MicroRNA-548a-5p Promotes Proliferation And Inhibits Apoptosis In Hepatocellular Carcinoma Cells By Targeting Tg737

【BACKGROUND】Primary hepatocellular carcinoma(HCC)is currently one of the most common and lethal malignancies,and the leading cause of death among patients with cirrhosis.Despite advances in surgical therapies,the reason for frequent recurrence remains mostly obscure.Several studies showed that some genes may play a major role in cell proliferation and migration in HCC cells.However,little information is available about the regulation of these genes.The Tg737 gene,first found in algae,is now identified as a tumor suppressor gene in multiple cancers,including cancers of the liver,kidney and pancreas.In liver tissues from HCC patients,a 59% down-regulation of the Tg737 was observed.This gene may participate in alterations in a series of human or rodent liver tumors and tumorigenic cell lines.In the previous investigation,we found that Tg737 contributed to hypoxia-induced invasion and migration in Hep G2 and MHCC97 H cells,and that Tg737 inhibition resulted in malignant transformation in fetal liver stem/progenitor cells by promoting cell cycle progression and differentiation arrest.However,the regulatory factor for Tg737 is not yet clear.In recent years,micro RNAs(mi RNAs)emerged as a group of important endogenous modulators of gene function at the posttranscriptional level.It has been revealed that many mi RNAs are underscored by the promotion of tumorigenesis andcancer progression.More importantly,in HCC,several mi RNAs have been demonstrated to contribute to the tumor regulation,including,but not limited to,migration,invasion and proliferation.Multiple mi RNAs have been identified as down-regulated tumor-suppressing genes involved in cellular processes,including mi R-34 a,mi R-122,mi R-199 a and mi R-200.Contrarily,mi R-21,mi R-148 a and mi R-221,as oncogenic mi RNAs,showed up-regulated expression which potentially targeted many tumor-suppressive genes.These results suggest the involvement of mi RNAs in HCC.Mi R-548 is a big,poorly conserved primate-specifc mi RNA family.There are 68 members of the hsami R-548 family recorded in the mi RBase database.The products of mi R-548c-5p and mi R-548c-3p show discrepant evolutionary patterns,which brings about great genetic distances between pre-mi RNAs to some extent and might contribute to dynamic expression profiles and regulatory network.Mi R-548c-3p was identified as a source of functional biomarkers for the primary prostate cancer progression.It also significantly increased in Helicobacter pylori-negative cancer tissues.In addition,our previous studies found the high expression of mi R-548c-3p and mi R-548c-5p in side population cells from HCC.However,like the homologous gene,the exact effect of mi R-548a-5p on HCC is still unknown.The direct aim of this study was to investigate whether Tg737 is regulated by mi R-548a-5p,and correlates with HCC cell proliferation and apoptosis.【PURPOSE】To investigate whether Tg737 is regulated by micro RNA-548a-5p(mi R-548a-5p),and correlates with hepatocellular carcinoma(HCC)cell proliferation and apoptosis.【METHODS】Assays of loss of function of Tg737 were performed by the colony formation assay,CCK-8 assay and cell cycle assay in HCC cell lines.The interaction between mi R-548a-5p and its downstream target,Tg737,was evaluated by a dual-luciferase reporter assay and quantitative real-time polymerase chain reaction.Tg737 was then up-regulated in HCC cells to evaluate its effect on mi R-548a-5p regulation.Hep G2 cells stably overexpressing mi R-548a-5p or mi R-control were also subcutaneously inoculated into nude mice toevaluate the effect of mi R-548a-5p up-regulation on in vivo tumor growth.As the final step,the effect of mi R-548a-5p on the apoptosis induced by cisplatin was evaluated by flow cytometry.【RESULTS】Down-regulation of Tg737,which is a target gene of mi R-548a-5p,accelerated HCC cell proliferation,and mi R-548a-5p promoted HCC cell proliferation in vitro and in vivo.Like the downregulation of Tg737,overexpression of mi R-548a-5p in HCC cell lines promoted cell proliferation,increased colony forming ability and hampered cell apoptosis.In addition,mi R-548a-5p overexpression increased HCC cell growth in vivo.Mi R-548a-5p downregulated Tg737 expression through direct contact with its 3’untranslated region(UTR),and mi R-548a-5p expression was negatively correlated with Tg737 levels in HCC specimens.Restoring Tg737(without the 3’UTR)significantly hampered mi R-548a-5p induced cell proliferation,and rescued the mi R-548a-5p induced cell proliferation inhibition and apoptosis induced by cisplatin.【CONCLUSION】MiR-548a-5p negatively regulates the tumor inhibitor gene Tg737 and promotes tumorigenesis in vitro and in vivo,indicating its potential as a novel therapeutic target for HCC.