| Background&ObjectiveHepatocellular carcinoma (HCC) is the fifth most common type of cancer affecting over one million people annually worldwide, with a mortality rate almost equal to its incidence [1,2]. In spite of recent advances in diagnosis and treatment, the long-term outcome of HCC patients after curative therapy remains very poor. After radical resection of HCC, its5-year recurrence rate is still as high as65%. Thus, to assess the progression and prognosis of HCC by elucidating the exact molecular mechanism of cell differentiation and proliferation, as well as the recurrence and metastasis pattern of HCC is of great importance. Recent studies showed that TRIM24, member of the tripartite motif protein family which was formerly known as transcription intermediary factor1-alpha (TIF1a), played an important role in the modulation of cell apoptosis and cycle. TRIM24was overexpressed in many types of cancers and was closely associated with cell differentiation, proliferation and the pathogenesis of tumors. However, either the expression profile or the clinicopathological correlation and prognostic significance of TRIM24in human primary HCC is still unknown.In this present study, immunohistochemical staining was employed in sections from both tumor and tumor-free tissues of HCC patients, to determine the expression pattern of TRIM24. Correlation between the expression profile of TRIM24and the clinicopathological features of patients was analyzed afterwards. The mRNA level of TRIM24in5HCC cell lines, including HepG2, SMMC-7721,97L,97H, HCCLM3and normal liver cell line LO2were detected by quantitative real-time PCR. Inhibition of TRIM24by small interfering RNA (siRNA) was conducted in HepG2cells. Changes in protein level of TRIM24, cell apoptosis, cell cycle and EMT before and after siRNA transfected were analyzed. The aim of our study was to investigate the potential role of TRIM24in the pathogenesis of HCC, which might finally lead us to a novel strategy of diagnosis and assess the progression and prognosis of HCC.Methods1. Eighty-three cases of HCC samples were obtained from the Department of Hepatobiliary Surgery of Nanfang Hospital during the period of November2010to July2012. All samples were surgically excised and fixed within2hours. The diagnosis of HCC was confirmed by pathology.2. Immunohistochemical staining was employed in sections from both tumor and tumor-free tissues of HCC patients to determine the expression pattern of TRIM24. Correlation between the expression profile of TRIM24and the clinicopathological features of patients was analyzed.3. The mRNA level of TRIM24in normal liver cell line LO2and5HCC cell lines of different metastasis potential, including HepG2, SMMC-7721,97L,97H and HCCLM3cells were detected by quantitative real-time PCR.4. Inhibition of TRIM24by small interfering RNA (siRNA) was conducted in HepG2cells.5. Changes in expressions of TRIM24protein, and cell apoptosis, cell cycle, EMT related proteins before and after inhibiting TRIM24with siRNA were analyzed by western blotting and flow cytometry. 6. The data were analyzed with SPSS software (SPSS13.0, Inc., Chicago, IL). Statistical analysis included the description of all variables by mean value and standard deviation. The comparison of TRIM24expression ratio between tumor and tumor-free tissue and the ratio among different clinicopathological status were analyzed by Pearson Chi-Square test. The influence factors of clinical outcomes of HCC patients were analyzed by Cox multivariate regression analysis. The correlation between TRIM24expression level and recurrence of the disease was analyzed by Kaplan-Meier survival analysis. The results of quantitative real-time PCR were calculated with2-ΔΔCt method and one-way ANOVA followed with post hoc analysis using LSD-t test were performed to compare TRIM24expression among cell lines.(The data of Quantitative real-time PCR were handled with2-ΔΔCT, each sample repeated trial for3times, then the mean Ct and ΔCT value was calculated). P<0.05was considered to be statistically significant.Results1. Eighty-three samples of HCC were tested by immunohistochemical staining. The positive rate of TRIM24in tumor and tumor-free samples were61.4%(51/83) and4.8%(4/83), respectively (χ2=60.065, P<0.001). Positive expression of TRIM24was found to be well correlated with patients’AFP level, the degree of differentiation of the tumor, post-surgery recurrence, intrahepatic metastases and tumor-free survival time. The positive rate of TRIM24in patients with high AFP level was significantly increased than that in those with lower AFP level (P=0.036). The positive rate of TRIM24protein expression was higher in poor-differentiated HCC than that in well-differentiated HCC (P=0.004). Higher TRIM24positive rate was observed in patients with intrahepatic metastases (P=0.004) or post-surgery recurrence (P=0.000006). Mean recurrence interval of TRIM24positive patients was remarkably shorter than those of TRIM24negative patients (P=0.002). Univariate regression analysis showed cirrhosis and expression of TRIM24were independent predictors of recurrence after resection(P<0.05), while Cox regression analysis showed that the risk of recurrence of TRIM24positive patients was5.327times that of the TRIM24negative patients (HR=5.327;95%Confidence interval2.218-12.795; P<0.01). Moreover, Kaplan-Meier survival analysis showed a tumor-free survival rate in TRIM24positive patients was significantly lower than that in TRIM24negative patients (P=0.000017), which suggested overexpression of TRIM24might indicate worse prognosis.2. Quantitative real-time PCR showed that mRNA level of TRIM24in HepG2cells was elevated10-fold, compared to normal control (LO2)(P<0.05). No statistical significance was noted among other HCC cell lines(P>0.05).3. After inhibiting TRIM24with siRNA, protein level of TRIM24was dramatically decreased in HepG2cells. An increase of pro-apoptotic proteins including p53, Bax and Caspase-8was found simultaneously with the decrease of anti-apoptotic proteins, including Bcl-2and Survivin. The expression of Cyclin D1and CDK4, which modulate the cell cycle, was decreased after the inhibition of TRIM24. The expression of representative EMT related proteins were also determined by western blotting, showing E-cadherin increased and Snail, Slug, Vimentin and β-catenin decreased.4. Cell apoptosis rate increased after down-regulation of TRIM24by siRNA. Cell cycle analysis showed that cells in G1phase significantly increased and those in S phase or G2phase decreased, which indicated cell cycle arrest was induced by down-regulation of TRIM24.Conclusions 1. Expression of TRIM24protein in HCC tissues might influence the biological behavior of HCC, which suggested that TRIM24might be important to the clinical outcomes of HCC.2. Overexpression of TRIM24might work as an anti-apoptosis protein in HepG2cells.3. Cell cycle might be promoted by TRIM24via modulation of important cell cycle related proteins.4. TRIM24might play a role in promoting the process of EMT and eventually lead to recurrence and metastasis of HCC.5. TRIM24might inhibit apoptosis of HCC cells by promoting the degradation of p53and accelerate cell cycle by modulating important cell cycle related proteins, which might eventually contribute to the progress of tumor. |
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