Preliminary Study On The Mechanism Of Long Chain Non Coding RNA GHET1 In Hepatocellular Carcinoma And Its Prognosis

Objective: To detect the expression of long non-coding RNA(GHET1)in human hepatocellular carcinoma(HCC)tissues and cells and to explore its role in the development and progression of HCC.Liver cancer and liver cancer patients with liver transplantation prognostic markers and clinical treatment of the target.Methods:(1)Twenty liver cancer tissues and adjacent tissues were collected.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of GHET1(2)To detect the expression of GHET1 in human hepatocellular carcinoma cell line Huh7,HepG2 and human liver immortal cell line LO2,and to screen suitable hepatocellular carcinoma cell model with high expression of GHET1 in vitro.(3)We designed a siRNA targeting small interfering RNA(GHET1-siRNA),siRNA-NC(control RNA interference)and control group(Con).Transfection efficiency was observed by inverted fluorescent microscope.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to test the expression of GHET1 after siRNA interference.(4)The hepatoma cell lines screened by GHET1-siRNA and siRNA-NC were constructed.CCK-8 assay was used to detect the effect of GHET1 on the proliferation of hepatocellular carcinoma cells.Transwell assay was used to detect the migration and invasion ability of cells.Western blot was used to detect the expression of EMT related genes.(5)The expression of GHET1 in 182 HCC tissues and 106 liver cancer liver transplantation tissues was detected by qRT-PCR.The expression of GHET1 in hepatocellular carcinoma was analyzed by clinicopathological data.Relationship between clinicopathological features and prognosis.Results:(1)The expression of GHET1 in hepatocellular carcinoma was significantly higher than that in adjacent non-tumor tissues(P <0.001),and the expression of GHET1 was also up-regulated in HCC cell lines(P <0.001)GHET1 expression was down-regulated in HepG2 cells compared with siRNA-NC in control group(p <0.001)by GHET1-siRNA transfection;(3)CCK-8 results showed that down-regulation of GHET1 expression in Huh7 and HepG2 cells inhibited cell proliferation(P <0.001).(4)Transwell chamber invasion,migration experiments showed that the number of HCC cell invasion and migration through silencing GHET1 significantly decreased,the difference was significant(P <0.001).(5)Western blot showed that knockdown of GHET1 in Huh7 cells significantly increased the expression of E-cadherin and reduced the levels of fibronectin and vimentin(P <0.01).(6)In the study of patients with liver cancer,the results of clinicopathological features suggested that the high expression of GHET1 was significantly associated with tumor size,vascular invasion,Edmonson classification,tumor envelope and serum AFP levels in patients with hepatocellular carcinoma(all p <0.05).There was no significant correlation between GHET1 expression and other clinicopathological features(all P> 0.05).(7)Statistical analysis using Kaplan-Meier method and rank sum test showed that the overall survival of GHET1-overexpressing HCC patients was significantly shorter than that of low expression patients(P <0.01).In order to determine the prognostic significance of the total recurrence(OR)and the overall survival(OS)clinical and pathological factors,multivariate analysis showed that GHET1 overexpression was all associated with HCC(HR = 0.584,95% CI: 0.352-0.968,P = 0.037)And OS(HR = 1.730,95% CI: 1.071-2.797,P = 0.025).(8)The ROC curve was used to evaluate the ability of GHET1 expression to predict the prognosis of HCC.The results showed that the cutoff at 4.255(AUC = 0.748,95% CI 0.677,0.819,p <0.001);whereas the cutoff at 4.740 correlated with OS(AUC = 0.746,95% CI 0.673,0.818,p <0.001).(9)In the study of liver cancer patients with hepatocellular carcinoma,the results of clinicopathological features showed that the expression of GHET1 was associated with tumor envelope(P <0.001),tumor diameter(P = 0.005),differentiation degree),Vascular invasion(P = 0.009)and TNM stage(P = 0.001),but not with age,gender,cirrhosis,serum AFP level,serum NLR,solid tumor location and number of solid tumors p> 0.05).At the same time,the expression of GHET1 correlated with the recurrence of liver cancer after transplantation(p <0.001).(10)Statistical analysis using Kaplan-Meier method and rank sum test showed that the cumulative survival rate of patients with GHET1-overexpressing liver cancer was significantly lower than that of patients with low expression(P <0.01).The cumulative recurrence rate of patients with GHET1-overexpressing liver cancer Significantly higher than GHET1 low expression patients(P <0.01).Conclusions:(1)GHET1 gene is up-regulated in hepatocellular carcinoma and in different hepatoma cell lines.(2)Silencing of GHET1 by siRNA also inhibited the proliferation of human hepatocellular carcinoma cells Huh7 and HepG2,and at the same time,the invasion and migration abilities decreased.(3)The knockdown of GHET1 in Huh7 cells affected EMT-related gene expression.(4)GHET1 plays an oncogene role in hepatocellular carcinoma and can be used as an index to predict and diagnose the poor prognosis of HCC patients.(5)GHET1 overexpression is associated with clinicopathological data and poor prognosis in patients with liver cancer and its significance in prediction of OR and OS in liver transplantation.The conclusion of this study suggests that GHET1 may be involved in the occurrence and development of hepatocellular carcinoma,which can be used as a marker and clinical target of prognosis in patients with liver cancer and liver cancer.