| Objective: hepatocellular carcinoma(HCC)is a multiple tumor in the world with high malignancy and poor survival and prognosis,although some progress has been made in the treatment of liver cancer through surgery,radiotherapy,chemotherapy and targeted therapy.However,the etiology of HCC has not been fully elucidated,and many molecular mechanisms remain to be explored.tripartite motif protein 11(TRIM 11)have recently become a hot topic in the field of cancer research,and many research results have been obtained.The aim of this study was to investigate the role of TRIM11 in hepatocellular carcinoma(HCC)tissues.After collecting the clinical data,we analyzed the relationship between the expression level and the occurrence,development,metastasis and prognosis of HCC patients.Gene silencing technique was used to transfect the small interfering RNA(Small interfering RNA,siRNA designed for TRIM11 into human hepatoma cell line SMMC-7721(called siRNA-TRIM11 cell),which silenced the expression of TRIM11 protein in HCC cells.SMMC-7721 with high expression of TRIM11 was used as control group.Both groups were cultured in vitro.Study the effect of TRIM11 expression on the proliferation,migration and invasion of hepatoma cells in vitro and explore the molecular mechanism of signal pathway of anoikis and apoptosis in hepatoma cells.Methods: 1.Real time polymerase chain reaction(RT-PCR)and semi-quantitative immunohistochemical technique were used to detect the expression of TRIM11 protein in tumor tissues and adjacent tissues of 117 patients with hepatocellular carcinoma after radical resection.Chi-square test was used to analyze the relationship between the expression of TRIM11 protein and metastasis,recurrence,survival and prognosis,and the correlation between TRIM11 protein and disease-free survival rate(DFC)and overall survival rate(OS)was analyzed by log-rank in Kaplan-Meier assay.Analysis of serum AFP and TRIM 11 by non-parametric Spearman correlation test.The expression of Bel7402,SMMC77,HepG2 and HuH7 in four kinds of human hepatoma cell lines were detected by Western-blot.siRNA-TRIM11 was used to transfect the hepatoma cells with high expression of TRIM11.After stable culture,the effect of siRNA-TRIM11 on the survival and proliferation of HCC cells was detected by CCK-8 assay.Caspase-3 activity assay kit was used to detect apoptosis rate of hepatoma cells;Transwell migration invasion assay was used to detect the migration and invasion ability of hepatoma cells.;Phosphorylation and activation of PI3K/Akt and MAPK signaling pathways associated with apoptosis in hepatocellular carcinoma cells;PI3K/Akt pathway inhibitor(LY294002)or MAPK pathway inhibitor(U0126)were added to siRNA-TRIM11 cells and SMMC7721 cells to detect the apoptosis rateof Bcl-2,Bad and P-Bad with flow cytometry.Expression of Bcl-2,phosphorylated Bad(P-Bad)andBad was tested with western blot in both groups.Results: the results of RT-PCR and semi-quantitative immunohistochemistry in 1117 patients showed that the expression of TRIM11 in tumor tissues was significantly higher than that in paracancerous tissues(P=0.007).The expression of TRIM11 was related to pathological stage(P=0.003),metastatic,(P=0.031),recurrence(P=0.022).There was a significant correlation between TRIM11 and AFP expression(P=0.002).Analysis of Survival Predication by Kaplan-Meier log-rank Test and the results showed that high expression of TRIM11 protein had lower overall survival rate(P=0.003)and disease-free survival rate(P=0.004).The correlation analysis showed that the level of TRIM11 was positively correlated with the level of serum AFP expression(r = 0.82,P<0.001).TRIM11 expression in four groups of hepatoma cell lines by western blot showed that SMMC-7721 expressTRIM11 best;The transfection efficiency of TRIM11 protein was 100% and the expression of TRIM11 protein was decreased in siRNA-TRIM11 cells,and the expression level of TRIM11 gene in SMMC-7721 cells was significantly lower than that in siRNA-TRIM11 cells.The OD of siRNA-TRIM11 measured by CCK-8 kit was significantly lower than that of SMMC7721 cells(P=0.007).After anoikis culture,the shearing of caspase-3 in siRNA-TRIM11 cells was significantly higher than that in SMMC7721 cells,which indicated the probability of apoptosis was significantly increased.Transwell migration assay:compare with SMMC7721 cells the number of siRNA-TRIM11 cells that could penetrate the superior ventricular membrane of Transwell decreased significantly when they were anoikis cultured(143.40 + 6.58 vs 42.34 ±7.28 TX 16.315 P= 0.007)and The number of siRNA-TRIM11 cells that could penetrate the upper compartment membrane of transwell was significantly decreased(45.30 + 5.68 vs 9.86 ±3.13 vs 13.629(P=0.006).Compare with SMMC7721 cells were inhibited in siRNA-TRIM11 cells.The number of apoptotic cells was measured by flow cytometry when cells cultured with PI3K/Akt pathway inhibitor LY294002 and MAPK/ERK pathway inhibitor U0126.The results showed that the number of apoptotic cells in the siRNA-TRIM 11 was higher than that in the control group.Inhibition of the pathway could promote apoptosis(13.05 ±2.89% vs 24.38 ±3.01)(P=0.03).While SMMC7721 cells lost their nest and added MAPK/ERK pathway inhibitor U0 126),compared with the control group without inhibitor,there was no significant change in apoptosis(13.05 ±2.89% vs 14.13 ±3.08,P=0.65).When SiRNA-TRIM11 cells and control SMMC-7721 cells culture in anoikis the expression of anti-apoptotic protein Bcl-2,p-Bad was lower in siRNA-TRIM11 cells.After adding PI3K/Akt blocker LY294002,the siRNA-TRIM11 cells were detected 24 hours after culturethe antiapoptotic protein Bcl-2,p-Bad of SMMC7721 cells was still decreased,and the inhibitor could not completely block the decrease of anti-apoptotic protein induced by silencing TRIM11.Conclusions: 1.The data of this study showed that TRIM11 was significantly increased in the tumor tissues of patients with HCC.The HCC patients with high expression of TRIM11 had a higher probability of metastasis and recurrence and a shorter disease-free survival and total survival time.2.TRIM11 protein plays an important role in metastasis and recurrence of HCC by resisting apoptosis.3.The effect of TRIM11 protein on apoptosis is related to the signaling pathway PI3K/Akt,which suggests that other pathways may be involved in the anti-apoptosis process mediated by TRIM11. |
PDF Full Text Request