Expression And Prognosis Of MicroRNA - 497 In Hepatocellular Carcinoma And Its Mechanism Of Invasion And Metastasis

Hepatocellular carcinoma(HCC) is one of the most common human Malignancies worldwide. Due To it’s highly malignant potential, HCC ranks as the third leading cause of cancer death in the world, resulting in almost600,000deaths each year.Despite great improvement in the treatment options, such as hepatic resection, liver transplantation and Liver cancer comprehensive treatment, survival of Patients with HCC remains unsatisfactory, with a5-year surivial rate of30%to40%reported in the literature. Carcinogenesis and progression of HCC are multistage proeess that involve lots of oncogenes and tumor suppressor genes.Making clear the molecular events underlied the tumorgenesis of HCC,espical the mechanism of metastasis, are important for its screening, prevention, treatment.MicroRNAs(miRNAs) are highly conserved small non-coding regulatory RNAs with sizes of17-25nucleotides.As posttranscriptional regulators, miRNAs can negatively regulate gene expression by binding directly to3’untranslated region(3’UTR)of corresponding target messenger RNAs(mRNAs) in a sequence-specific manner,which induces mRNA degradation or protein translation repression. Currently, more than1000human miRNAs have been identified, and aberrant expression of these miRNAs is involved in various physiological and pathological processes,including human carcinogenesis.Recently,emerging evidence links the biological function of miRNAs to the initiation,promotion and progression of HCC, and studies have confirmed that miRNAs played multiple roles in Pathological processes of HCC, such as cell proliferation, celldeath, metastasis and DNA methylation,etc.Although the importance of miRNAs in hepatocarcinogenesis has attracted much attention in recent years,the pathological revevance and significance of the majority of miRNAs in HCC remain unclear.MiR-497belongs to miR-15family. Which includes miR-15,miR-16, miR-195et al. In human tumors, the cluster of miR-15were shown downregulated in a variety of tumors. They exerted potential tumor suppressor in these tumors. Recently,some evidence also show that miR-15family are up-regulated in some other cancers such as cervical carcinoma and head and neck squamous cell carcinomas. MiR-497is also overexpressed in pancreatic cancer. Now,the role of miR-15cluster in hcc is limited.Before the study,we analyse the expression level of miR-195and miR-497in hcc and the vale of prognosis. We found that the expression of miR-497was obviously raised in hcc and high level of miR-497was significantly related to hcc metastasis.The study will focus on the role of miR-497in hcc, such as the expression level,prognosis,function and the mechanism of invasion and metastasis. Methods and Results1.MiR-497is frequently up-regulated in HCC and associated with tumor invasion and metastasis.We first determined the expression of miR-497in50cases of HCC and adjacent non-cancerous liver tissues using quantitative reverse-transcription PCR. The expression of miR-497is significantly upregulated in HCC tissues when compared with non-cancerous liver tissues. Furthermore we found that there is close relationships between miR-497expression and Vascular invasion and TNM stage. Hcc with advanced stages (stage Ⅲ) had a higher miR-497expression than hcc with early stages,Furthermore, the expression levels of miR-497were significantly higher in metastatic HCC tissues and recurrence patients, we also confirmed that miR-497levels is higher in high-metastatic HCC cell lines than that in the low-metastatic HCC cell lines. These results suggested that up-regulation of miR-497might be related to the increase of HCC metastasis, and may play an important role in the pathological process.2. The association of miR-497with prognosis of HCC patientsIn the Kaplan-Meier analyses, high level of miR-497exhibited a decreased postoperative OS and a shorter TTR compared those with low level. Moreover, the prognostic value of miR-497is remarkably more significant in the patients with small HCCs (≤5cm) and is independent prognostic indicators for both OS and TTR of HCC.3、MiR-497promotes HCC cell invasion and metastasis in vitro and in vivoWe first constructed a lentivirus vector expressing miR-497and established two stable cell lines of SMMC-7721,Huh-7and transfected miR-497inhibitor into MHCC-97H cells. In cell proliferation and colony formation assays, ectopic expression or knockdown of miR-497showed no obvious impacts on in vitro HCC cell proliferation.. While,overexpression of miR-497significantly promote the migratory and invasive abilities of Hcc cells. In vivo assays,we transplanted SMMC-7721cells stably expressing miR-497into nude mice through the lateral tail vein and found the number of the metastatic nodules in the lung were dramatically increased in miR-497groups.4. ARHGDIA is the direct target gene of miR-497We used TargetScan algorithm to search for putative protein-coding gene targets of miR-497, especially for those that have the abilities to promote tumor cell invasion and metastasis.The relative luciferase activity was remarkably reduced by miR-497when the full-length wild-type3-UTR of ARHGDIA was present.The relative luciferase activity did not drop as sharply in UTRs that contained mutant binding sites as in those that contained wild-type binding sites. Western blot analysis showed that enforced expression of miR-497in SMMC-7721and Huh-7cell lines triggered a silencing effect on the endogenous ARHGDIA protein expression.On the contrary, reduced miR-497expression enhanced ARHGDIA protein amounts in MHCC-97H cell lines. qRT-PCR assay showed miR-497had no significant effect on ARHGDIA mRNA level. We also found downregulation of ARHGDIA is inversely correlated with miR-497expression.Taken together, these results indicate that miR-497downregulates ARHGDIA expression by directly targeting its3’UTR.5. The biopathological and prognosis of ARHGDIA in HCCImmunohistochemical staining revealed ARHGDIA expression was very low in more than half of the HCC tissues. Moreover,we also found the expression level of ARHGDIA was significantly associated with OS and TTR. The patients with low level of ARHGDIA exhibited a decreased postoperative OS and a shorter TTR compared those with high level..Further more, the level of ARHGDIA was closely associated with Vascular invasion when analyzing the clinicalpathological features of HCC patients,which confirmed once again the important role of miR-497-ARHGDIA-mediated tumor invasion and metastasis.6. Down-regulation of ARHGDIA directly mediates miR-497-initiated hcc cell invasion and metastasis.Migration and invasion assays showed that si-ARHGDIA could promote hcc cell migration and invasion, which resembled the stimulative effects of miR-497on the hcc cell..We next reintroduced ARHGDIA in SMMC-7721-miR-497stable cell lines. Transwell assays indicated that the restoration of ARHGDIA significantly reduced the hcc cell migration and invasiveness that initiated by miR-497. Similarly,we knocked down ARHGDIA expression in MHCC-97H-anti-miR-497cell and found that the migration-and invasion-suppressing effects of anti-miR-497were partially attenuated by siRNA of ARHGDIA. In summary, these findings indicated that ARHGDIA is indeed a functionally important target of miR-497involving in hcc migration and invasion.7. MiR-497increase the activities of Racl,Cdc42and RhoAImmunoprecipitation and Immunofluorescence assay show that increased miR-497significantly induced Racl, Cdc42and Rho activation in SMMC-7721cells.Numerous studies had indicated that activing signaling of Rho GTPases plays an important role in cancer progression and metastasis.these results show that miR-497promotes HCC cell migration and invasion by activating Rho GTPase proteins in HCC cells.ConclusionIn summary, our results show that miR-497is up-regulated in HCC and positively related to tumor recurrence and metastasis,and is an independent prognostic factor for HCC with r≤5cm. Using both in vitro and in vivo studies, miR-497was confirmed to be oncogenes, which can promote invasion and metastasis of HCC through directly targeting ARGHDIA and in turn activate Racl, Cdc42and Rho GTPases. This newly identified miR-497/ARHGDIA module provides a new avenue to an understanding of the processes of hepatic tumorigenesis, especially invasion and metastasis which may provid a potential therapeutic target in HCC.Main Innovative Pointsl.For the first time,the present study documented that miR-497was up-regulated in HCC and correlated significantly to clinical pathologic characteristics as well as prognosis of HCC, which implicated miR-497as a potential prognostic marker of HCC and extend the research of miR-497to HCC.2. For the first time,The present study has showed important role of miR-497in promoting hcc metastasis by directly targeting ARHGDIA, which help understanding the molecular mechanism of carcinogenesis and development of HCC.3. we firstly documented the role of ARHGDIA in the prognosis of HCC and the relationship to clinical pathologic characteristics of HCC.We confirmed that ARHG DIA exerted a tumor suppresson effect on hcc.