MiR-194 Inhibits Liver Cancer Stem Cell Expansion By Regulating RAC1 And Predicts The Prognosis Of HCC Patients

Background and Aims:Hepatocellular carcinoma(HCC)is a major global disease.Postoperative recurrence and drug resistance are important reasons for its poor prognosis.Liver cancer stem cells(LCSCs)play an important role in the progression of tumors.The specific mechanism of stem cell expansion has not yet been fully elucidated.Micro RNA is a kind of small molecule that plays an important role in tumor growth,apoptosis,as well as other tumor activities.Micro RNA 194(miR-194)is one of these microRNAs.In recent years,there have been many studies on the role of microRNA-194.However,it is still unknown about its role in LCSCs and in the prognosis of hepatocellular carcinoma.This study aims to find out the expression pattern of miR-194 in hepatocellular carcinoma and LCSCs,to determine the effect of miR-194 on LCSC function,to elucidate the mechanism of miR-194 on regulating cancer stem cell activity,and to explore its role in the prognosis of hepatocellular carcinoma and in sorafenib treatment resistance.Methods and Results: The experiment was divided into four parts.In the first part,we use paired tissue samples from 110 HCC patients and LCSCs which were enriched by sphere formation from HCCLM3,Huh7 and primary HCC cell lines.Total RNA was extracted from tissue samples and LCSCs,and the expression of miR-194 was analyzed by real-time PCR.Results: The expression level of miR-194 in HCC tissues was significantly lower than that in para-cancerous controls,p<0.05.In HCCLM3,Huh7 and primary HCC cell lines,the expression of miR-194 in stem cell spheres was significantly lower than that of non-spherical cells,p<0.05.In the second part,lenti-vector expressing miR-194,miR-194 inhibitor,as well as their controls were transfected into HCCLM3 and Huh7 cell lines,respectively.The stem cell characteristics of these cells were detected by a series of experiments,including spheroid formation assay,limiting dilution assay,q PCR analysis of LCSCs surface marker(Ep CAM,CD133,CD24 and CD90),as well as flow cytometric analysis of Ep CAM+ and CD133+ cells.Results: Compared with control cells,the proportion of Ep CAM+ and CD133+ HCC cells were significantly decreased in cells overexpressing miR-194 and significantly increased in cells with miR-194 inhibitor,p<0.05.In spheroid formation assay,the number of spheres were significantly reduced in cells overexpressing miR-194,while the number were significantly increased in cells transfected with miR-194 inhibitor,p<0.05.The expression LCSC markers such as Ep CAM,CD133,CD24 and CD90 was significantly decreased in cells with miR-194 overexpression,and significantly increased after miR-194 was inhibited,p<0.05.The in vitro limiting dilution method found that the proportion of LCSCs was significantly decreased in miR-194 overexpressing cells and significantly increased in cell with miR-194 inhibitor,compared with controls,p<0.05.The results indicate that miR-194 plays an indispensable role in inhibiting the self-renewal of liver cancer stem cells.In the third part,lenti-vector overexpressing miR-194 and controls were transfected in HCC cells firstly,and the expression of RAC1 of these cells were detected by real-time PCR and Western Blotting.Luciferase reporter assay were conducted to test the binding of miR-194 and RAC1 3’UTR.Afterwards,RAC1 special si RNA and negative controls were transfected in miR-194 overexpressing HCC cells and control cells,respectively.The stem cell characteristics of these cells were detected by a series of experiments,including spheroid formation assay,limiting dilution assay,q PCR analysis of LCSCs surface marker,as well as flow cytometric analysis of Ep CAM+ and CD133+ cells.Results: Both the m RNA level and protein level of RAC1 were downregulated in liver CSCs overexpressing miR-194.The luciferase activity of the wild-type RAC1 3’UTR reporter was inhibited markedly in the presence of miR-194.RAC1 silencing resulted in a decreased number of spheres in spheroid formation assay,a reduced portion of Ep CAM+ and CD133+ cells by FACS.In the absence of special RAC1 si RNA,overexpressing miR-194 resulted in a decreased number of spheres in spheroid formation assay,a reduced portion of Ep CAM+and CD133+ cells by FACS,a reduced proportion of LCSCs by limiting dilution assay,and an upregulated expression of stem cell markers by q PCR test,with significant difference p<0.05.But when RAC1 si RNA was transfected in,the difference between miR-194 overexpressing cells and controls was diminished.In the last part,110 HCC patients were divided into low(n=56)and high(n=54)miR-194 expression groups.The overall survival(OS)and Disease-free survival(DFS)of HCC patients in low and high miR-194 groups was studied by Kaplan–Meier analysis.The prognosis of these patients was analyzed by multivariate analysis.Afterwards,the effect of miR-194 on drug resistance of HCC to sorafenib was studied.The miR-194 expression difference was detected by cell real-time PCR between sorafenib-resistant HCC cell lines and controls.Then a certain does of sorafenib and DMSO controls were added to miR-194 overexpressing HCC cells and negative control cells,and CCK8 cell proliferation assay and apoptosis assay was conducted to these cells.The expression of apoptosis-related molecule PARP was also detected in them.Results: miR-194 expression was significantly reduced in sorafenib-resistant hepatoma cells.When exposed to sorafenib,a reduced cell proliferation,increased apoptosis cells and upregulation of PARP was found in miR-194 overexpressing cells compared with control.While exposed to DSMO control there were no difference between miR-194 overexpressing cells and controls in terms of cell proliferation,apoptosis cells or PARP expression level.Conclusion: Mi R-194 expression was decreased in LCSCs compared with non-stem cells.Mi R-194 can inhibit the expression of RAC1 by direct targeting and reduce the self-renewal ability of LCSCs as a result.The expression of miR-194 was decreased in hepatocellular carcinoma tissue compared with para-cancerous controls,and the level of miR-194 in tumor was an independent protective prognostic factor for HCC patients.Besides,miR-194 overexpression can increase the sensitivity of sorafenib treatment to HCC cells.These results suggest that microRNA-194 can provide potential therapeutic targets for hepatocellular carcinoma therapy and provide new ideas for improving the mechanism of stem cell renewal in hepatocellular carcinoma.