MCM6 Promotes Metastasis Of Hepatocellular Carcinoma Via MEK/ERK Pathway And Serves As A Novel Serum Biomarker For Early Recurrence

BackgroundHepatocellular carcinoma(HCC)is the second cause of cancer death in global worldwide.Half of the new HCC cases and deaths were estimated to occur in China and its incidence and mortality are still increasing every year,so HCC is a serious damage to people's life and health.Most patients were already at advanced stages at the time they were diagnosed.The high mortality rate was mainly attribute to its high frequencies of tumor recurrence and distant metastasis after curative resection.Intrahepatic recurrence or extrahepatic metastasis may ocurr in most of patients who have had received surgeical treatment.It is important to screen out the patients who are more likely to suffer early recurrence before or after the operation.Therefore,exploring the molecular mechanisms underlying invasion and metastasis and finding biomarkers that can predict early recurrence are of great clinical significance for improving the overall survival of liver cancer.Minichromosome maintenance complex component 6(MCM6),which has been reported to up-regulate in multiple malignancies.However,little is known about its functional contributions in migration or its predictive significance of early recurrence in HCC.Aims1.To evaluate the expression of MCM6 in HCC cell lines,tissues and matched adjacent non-tumorous tissues;2.To explore the role of MCM6 in hepatocarcinogenesis,and provide a new theory for metastasis of hepatocellular carcinoma;3.To analyze the value of serum MCM6 in predicting the early recurrence after curative hepatectomy.Contents and methods1.The MCM6 expression in HCC tissues and correlation of MCM6 with clinicopathological parameters(1)MCM6 expression in 70 HCC specimens(tumour and matched adjacent non-tumorous tissues),and 30 normal hepatic tissue specimens were assayed by immunohistochemistry(IHC).(2)Statistical analysis to determine the prognostic value of MCM6 in HCC patients.Survival curves were constructed using the Kaplan-Meier curve and analyzed by the log-rank test.Significant prognostic factors identified by univariate analysis were entered into multivariate analysis using the Cox proportional hazards model.The two-tailed chi-squared test was used to analyse the association of MCM6 expression with some clinicopathological parameters.(3)mRNA expression of MCM6 in liver carcinoma(291 cases)were performed from TCGA database and survival curves were constructed using the Kaplan-Meier method.Expression of MCM6 in liver carcinoma(291 cases)was also performed,then the relationship between MCM6 expression and clinical pathological parameters,and its prognostic value were evaluated by statistical analysis.Based on the relative expression of MCM6,patients were classified into high-and low-expression groups,respectively.2.The effect on cell biology of HCC by MCM6 and the molecular mechanism.(1)Western blot and qPCR were used to detect the expression of MCM6 in five HCC cell lines.(2)Establishment and identification of HCC cell clones with stable knockdown MCM6 expression.(3)We explore the influence of MCM6 on cell proliferation and migration in vitro.(4)To verify the above findings in vivo,we established subcutaneous xenograft tumor and orthotopic xenograft tumor models in nude mice.Xenografts model in nude mice was conducted to detect the tumor growth capability in vivo,the expression of Ki67 and EMT markers in xenograft tumor was examined by immunohistochemistry.(5)Western blotting,qPCR and Immunofluorescence staining were conducted to detect the expressions EMT-related markers.The activation status of Ras-Raf-MEK-ERK pathway was detected by Western-blot.Immunofluorescence assay was used to detect the localization and expression of ERK protein.3.The predictive value of serum MCM6 for the early recurrence after curative hepatectomy.Blood samples were collected from HCC,cirrhotic patients,and healthy individuals.The serologic level of MCM6 was detected using the human MCM6 ELISA kit.Receiver operating characteristic(ROC)curve analysis was used to determine the predictive value of the parameters,and the differences in the area under the curve(AUC)were detected.We compared the clinical value between the role of MCM6 serum levels and AFP in monitoring the postoperative recurrence and metastasis of patients who underwent hepatectomy.Results1.MCM6 is primarily localized in the nucleus,and its expression levels is much higher in HCC tissues than that in adjacent non-tumourous and normal tissues.High expression level of MCM6 correlated with poor prognosis and high recurrence rate.2.The expression of MCM6 is positively correlated with invasiveness in five cell lines;MCM6 promotes HCC cell proliferation,migration and invasion in vitro.The subcutaneous xenograft tumor and orthotopic xenograft tumor models in nude mice showed that MCM6 could promote HCC growth and metastasis in vivo.3.The mechanism research of MCM6 in migration and invasion.To figure out the specific mechanism which MCM6 modulates migration and invasion,we examined levels of EMT-related proteins,further figured out the specific signaling.The results showed that MCM6 expression inversely correlated withE-cadherin but positively correlated with vimentin expression.Moreover,the activation of ERK1/2 was strongly suppressed when MCM6 was knocked down.Taken together,these data suggest that MCM6 promotes EMT,at least in part,through the upregulation of MEK/ERK signaling pathways.4.To further explore its potential value in early recurrence non-invasive serologic detection,we examined MCM6 expression in patient serum.ROC curve indicated that postoperative serum MCM6 has a significant predictive ability of HCC early recurrence than traditional biomarker AFP,and patients with dual positivity of postoperative serum MCM6 and AFP should be supplied with more intensive care and close follow-up after they undergo tumor resection.ConclusionsMCM6 functions as a tumor promotor by activating MEK/ERK signaling and subsequently regulates metastasis and EMT in HCC.In addition,MCM6 can be a potential indicator to predict poor prognosis and early recurrence after receiving curative hepatectomy.These efforts to provide personalized surveillance are expected to improve the overall clinical management of HCC.