European Review for Medical and Pharmacological Sciences 2020; 24: 12131-12143 Long noncoding RNA MAFG-AS1 facilitates the progression of hepatocellular carcinoma via targeting miR-3196/OTX1 axis Z.-Q. HU1,2, H.-C. LI2,3, F. TENG1,2, Q.-M. CHANG1,2, X.-B. WU1,2, J.-F. FENG1,2, Z.-P. ZHANG1,2 1Department of Hepatobiliary-Pancreatic Surgery, Minhang Hospital, Fudan University, Shanghai, China 2Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, Shanghai, China 3Department of Surgery, Minhang Hospital, Fudan University, Shanghai, China Zhiqiu Hu and Hongchang Li contributed equally to this work Abstract. – OBJECTIVE: Hepatocellular carci- Key Words: noma (HCC) is an invasive malignant tumor with MAFG-AS1, MiR-3196, OTX1, Hepatocellular carci- high mortality rate. Long non-coding RNA (ln- noma (HCC). cRNA) MAFG-AS1 has been showed to play an oncogenic role in several malignant tumors. Nonetheless, the exact role of MAFG-AS1 in the progression of HCC has not been fully elucidated. PATIENTS AND METHODS: Real-time quanti- Introduction tative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the mRNA and Primary liver cancer is the second leading protein expression of MAFG-AS1 in HCC tissues cause of death around the world1. In recent years, and cells. Cell counting kit-8 (CCK-8), transwell the incidence of liver cancer has been on the rise, and tubule formation assays were applied to un- 2 cover the proliferation, migration, invasion and with about 3% in women and 4% in men . Hepa- tumor angiogenesis of HCC cells, respectively. tocellular carcinoma (HCC), the most typical sub- RNA binding protein immunoprecipitation (RIP) type of primary liver cancer, possesses high mor- assay and Luciferase reporter gene assay were bidity and mortality worldwide3. Currently, tran- employed to explore the molecular mechanism. scatheter arterial embolization (TAE) and tran- In addition, Xenograft assay was used to investi- scatheter chemotherapy embolization (TACE) are gate the effect of MAFG-AS1 in vivo. RESULTS: MAFG-AS1 was highly expressed the primary interventional therapies for HCC pa- in HCC tissues and cells. Attenuation of MAFG- tients. Hepatectomy is more effective for patients AS1 evidently suppressed the proliferation, mi- with HCC at an early stage and those with sat- gration, invasion and tumor angiogenesis of isfactory liver function, while TACE is recom- HCC cells, suggesting that MAFG-AS1 played an mended for patients with advanced HCC4. Al- oncogenic role in HCC. MiR-3196 was sponged though it has been reported that TACE is more by MAFG-AS1, and OTX1 was a downstream tar- 5 get of miR-3196 in HCC. In addition, OTX1 ex- effective in the treatment of 3-5 cm tumors , the pression was negatively associated with miR- long-term survival rate of patients is still far from 3196 but positively associated with MAFG-AS1 satisfactory. TACE or TAE cannot significant- in HCC tissues. Overexpression of OTX1 could ly improve the survival rate of patients with liv- abolish the repressive influence of MAFG-AS1 er cancer in the middle stage6. Besides, the 5-year inhibition on the proliferation, migration, inva- survival rate of HCC patients is lower than 10%, sion and tumor angiogenesis of HCC cells. which remains dismal7. Therefore, it is meaning- CONCLUSIONS: MAFG-AS1 facilitated the progression of HCC via targeting miR-3196/ ful to explore the potential molecular mechanism OTX1 axis, which might be used as a new insight of HCC development and to uncover more effi- for HCC treatment. cient therapeutic targets. Corresponding Author: Ziping Zhang, MD; e-mail: [email protected] 12131 Z.-Q. Hu, H.-C. Li, F. Teng, Q.-M. Chang, X.-B. Wu, J.-F. Feng, Z.-P. Zhang Long noncoding RNAs (lncRNAs) are a type Cell Culture of RNA transcripts implicated in various bio- Human normal liver cell line L02 and human logical processes, such as cell proliferation, mi- HCC cell lines (HCCLM3, Hep3B, Huh7 and gration, invasion and apoptosis8,9. They serve as MHCC97-H) were obtained from Shanghai In- vital factors in malignant tumors. Of note, ln- stitutes for Biological Sciences, Chinese Acade- cRNA LINC01197, regulated by FOXO1, inhib- my of Sciences (Shanghai, China). All cells were its the proliferation of pancreatic adenocarcinoma cultured in Dulbeccoʼs Modified Eagleʼs Medi- cells via inactivating Wnt/β-catenin signaling10. um (DMEM) medium (Invitrogen, Carlsbad, CA, LINC00612 promotes the proliferation and in- USA) containing 100 μg/mL streptomycin, 100 U/ vasion of bladder cancer cells via sponging miR- mL penicillin, and 10% fetal bovine serum (FBS) 590 to upregulate PHF14 expression11. LncRNA (Gibco, Rockville, MD, USA) in an incubator at ZEB1-AS1 promotes the migration, invasion and 37°C with 5% CO2. proliferation, and inhibits the apoptosis of blad- der cancer as a competing endogenous RNA via Cell Transfection elevating the expression of fascin-1 by spong- Short hairpin RNA targeting MAFG-AS1 (sh- ing miR-200b12. HOXD-AS1 facilitates the epi- MAFG-AS1#1, sh-MAFG-AS1#2 and sh-MAFG- thelial to mesenchymal transition of ovarian can- AS1#3) and sh-NC were synthesized from San- cer cells through sponging miR-186-5p and regu- gon Biotech (Shanghai, China). For overexpres- lating PIK3R313. LncRNA MAFG-AS1 has also sion of miR-3196, miR-3196 mimics and NC mim- been confirmed to play a significant role in sever- ics were transfected into HCC cells. For overex- al malignancies. Sui et al14 has demonstrated that pression of OTX1, pcDNA3.1 vector (Genechem MAFG-AS1 aggravates the proliferation of lung Company, Shanghai, China) was subcloned with adenocarcinoma cells through targeting miR- OTX1 to construct pcDNA3.1/OTX1 vector. 744-5p/MAFG axis. MAFG-AS1 enhances the Empty pcDNA3.1 vector was employed as a neg- development of breast carcinoma via regulating ative control. Cell transfection was performed ac- miR-339-5p/MMP15 axis15. MAFG-AS1 drives cording to the instructions of Lipofectamine 2000 the progression of colorectal cancer by sponging (Invitrogen, Carlsbad, CA, USA). After 48 h of miR-147b and enhancing the expression of NDU- transfection, the cells were collected for subse- FA416. Nevertheless, the exact role of MAFG-AS1 quent experiments. in the progression of HCC and the possible under- lying mechanism have not been fully elucidated. Cell Proliferation Assay In the present study, we aimed to unveil the The viability of MHCC97-H and Huh7 cells role and function of MAFG-AS1 in HCC progres- was detected in strict accordance with cell count- sion. The results indicated that MAFG-AS1 facil- ing kit-8 (CCK-8) assay. Briefly, transfected cells itated the progression of HCC via targeting miR- were first seeded into 96-well plates, and cultured 3196/OTX1 axis. All our findings suggested that for 0 h, 24 h, 48 h, 72 h, and 96 h, respectively. MAFG-AS1 might become a promising therapeu- At appointed time points, 10 μL of CCK-8 reagent tic target for the treatment of HCC. (Dojindo Molecular Technologies, Kumamoto, Japan) was added to each well, followed by incu- bation for 4 h in the dark. Absorbance at 450 nm was finally evaluated by a micro-plate reader. Patients and Methods Transwell Assay Patients and Tissue Specimens The migration and invasion of HCC cells This investigation was approved by the Re- were examined by the use of chambers coated or search Ethics committee of Minhang Hospital, non-coated with Matrigel, respectively. Briefly, Fudan University. HCC tissues and adjacent nor- cells were plated into the upper chamber with se- mal tissues were obtained from patients. The se- rum-free DMEM. Meanwhile, complete DMEM lection of patients was based on the guideline pro- with 10% FBS was added to the lower chamber as posed by the Union for International Cancer Con- chemoattractant. After 24 h of incubation, cells trol (UICC). All patients did not receive adjuvant migrated or invaded through the membrane to the radiotherapy, chemotherapy, or immunotherapy lower chamber were stained with 0.1% crystal vi- before surgery. Informed written consent was ob- olet. Finally, the number of migrated or invaded tained from patients before the study. cells was counted under an inverted microscope 12132 Long noncoding RNA MAFG-AS1 in hepatocellular carcinoma (Olympus, Tokyo, Japan). 5 fields of view were tion Kit (Applied Biosystems, Foster City, CA, randomly selected for each sample. USA). Glyceraldehyde 3-phosphate dehydroge- nase (GAPDH) and U6 were used as internal refer- RNA Binding Protein ences for MAFG-AS1 and OTX1, and miR-3196, Immunoprecipitation (RIP) Assay respectively. The relative expression of genes was RIP assay was conducted according to the in- calculated by the 2-ΔΔCt method. Primer sequenc- structions of a Magna RIP RNA-Binding Pro- es used in this study were as follows: MAFG- tein Immunoprecipitation reagent kit (Merck AS1 forward: 5’-ATGACGACCCCCAATA- Millipore, Billerica, MA, USA). HCC cells were AAGGA-3’, and reverse: 5’-CACCGACATG- lysed in RIP buffer and incubated with magnetic GTTACCAGC-3’; miR-3196 forward: 5’-CCT- beads conjugated to Ago2 antibody (ab32381; Ab- GTGTATGCATCCTCGACTG-3’, and reverse: cam Inc., Cambridge, MA, USA) or IgG antibody 5’-CTGGCGTGTAATGGAGTCG-3’; OTX1 (ab172730; 1:5000; Abcam Inc., Cambridge, MA, forward: 5′-CTGCTCTTCCTCAATCAAT- USA). Finally, immunoprecipitated RNAs were GG-3′, and reverse: 5′-ACCCTGACTTGTCT- evaluated by real-time quantitative polymerase GTTTCC-3′; GAPDH forward: 5′-ATGGGAC- chain reaction (RT-qPCR). GATGCTGGTACTGA-3′, and reverse: 5′-TGCT- GACAACCTTGAGTGAAAT-3′;