Upregulation Of MicroRNA-122 By Farnesoid X Receptor Suppresses The Growth Of Hepatocellular Carcinoma Cells

Hepatocellular carcinoma(HCC)is one of the most common cancers and one of the deadliest cancers in the world.There are many predisposing factors,including viral infection,activation of oncogene,abnormal expression of tumor repressors and so the pathogenesis of hepatocellular carcinoma is needed to be further studied,and genes involved in the pathogenesis of HCC and their intereaction is waiting to be clarified,which will undoubtedly help to deepen the understanding of the pathogenesis of HCC,and finally find out novel targets for curing HCC.In recent years,farnesoid X receptor and microRNAs which are closely related with HCC are getting increasing attention.Farnesoid X receptor(FXR),as a member of the nuclear receptor superfamily,is a transcription factor with multiple functions which is mainly expressed in the liver,instestine,kidneys and adrenal glands.FXR plays an important role in regulating bile acid synthesis and lipid and glucose metabolism,and it has been found to regulate liver regeneration,hepatic fibrosis,cholestasis and liver inflammation.A large number of studies found that FXR is closely related to occurrence and development of HCC,but the mechanism remains unclear,which is needed to be further studied.miR-122 is a liver-specific microRNA,which accounts for about 70% of all the microRNAs in the liver.miR-122 plays an important role in regulating development of liver cells,inducing cell differentiation,regulating cell metabolism and participating in the emergency response of hepatocytes.miR-122 is closely related to HCC in pathological state.It acts as an important tumor suppressor in hepatocellular carcinoma by repressing expression of tumor related genes.In view of the important role of miR-122 in anti-hepatoma effects,increasing the expression of miR-122 will undoubtedly contribute to prevention and treatment of HCC.Most of the current studies focus on the mechanism by which mi R-122 regulates expression of its target genes,but there are few reports about the regulation of mi R-122 itself.Therefore,combined with relevant literature and our previous research results,our study first identified miR-122 as a new target gene of FXR,and announced the mechanism of upregulation of miR-122 by FXR;then we clarified the new mechanism of “FXR-miR-122” pathway in anti-hepatoma effects,which accumulated new data of theory for revealing the anti-HCC role of FXR and provided new scientific basis for the exploration of “FXR-mi R-122” pathway as new target of anti-HCC therapy.Purpose: to verify the molecular mechanism of mi R-122 upregulation by FXR in vivo and in vitro,and investigate the importance of “FXR-miR-122” pathway in anti-HCC effects.Methods:1.Collect 20 patient-derived tumor and corresponding adjacent tissues,and seven HCC cell lines,then detect the expression of FXR and miR-122 using Real-time PCR and Western blot,then analyze dependency between them.2.Treat HCC cells with different concentrations of FXR agonists,as well as RNA interference.Then detect expression of miR-122 and its target genes(IGF-1R,cyclin G1)using Real-time PCR and Western blot,analyze the effect of FXR on the expression of mi R-122.3.Use bioinformatics methods to predict possible binding sites of FXR in miR-122 5’flanking region,then construct reporter plasmids containing potential binding sites.After transfect cells with these plasmids,conduct reporter assay to preliminarily confirm the region of binding sites.Then using EMSA and ChIP to explore the specific molecular mechanism of miR-122 regulation by FXR.4.Transfect HCC cells with antagomir-122 to inhibit miR-122,then using CCK-8 to detect the proliferation.5.Treat HCC xenografts with FXR ligands for some days,monitore growth condition of tumor.Harvest tumors after the mice were sacrificed,then use Real-time PCR and Western blot to measure expression of miR-122,IGF-1R,cyclin G1.Operate immunohistochemical staining to measure expression of Ki67.Results:1.The expression of both FXR in HCC tissues is lower than that in the adjacent noncancerous tissues,as while as that of miR-122,and FXR expression is positively correlated with that of miR-122 in HCC tissues,as well as in HCC cell lines.2.FXR upregulates miR-122 expression and in turn downregulates the expression of mi R-122 target genes in HCC cells.3.FXR enhances he transcriptional activity of miR-122 promoter,and binds directly to the FXRE/DR2 in miR-122 promoter region.4.Inhibition of miR-122 dramatically attenuates the FXR-mediated growth suppression of HCC cells.5.FXR-induced miR-122 is involved in the growth suppression of HCC xenografts in vivo.Conclusions:mi R-122 is a novel target gene of FXR,and the upregulation of miR-122 by FXR represses the growth of HCC cells,suggesting that FXR may serve as a key transcriptional regulator for manipulating miR-122 expression,and the FXR/miR-122 pathway may therefore be a novel target for the treatment of HCC.