The Role And Mechanism Of MicroRNA-221 In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common cancers due to the association with chronic liver disease,particularly in patients who have been infected with hepatitis B or hepatitis C virus in Eastern Asia and China.The overall prognosis of HCC is still very poor.One of the main reasons is the high rate of postoperative recurrence and metastasis.Therefore,it is very important to explore the mechanism of metastasis and recurrence of HCC.It is also very important to explore the key regulatory remission which can improve the prognosis of HCC patients and provide a new therapeutic target for HCC treatment.MicroRNA is a non-encoded single-stranded RNA molecule with a length of about 22nt encoded by an endogenous gene,which are caused by complementary pairing with the 3' UTR(3' Untranslational region)of the target gene.Therefore,miRNA can negatively regulate the expression of genes at the post-transcriptional level,and thus participate in various biological processes such as development,proliferation,differentiation and apoptosis.Meanwhile,the abnormal expression of miRNA is also involved in the occurrence and development of some tumors.Many studies have confirmed that some miRNAs play an important role in the occurrence of HCC,cell proliferation and apoptosis,tumor angiogenesis,invasion and metastasis.Therefore,miRNA may serve as a new therapeutic target and predictive treatment for HCC metastasis.MiRNA221 is a kind of tumor-related miRNA which has attracted much attention in recent years,and its regulated target genes and the functions of these target genes have been revealed in succession,and some studies have been used miRNA microarray technology to find the high expression of miR-221 in primary hepatocellular carcinoma.It is suggested that miR-221 plays an important role in the occurrence of hepatocellular carcinoma,However,the potential molecular mechanism of miR-221 in HCC is still not very clear.In this study,we investigated the predictive value of miR-221 to HCC patients and its function in occurrence and development,and further elucidated the underlying mechanism by which miR-221 regulates HCC.Part One Expression level of miR-221 in HCC and its effect on HCC prognosisObjective:In this study,the relationship between miR-221 and HCC metastasis recurrence and its effect on HCC prognosis were evaluated by detecting the expression level of miR-221 in HCC tissue and HCC cell lines.Methods:Real-time PCR was used to detect the expression levels of 156 HCC tissues(including 40 paired adjacent cancerous tissues)and the miR-221 of HCC cell lines,and the expression of miR-221 in HCC tissue and different metastatic potential cell lines were analyzed.The overall survival time and desease-free survival time curves of HCC patients were plotted by kaplan-meier method,and the comparison between groups was performed by Log-rank method.Univariate and multivariate survival analyses were performed using the Cox proportional hazards model to determine whether miR-221 expression level was an independent prognostic indicator for HCC.Results:(1)The expression of miR-221 in HCC cell lines increased significantly,and the expression was positively correlated with the metastasis potential of HCC cell lines,and the expression level of HCC cell lines increased significantly compared with normal liver cell line(P<0.01),The expression level of miR-221 in the low metastatic potential group was significantly lower than that in the high metastatic potential group(P<0.01).(2)The level of miR-221 in HCC tissues was positively correlated with the metastasis and recurrence of HCC.Comparing with the corresponding adjacent tissues,the expression level of miR-221 in HCC tissues was significantly increased(P<0.01).Comparing with non-metastatic HCC tissues,the expression level of miR-221 was significantly increased in HCC tissues with metastasis(P<0.01),and the expression level of miR-221 in recurrent HCC tissues was significantly higher than that in non-recurrent HCC tissues(P<0.01).(3)Patients with high expression of miR-221 in HCC tissues showed shorter overall survival time,and there was a significant difference between the two groups(P=0.0002).Patients with high expression of miR-221 showed a higher recurrence rate,and there was a significant difference between the two groups(P<0.0001).(4)Univariate and multivariate analysis suggested that miR-221 is an independent predictor of prognosis in patients with HCC.Conclusion:miR-221 is up-regulated in HCC patients and positively correlated with HCC metastasis and recurrence.miR-221 is an independent predictor of HCC prognosis.Part Two miRNA221 regulates the mechanism of invasion and metastasis in hepatocellular carcinoma through REDD1 and mTOR signaling pathwayObjective:In this study,the molecular mechanism of miR-221 participating in the regulation of HCC growth?migration and invasion phenotype was further verified and expounded by studying the functional tests of miR-221 in vitro and in vivo.Methods:Silencing miR-221 by lentiviral transfection,to obtain stable miR-221 inhibition cell line HCCLM3,using Transwell chamber system and scratch experiment to evaluate cell invasion and migration ability;Using flow cytometry to detect cell cycle and apoptosis;MTT method was used to detect the change of cell growth inhibition rate;Using colony formation assay to verify cell cloning ability.Meanwhile the target gene REDD1 of miR-221 was found by using database target gene prediction and dual luciferase reporter gene detection.The tumor-forming ability of cells was verified by subcutaneous tumor model.The expression of related molecules in mTOR pathway were detected by RT-PCR,Western Blot and immunohistochemistry.Results:(1)Silencing miR-221 inhibited the proliferation and invasion of HCC cells in vitro,induces cell cycle block and promotes apoptosis.(2)REDD1 was the downstream target gene of miR-221,and miR-221 expression silenced significantly up-regulates luciferase activity in wild-type REDD 1 3'UTR,whereas luciferase activity in mutant-containing REDD1 3' UTR had no influence.(3)miR-221 regulated HCC invasion through mTOR signaling pathway.The key molecules of mTOR signaling pathway including PI3K,Akt,mTOR and PTEN were regulated by miR-221.Silencing miR-221 increased the mRNA expression of Akt,PI3K,S6K1 and decreased the mRNA expression of PTEN,4EBP1.(4)Silencing miR-221 promoted HCC tumor growth in vitro.Conclusion:miR-221 regulates the growth,migration,invasion and metastasis of HCC in vivo and in vitro,and regulates the regulation of HCC through REDD1 and mTOR signaling pathways.