changes of TGF-β1 were tested using qRT-PCR and Western blot. Targeted binding among LINC01116, miR-744-5p and TGF-β1 was verified using double luciferase reporter, RNA Immunoprecipitation (PIR) and RNA pull-down experiments. The effect of LINC01116 on tumor growth was deter- mined by tumor allografting test. Results: GEO database and clinical research revealed that the expression level of LINC01116 in glioma increased, and the elevation of LINC01116 was closely related to the adverse prognosis of clinical patients. Functional experiments showed that the inhibition of LINC01116 could up-regulate miR-744-5p- mediated proliferation and metastasis of glioma cells. Western blot analysis and qRT-PCR analysis showed that LINC01116 regulated TGF-β1 by mediating miR-744-5p. Further cell behavior experiments showed that LINC01116 acted as miR-744-5p sponge to inhibit proliferation and metastasis caused by TGF-β1. Finally, the analysis of animal models in vivo showed that LINC01116 could regulate the tumor growth of glioma. Conclusion: LncRNA LINC01116 acts as an oncogene and promotes TGF-β1 mediated proliferation and metastasis by acting as competitive endog- enous RNA (ceRNA) in glioma. Keywords: LINC01116, miR-744-5p, TGF-β1, glioma, ceRNA Introduction glioma patients is still poor [5]. The data have shown that the median survival time of glioma Gliomas are common primary malignancies in patients is usually within 12 months after diag- brain, and it accounted for 70% of primary nosis, and even under the most favorable con- malignancies in brain [1]. There are data show- ditions, the estimated survival time will not ing that 4-5 out of 100,000 people suffer from exceed 2 years [6]. Therefore, an increasing the disease every year [2]. At present, the clini- number of researches are devoted to exploring cal therapy of glioma mainly includes surgery, the potential mechanism of gene regulation of radiotherapy and chemotherapy [3]. Among glioma and developing more effective thera- them, surgical resection combined with radio- peutic schemes. therapy and temozolomide adjuvant chemo- therapy is the first choice [4]. Although a diag- Long-chain non-coding RNA (LncRNA) is one of nosis and treatment plan for glioma has been the members of non-coding RNA, with the formulated in recent ten years, the prognosis of length exceeds 200nt [7]. Studies have found LINC01116 inhibits the growth of glioma that LncRNA can regulate the expression level Hospital from May 2012 to May 2014 were col- of transcribed and post-transcribed genes, lected during surgery. The samples were then resulting in protein inactivation or instability [8]. transported in liquid nitrogen at -80°C for later Many studies have proved that LncRNA can use. The informed consent forms were obtained regulate the growth and metastasis of glioma. from the patients and their families. This study For example, [9] LncRNA UCA1 interacts with conformed to the ethics committee of Kaifeng miR-182 to regulate glioma proliferation and Central Hospital and was in accordance with migration by targeting iASPP. Other studies Helsinki Declaration. have found that [10] LncRNA HSP90AA1-IT1 promotes glioma through targeted miR-885- Cell culture 5p-CDK2 pathway. LINC01116 is a newly dis- covered LncRNA and is found to be highly NHA (normal human astrocytes), U251, H4, expressed in mammary carcinoma [11], ovari- SW1783 and LN229 (glioma cells) were an carcinoma [12] and osteogenic sarcoma obtained from ATCC (USA), and the collected [13]. Moreover, some studies found that [14] cells were cultured in DMEM (FBS; Sigma- LINC01116 can regulate the tumorigenesis of Aldrich, St.Louis, MO, USA) including 10% fetal glioma by targeting VEGFA, indicating that bovine serum. The cells were cultured in an LINC01116 plays an important role in the incubator at 37°C with 5% CO2. occurrence of glioma, but we are still unclear about its in-depth mechanism. Cell transfection Competing endogenous RNA (ceRNA) is a newly Cells were transfected using Lipofectamine proposed theory, and it combines with microR- 2000 (Invitrogen, Carlsbad, CA, USA). Glioma NA (miR) through response elements to affect cell lines were transfected with siRNA. siRNA miR-induced gene silencing [15]. Studies (si-NC, si-LINC01116, si-TGF-β1), miRNA mi- revealed that [11] LncRNA can restrain the metic inhibitors (miR-NC, miR-744-5p-mimics, growth of tumor by binding to miR with specific miR-744-5p-inhibit) and over-expression plas- binding sites. In this research, we found that mids (pcDNA, pcDNA-LINC01116) were syn- miR-744-5p and LINC01116 have targeted thesized by Shanghai Gene Pharmaceutical binding sites through online prediction website Co., Ltd. In order to establish a nude mouse for LINC01116 to bind miR. Previous reports model, we also constructed a human recombi- indicated that miR-744-5p can target down- nant lentivirus vector (SH-LINC01116) stably stream target genes to inhibit the development expressed by LINC 01116, packaged the lenti- of glioma. virus carrying sh-LINC01116 in human embry- onic kidney cell line 293T, and collected the Therefore, this study aimed to explore whether virus. U87, U251 and LN229 cells were infected LINC01116 can inhibit the growth of glioma by with lentivirus, then puromycin was selected to regulating miR-744-5p and can provide poten- establish a stable cell line, and the blank vector tial targets for clinical treatment. was used as the control (sh-NC). Methods and data qRT-PCR GEO database analysis The obtained samples (tissues, cells) were extracted with TRIzol reagent to obtain total We logged into https://www.ncbi.nlm.nih.gov/ RNA. The purity, concentration and integrity gds/to get GSE103227 chip and obtain matrix were detected using UV spectrophotometer file Series Matrix File(s). LINC01116 in matrix and agarose gel electrophoresis. cDNA was file was extracted and the expression level of synthesized from 1 µg RNA of random primers LINC01116 in glioma was further analyzed. In addition, GSE103229 chip was selected to using a high capacity cDNA reverse transcrip- extract miR-744-5p in matrix file and further tion kit (Applied Biosystems, USA). One-step analyze its expression level in glioma. SYBR PrimeScript RT-PCR and ABI 7500 PCR were used for amplification. The amplification Collection of clinical samples system and amplification conditions were con- figured and operated according to the kit A total of 50 pairs of glioma samples and PTBE instructions. PCR amplification was carried out tissues of patients treated in Kaifeng Central for a total of 40 cycles, with 3 repeat wells for 5703 Am J Transl Res 2021;13(5):5702-5719 LINC01116 inhibits the growth of glioma each sample, and the experiment was carried fected cells were collected, adjusted to 1 * 103 out for 3 times. In this study, GAPDH was cells/well, and inoculated in a 6-well plate. applied as internal reference for LncRNA and After incubation for 24 hours, cells were har- mRNA, U6 for miR. 2-ΔΔct was used to analyze vested and rinsed twice with phosphate buff- the data [16]. ered saline. Then the cells were resuspended in Annexin V-fluorescein isothiocyanate (Annexin Western blotting (WB) V-FITC; 5 μL) and propidium iodide (5 μL) by Annexin V-FITC cell apoptosis detection kit. The The collected cells were extracted with RIPA apoptosis rate was measured by FACS Calibur. buffer solution to obtain total protein, the pro- tein concentration was adjusted to 4 μg/μL Double fluorescein report using BCA kit, and 12% SDS-PAGE was us- ed for electrophoresis separation. The protein LINC01116 and TGF-β1 fragments containing was transferred to PVDF membrane after ion- miR-744-5p binding sites and wild-type or ization, stained with Ponceau S working solu- mutant sequences of binding sites were tion, rinsed in PBST for 5 min, washed, and cloned into pmirGLO bifluorescase vectors to sealed with 5% defatted milk powder for 2 h. obtain report vector wild-type (LINC01116-WT, anti-TGF-β1 and anti-GAPDH (overnight at 4°C) TGF-β1-WT) and mutant (LINC01116-MUT, were incubated with HRP coupled secondary TGF-β1-MUT). LINC01116/TGF-β1 wild-type antibody (1:5000) for 2 h to test the blot. WB and mutant vectors and miR-744-5p-mimics or was observed by enhanced chemilumines- miR-NC were co-transfected into glioma using cence system.

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