Effect Of Phospholipase C?1 Expression On Proliferation And Prognosis Of Hepatocellular Carcinoma

Objective:To investigate the effect of PLC?1 on the proliferation and prognosis of HCC cells by studying the expression in liver cell carcinoma cells and tissues and its relationship with clinical features.Methods:(1)The cells were cultured and extracted RNA and protein from 5 types of hepatocellular carcinoma cell lines(H7402,Hep3B,LM3,HepG2,Huh7)and normal liver cell lines(L02).By RT-PCR and WB,the expression of mRNA level and protein level corresponding to PLC?1 in the cells was detected,and the differences in the expression of PLC?1 between cancer cells and normal liver cells were compared.(2)RNA interference technique was used to construct HepG2 and Huh7 cell line which overexpressed PLC?1 gene.The cell viability was detected by CCK-8,and the vitality of liver cancer cell line and control group were compared.At the same time,caspase-3/7 activity was detected,and cell apoptosis and growth status were determined.Finally,the role of PLC?1 in the growth of hepatocellular carcinoma cell line was verified by clone formation assay.(3)The Hep3B and LM3 cell lines of the silent PLC?1 gene were constructed by RNA interference technique,through the WB screening the ShRNA which can effectively silence the PLC?1 gene,then detecting the cell viability through CCK-8 and comparing the vitality between the liver cancer cell line and the control group.Finally,the effect of the silent PLC?1 gene on the growth of the two groups of cells was detected by the colony formation assay.(4)After constructing the HepG2 and Huh7cell line overexpressing PLC?1 and Hep3B cell line silencing PLC?1,the activation state of ERK was detected by WB,and the carcinogenic mechanism of PLC?1 was further studied.(5)A total of 141 patients with hepatocellular carcinoma were collected from January 2010 to January 2017,who were hospitalized in our hospital and diagnosed as hepatocellular carcinoma by pathology.The expression of PLC?1 in tumor tissues and adjacent tissues of 141 patients with HCC was analyzed by immunohistochemical method.The relationship between the expression of PLC?1 and clinicopathological features was investigated by using the Kaplan Meier curve and the total survival time(OS)analysis and Cox regression analysis were conducted.(6)SPSS 19.0 statistical software(IBM USA)and GraphPad Prism 5 were used for statistical analysis and graphical display.The measurement data is expressed as mean±standard deviation((?)±s).The comparison of the two groups of mean is analyzed by paired t test,and the rate of counting data is expressed,and the?~2 test or Fisher exact probability method is used to analyze the data.When P<0.05,the difference is statistically significant.Results:(1)The mRNA expression level of PLC?1 in HCC cell lines was higher than that in normal hepatocytes(t=4.50,p<0.05),and the protein expression level was higher than that in normal hepatocytes(t=4.30,p<0.05).(2)The cell viability of over-expressed PLC?1 gene cells was significantly higher than that of control group(t=4.58,p<0.05).The growth ability of HepG2 and Huh7 cells over-expressing PLC?1 was significantly higher than that of the control group(t=4.20,p<0.05).The activity of caspase-3/7 in PLC?1 overexpression group decreased significantly(t=4.40,p<0.05).The Caspase-3/7 activity of SH-1 and SH-2 cells was higher than that of control shRNA cells(t=4.92,p<0.05).(3)Silencing of PLC?1 inhibited malignant expression of hepatocellular carcinoma cells.(4)PLC?1 can regulate ERK activation signal transduction in hepatocellular carcinoma cells and activation of ERK promoted the carcinogenicity of PLC?1.(5)PLC?1 protein was detected in the nuclei and cytoplasm of HCC tissues,and the expression of PLC?1 protein in HCC tissues was significantly higher than that in paracancerous tissues(t=5.05,p<0.05).The expression of PLC?1 was significantly correlated with the stage of liver cancer in Barcelona.(?~2=6.690,p=0.0145).The survival rate of tumor patients with positive expression of PLC?1 was lower than that of patients with negative expression,and the mean survival time was significantly shorter than that of patients with negative expression of PLC?1(t=5.01,p<0.05).The expression of PLC?1 was an independent prognostic factor associated with survival time of HCC patients.Conclusion:(1)The overexpression of PLC?1 can promote the proliferation and inhibit the apoptosis of hepatocellular carcinoma cells.(2)The PLC?1 may affect the growth and apoptosis of hepatocellular carcinoma cells by activating ERK signaling pathway.(3)The overexpression of PLC?1 in hepatocellular carcinoma was closely related to the staging and survival of HCC,and the PLC?1 may be an independent prognostic factor for HCC.It needs to be further studied if the PLC?1 can be used as a new candidate target for HCC molecular targeted therapy.