HIF-1α-activated TM4SF1-AS1 Promotes The Proliferation,Migration,and Invasion Of Hepatocellular Carcinoma Cells By Enhancing TM4SF1 Expression

Objective: Liver cancer is the sixth most common form of cancer diagnosis globally and the third leading cause of cancer-related death worldwide,with about 90% of these cancers being hepatocellular carcinoma(HCC).Long Non-Coding RNA(lnc RNA)is a kind of RNA molecules by the genome transcribed,that is more than 200 bp in length and usually lack of coding protein ability.The dysfunction of lnc RNA expression is closely related to the occurrence and progression of cancer.Hypoxia is not only a common feature of solid tumors,but also one of the causes of malignant behavior and drug resistance of hepatocellular carcinoma.Hypoxia-inducible factors(HIFs)is the main transcription factor that activates the expression of target genes,which mediates the proliferation and metastasis of hepatocellular carcinoma induced by hypoxia.The purpose of this study is to find the effect of hypoxia on lnc RNA TM4SF1-AS1 in hepatocellular carcinoma and the effect of lnc RNA TM4SF1-AS1 on proliferation,migration,invasion of hepatocellular carcinoma.To explore the potential mechanism of lnc RNA TM4SF1-AS1 affecting the biological function of hepatocellular carcinoma,in order to develop new biomarkers and new therapeutic targets for hepatocellular carcinoma.Methods:(1)Through bioinformatics analysis,to explore the difference of lnc RNA TM4SF1-AS1 expression level between hypoxia and normoxia.(2)The expression of lnc RNA TM4SF1-AS1 was detected by real-time quantitative PCR(q RT-PCR)in normal cell line of liver MIHA and HCC cell line Hep3 B,Huh7,Hep G2.(3)Western blotting test(WB)and q RT-PCR were used to determine the changes of lnc RNA TM4SF1-AS1 expression under hypoxia and the condition of inhibiting HIF-1α degradation.(4)HIF-1αand HIF-2α were silenced respectively in Hep3 B cell line,and the expression of lnc RNA TM4SF1-AS1 was detected by q RT-PCR.(5)Construction of Hep3 B and Huh7 cell lines with stable downregulation of lnc RNA TM4SF1-AS1,the changes of cell proliferation,migration and invasion were detected by cell proliferation test(CCK-8),Ed U test and Transwell test.(6)CCK-8 test,Ed U test and Transwell test were used to detect whether knocking down lnc RNA TM4SF1-AS1 could block the malignant behavior of Hep3 B cells induced by hypoxia.(7)Explore whether TM4SF1 is a downstream target of lnc RNA TM4SF1-AS1 by bioinformatics analysis and WB and q RT-PCR.(8)Construction of Hep3 B cell line overexpressing TM4SF1,Through WB,q RT-PCR,CCK-8 tests,Ed U tests,and Transwell tests to explore whether TM4SF1 is a functional effector of lnc RNA TM4SF1-AS1 in hepatocellular carcinoma.Results: In this study,we found that lnc RNA TM4SF1-AS1 was highly expressed in hepatocellular carcinoma tissue samples and cell lines.The expression of lnc RNA TM4SF1-AS1 was up-regulated under hypoxia,and further,we found that HIF-1α rather than HIF-2α activated the transcription of lnc RNA TM4SF1-AS1 in hepatocellular carcinoma cells.In addition,the ability of migration,invasion and proliferation of hepatoma cells was significantly decreased after knocking down lnc RNATM4SF1-AS1,and the silencing of lnc RNA TM4SF1-AS1 counteracted the malignant behavior of hepatoma cells enhanced by hypoxia.We demonstrated that lnc RNA TM4SF1-AS1 silencing significantly down-regulated the expression of TM4SF1 in hepatocellular carcinoma cells,but knocking down lnc RNA TM4SF1-AS1 did not change the m RNA degradation of TM4SF1.Then we verified that the up-regulated TM4SF1 induced by hypoxia can be significantly reversed by knocking down lnc RNA TM4SF1-AS1.According to the analysis of TCGA data,we determined that the expression of TM4SF1 m RNA and lnc RNA TM4SF1-AS1 was positively correlated in hepatocellular carcinoma.In Hep3 B cells,overexpression of TM4SF1 significantly restored the inhibition of proliferation,migration and invasion of hepatoma cells produced by knocking down lnc RNATM4SF1-AS1.Conclusion: lnc RNA TM4SF1-AS1 is a hypoxia response factor regulated by HIF-1α transcription.lnc RNA TM4SF1-AS1 promotes the migration,invasion and proliferation of hepatocellular carcinoma by up-regulating the level of TM4SF1.This article provides a new perspective for the study of the pathogenesis and progress of hepatocellular carcinoma.