The Biological Function And Mechanism Of Circ3596 In Hepatocellular Carcinoma

Background and aim:Liver cancer is the fifth most common cancer in the world and the third leading cause of cancer-related deaths.And at the same time,China is with a high incidence.Among the primary liver cancer,hepatocellular carcinoma(HCC)is the major histological subtype and account for 80-90%.At present,various therapeutic methods have made great progresses,but the recurrence and metastasis of HCC has always been an important factor affecting the survival rate of patients.About 90% of the deaths of cancer patients are caused by metastasis.Therefore,blocking HCC metastasis is a key point to reduce mortality and improve prognosis.In-depth study of the mechanism of HCC metastasis is the basis for finding effective blocking targets.But tumor metastasis is the final product of a multi-steps biological process,including invasion and migration from the primary focus,survival and diffusion in the circulatory system,and reaching and proliferation in metastatic organ.Each step is regulated by complex mechanisms,which brings difficulties and challenges to the study of tumor metastasis.Circular RNAs(circ RNAs)is one kind of non-coding RNAs,and are important epigenetic regulatory factors.With the rapid development of sequencing technology and bioinformatics technology,a large number of circ RNAs have been identified.And action mode of them has been revealed,including function as mi RNA sponge,acting as a molecular scaffold to provide a reaction platform,regulating the expression of parental genes,acting as m RNA to encode peptides.Circ RNAs play important regulatory roles in tumor formation and progression,but its roles in tumor metastasis need to be further explored.This study aims to find a circ RNA molecule that have an important role in the HCC metastasis and explore its biological function as well as action mode.It may provides a new therapeutic target for metastasis blocking in HCC patients.Methods:Cancer stem cells are the key population for tumor metastasis.Firstly,we used suspension culture to enrich cancer stem cells from the primary tumors isolated from HCC patients.And then we performed RNA-seq to select circular RNAs which abnormally express.Besides,we treated total RNA with RNAase R.Specific divergent and convergent primers combined with quantitative real-time PCR(q RT-PCR)were employed for structural verification.Among them,hsa?circ?0003596(hereinafter referred to as circ3596)significantly down-regulate in cancer stem cells.Then RNA Immunoprecipitation assay and si RNA interference technique were employed to explore the regular factor of the circ3596 biogenesis.Next,we constructed circ3596 overexpression vector to overexpress circ3596 in HCC cell lines,and verified the effect of overexpression by q RT-PCR and fluorescence microscope.Next,we tested the effects of circ3596 overexpression on the biological phenotype of HCC cell lines through experiments such as CCK8,transwell,and wound healing assay in vitro.We also employed q RT-PCR and western blot to examine the effect of circ3596 on COL5A1 expression.Finally,we explore the biological function of COL5A1 by bioinformation analysis and experiments in HCC cells.Results:Through second-generation sequencing technology,we found that a total of 193 circ RNAs are differently expressed in cancer stem cells enriched from suspension culture compared with adherent culture.In details,82 circ RNAs were up-regulated and 111 were down-regulated,and among them circ3596 was significantly down-regulated.After interfering with the expression of RNA-binding protein TDP43,the expression of circ3596 was significantly down-regulated.In addition,TDP43 RNA immunoprecipitation can significantly enrich the linear precursor of circ3596.In addition,circ3596 was overexpressed in HCC cell lines using lentiviral transfection.We found that although circ3596 had no statistically significant effect on the proliferation of HCC cells in vitro,it could significantly reduce the invasion and migration ability in HCC-LM3 and huh7 cell lines.When exploring the mechanism of circ3596 influencing cancer cells,we found that circ3596 could down-regulate the protein level of COL5A1.Bioinformatic analysis and experiments showed that COL5A1 has potential to inhibit the metastatic ability of HCC cells,which may be the mechanism of circ3596 inhibiting HCC metastasis.Conclusion:The results showed that circ3596 could inhibit the invasion and migration ability of HCC cells and inhibit the protein expression of its parental gene COL5A1 in vitro.These results suggest that circ3596 could inhibits HCC metastasis and has potential for being developed into drugs for blocking metastasis of HCC.