Primary Research On Relationship Between Galectin-4 Expression And The Development Of Hepatocellular Carcinoma And Its Related Molecular Mechanism

Background:Primary hepatocellular carcinoma is one of the most common malignancies in China.The mortality rate ranks third in China and easy to relapse after surgery.Therefore,identifying novel molecular biomarkers that could explore the related factors about occurrence/development and recurrence/metastasis of liver cancer,find early detection and predict the recurrence of liver cancer,will greatly benefit many HCC patients.Galectin-4 protein belongs to the family of adhesins,a large number of studies found that Galectin-4 was expressed abnormally in colon cancer,pancreatic cancer,breast cancer and other tumors,and confirmed to be closely associated with the occurrence/development of these cancers.Our preliminary data suggests that Galectin-4expression in hepatocellular carcinoma(HCC)has been significantly lower than in patients without recurrence and metastasis.However,its biological function and molecular mechanism in the occurrence/development of hepatocellular carcinoma is still unclear.Based on the previous research,the mechanism of Galectin-4 in the occurrence of hepatocellular carcinoma was studied from the molecular,cellular,tissue and in vivo experiments,through molecular cloning,gene knockout,and other methods.Purposes:1.To compare the expression of Galectin-4 in hepatocellular carcinoma and adjacent tissues,and to compare the expression of Galectin-4 in human normal hepatocytes and hepatocellular carcinoma cell lines.To investigate the relationship between Galectin-4and hepatocellular carcinoma2.To study the effect of Galectin-4 gene on cell cycle transition,proliferation and apoptosis of hepatocellular carcinoma from cell and animal levels and possible molecular mechanisms.Methods:1.Collecting 20 clinical specimens of liver cancer and adjacent tissues that confirmed by pathology after surgery,the expression of Galectin-4 in hepatocellular carcinoma tissues and adjacent tissues was examined by Q-PCR and Western blot to evaluate its relationship with the development of hepatocellular carcinoma.Western blot was used to detect the expression of Galectin-4 in different hepatocellular carcinoma cells.2.The transfection of hepatocellular carcinoma cell line MHCC-LM3 by lentiviral transfection and sh RNA-mediated RNAi technique was used to construct the hepatocellular carcinoma cell line with stable overexpressed / knockdown Galectin-4gene in vitro and was divided into 4 groups: empty vector group 1(Mock): transfected with PCDH-CMV-EF1-PURO-EGFP empty plasmid,overexpressing Galectin-4 group(Gal4),empty vector group 2(sh Non): transfected with p Green-puro,knockout Galectin-4 group(sh Gal4).3.The effects of Galectin-4 gene on the cell cycle transformation,proliferation,apoptosis and migration of hepatocellular carcinoma were detected by flow cytometry and CCK-8.The effect of Galectin-4 gene overexpression and silencing on the proliferation of hepatoma cells was observed in nude mice.4.Statistical analysis: Data analysis using student's test and analysis of variance,using Gragph Pad Prism6 software,P <0.05 was statistically significant.Results:1.The expression of Galectin-4 in hepatocellular carcinoma was significantly higher than that in adjacent tissues(P<0.05).The results of Western blot showed that Galectin-4 expression level was various in different liver cancer cells.Galectin-4expression was the lowest in the nomal hepatocyte HL7702 and HCC cell lines(Hep G2,Sk-hep-1),and was moderate in MHCC-LM3 and Huh7 cells,and was the highest in MHCC-97 L cells2.On the basis of previous studies,the hepatocarcinoma cell line MHCC-LM3 was successfully constructed by plasmid transfection and RNAi technique.3.The result of cell cycle assay by flow cytometry showed that the expression of G1 phase cells in the Galectin-4 overexpression group was less than that in the control group(P <0.05),and the S,G2/M phase in the Galectin-4 overexpression group was higher than that in the control group(P <0.05),vice versa.The result of cell proliferation assay by CCK8 test showed that the proliferation of Galectin-4 overexpression group was higher than the control group(P <0.05),vice versa.The result of cell apotosis assay by flow cytometry showed that the apotosis ratio of Galectin-4 overexpression group was lower than the control group(P <0.05),vice versa.The research found Galectin-4can reduce the expression of P21 and increase the expression of Cyclin-D1.The results of subcutaneous transplantation of nude mice showed that Galectin-4 overexpression of MHCC-LM3 cells has the effect of promoting the growth of subcutaneous xenografts,and vice versa.Conclusions:1.Galectin-4 is highly expressed in hepatocellular carcinoma and hepatocellular carcinoma cell lines,which may be related to its development.2.Galectin-4 can regulate the expression of P21 and Cyclin-D1,promote the proliferation and cell cycle conversion of hepatocarcinoma cells,and inhibit the apotosis of HCC cells.