Effects Of RASSF6 On The Proliferation,Invasion And Metastasis Of Hepatocellular Carcinoma And Related Mechanisms

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world.Epidemiological data showed that nearly half a million people died of liver cancer each year,ranking the second cause of cancer death.China,with high incidence of liver cancer,the number of patients increased every year.Although with the development of medical technology,liver cancer-related diagnosis and treatment programs have made great progress,leading to improved survival rate.However,due to the fast progress and high recurrence of liver cancer,the overall survival of patients with liver cancer is still not ideal.Therefore,to explore the molecular mechanism of liver cancer is important for the reduction of mortality rate in clinical work.It has been found that RASSF6 protein is highly expressed in many kinds of cancer cells.Research on the function of RASSF6 protein shows that overexpression of RASSF6 by gene transfection can effectively inhibit the proliferation and metastasis of cancer cells.At present,RASSF6 is one of the key regulatory proteins in the process of proliferation and metastasis in tumor cells.A large number of studies have confirmed that RASSF6 played crucial roles in the colon cancer,leukemia,nasopharyngeal carcinoma and renal cell carcinoma.However,the role of RASSF6 in hepatocellular carcinoma and its mechanism have not been reported.?Objective?To detect the expression of RASSF6 in clinical samples and corresponding adjacent tissues,and to analyze the correlation between the expression of RASSF6 and clinicopathologic parameters of HCC.To investigate the regulation of RASSF6 protein on the proliferation,apoptosis and invasion and metastasis of HCC cells.To investigate the specific mechanism of RASSF6 protein in regulating the malignant biological behavior of HCC cells.?Method?1,Real-time PCR and western blot were used to detect the expression of RASSF6 in32 HCC tissues and corresponding adjacent tissues.The clinical data of patients were collected and analyzed.The expressions of RASSF6 in HepG2,SMMC-7721 and MHCC-97 and HL-7702 cells were detected and analyzed.2.The RASSF6 overexpression plasmid vector was constructed for the transfection of human hepatocellular carcinoma cell line HepG2 to increase the expression level of RASSF6.The effects of RASSF6 on the proliferation,apoptosis and invasion and metastasis of HCC cells were detected by CCK-8,Transwell invasion,flow cytometry,migration assay and subcutaneous tumor transplantation in nude mice.The RASSF6 overexpression plasmid vector was constructed for the transfection of human hepatocellular carcinoma cell line HepG2 to increase the expression level of RASSF6.The effect of FAK on the proliferation,apoptosis,invasion and migration of hepatocarcinoma cells was detected by FAK inhibitor.The molecular mechanism of RASSF6 was further explored.?Results?1.The expression of RASSF6 mRNA in hepatocellular carcinoma tissue decreased to0.365±0.042,and the difference was significant(P<0.05).The results of western blot showed that the expression of RASSF6 protein in HCC tissues was significantly lower than that in the adjacent non-cancerous tissues(0.215±0.019).The expression of RASSF6 in HepG2,SMMC-7721 and MHCC-97H cells was significantly lower than that in HL-7702 human hepatocytes(p<0.05),and the expression of RASSF6 in different hepatoma cell lines was different.The m RNA and protein levels of RASSF6were the lowest in HepG2 cells.The relative expression level of RASSF6 mRNA in hepatocellular carcinoma was significantly correlated with HBsAg,tumor volume,TNM stage,histological grade,tumor thrombus and recurrence(P<0.05),but there were no relationships between RASSF6 mRNA and age,gender,serum AFP level,degree of tumor differentiation(P>0.05).There were no significant correlations between RASSF6 mRNA expression and clinicopathological parameters in 32 patients with HCC(P>0.05).Therefore,it is important to study the mutation mechanism of RASSF6 in HCC tissues.3.After transfection of pcDNA3.1-RASSF6,the expression level of RASSF6 in HepG2 hepatoma cells was significantly up-regulated.The proliferation ability of HepG2 cells transfected with pcDNA3.1-RASSF6 was significantly lower than that of negative control cells(P<0.05),and the proliferation ability of HepG2 cells was more obvious with the increase of the time.Flow cytometry showed that the percentage of apoptotic cells in HepG2 cells was significantly increased to 15.69±1.78%after transfection with pcDNA3.1-RASSF6 compared with that of negative control cells(5.62±0.55%)with statistical significance(P<0.05).Transwell assay showed that the migration of HepG2 cells was significantly decreased by RASSF6(P<0.05).Compared with negative control cells,the number of invasive cells in HepG2 cells overexpressing RASSF6 was significantly decreased(P<0.05).The expression of E-cadherin was significantly increased in HepG2 cells transfected with pcDNA3.1-RASSF6,but the expression of N-cadherin was significantly decreased.In vivo,the volume and weight of subcutaneous tumors in both groups were increased with time.The tumor volume of HepG2 cells overexpressing RASSF6 cells was smaller than that of negative control cells.From the 10th day,the tumor volume of the pcDNA3.1-RASSF6 group was 401.3±43.9mm~3 at the end of the 25th day,which was significantly smaller than that of the negative control group(956.4±93.4mm~3,p<0.05).The tumor weight of nude mice was 0.647±0.055g in the pcDNA3.1-RASSF6 group and 0.212±0.019g in the negative control group(P<0.05).The phosphorylation of FAK protein in HepG2 cells was significantly lower than that in negative control group(P<0.05).The expression of MMP-2 and MMP-9 protein was also significantly decreased in HepG2 cells after overexpression of RASSF6.It is possible that HepG2 negatively regulated the activation of the FAK pathway.FAK protein inhibitor FA-562271 was used to inhibit the activity of FAK protein,and the function of HepG2 cells was detected.CCK-8 proliferation assay showed that PF-562271 significantly inhibited the proliferation of Hep G2 cells compared with the control group(P<0.05).The expression of RASSF6 in HepG2 cells was significantly higher than that in the control group(P<0.05).Compared with the control group,the invasion ability of HepG2 cells was significantly decreased after FAK protein was inhibited by PF-562271(P<0.05),and the invasion ability was further decreased in RASSF6overexpressing hepatocellular carcinoma cells(P<0.05).Flow cytometry showed that the apoptosis of HepG2 cells was significantly increased by PF 562271(P<0.05),and the percentage of apoptotic cells in RASSF6 overexpressed hepatocarcinoma cells was significantly increased(P<0.05).These results suggested that RASSF6 might regulate the expression of FAK protein.The effect of FAK protein inhibitor PF-562271 on proliferation,invasion and apoptosis-related proteins in HepG2 cells was detected by western blot.Compared with the control group,PF-562271 inhibited the expression of Bcl-2 protein in hepatocellular carcinoma HepG2 cells,decreased the expression of Bax and the expression of MMP-2 and MMP-9(P<0.05),and the expression of these proteins was significantly increased in RCCF6 overexpressing hepatocarcinoma cells(P<0.05).These results suggest that RASSF6 may inhibit the malignant phenotype of hepatocellular carcinoma cells by regulating the related proteins of proliferation,apoptosis and invasion.?Conclusion?1.The expressions of RASSF6 were significantly lower in three human hepatocarcinoma cell lines and in clinical HCC tissues,and the expression level of RASSF6 was closely correlated with the clinical parameters of patients.2.Enhanced RASSF6 expression in vitro and in vivo resulted in the inhibition of liver cancer cell proliferation,invasion and metastasis,and increased apoptosis of liver cancer cells.3.FAK is the downstream target gene regulated by RASSF6.RASSF6 can inhibit the proliferation,invasion and metastasis of hepatocarcinoma cells and promote the apoptosis of hepatoma cells by inhibiting the activation of FAK pathway.Therefore,RASSF6 protein may become a potential target for the treatment of liver cancer.