| Objective:Transarterial Chemoembolization（TACE）has become an increasingly important postoperative palliative treatment for hepatocellular carcinoma（HCC）,but postoperative adjuvant transarterial chemoembolization（pa-TACE）treatment does not show significant adjuvant anticancer effects on certain HCC with specific genetic profiles.Therefore,the strategy how to screen patients with HCC to increase the efficacy of pa-TACE treatment is an critical topic in this certain research field.Recent studies have shown that microRNAs（miRNAs）not only play a key role in the development and progression of HCC,but also serve as biomarkers for HCC diagnosis and prognosis.Our previous research also suggested that serum miRNA-4651 serve as a potential early-stage diagnosis and prognostic biomarker,but the significance of miRNA-4651 expression in tumor tissue in the prognosis of HCC is still unclear.This project aims to investigate whether miRNA-4651 has an effect on pa-TACE treatment to improve the prognosis of HCC.Methods:（1）This project is a hospital-based retrospective survey study,including 302patients with advanced-stage hepatocellular carcinoma who received primary treatment in the affiliated hospitals of the Youjiang Medical University for Nationalities and Guangxi Medical University between January 2005 and December 2009 according to the inclusion and exclusion criteria on cases.Among these cases,150 patients who received pa-TACE as their initial treatment were defined as the pa-TACE treatment group,while the other patients who only underwent tumor resect as initial treatment were defined as the control group.All patients were confirmed by histopathological examination as HCC without history of radiotherapy and chemotherapy before surgery;（2）Collect appropriate amount of fresh tumor tissue samples from all patients and place in liquid nitrogen for analysis of miRNA-4651expression level;（3）The expression level of miRNA-4651 in tumor tissues was detected by real-time quantitative PCR（TaqMan probe technique）,and the relative expression of each sample was calculated by 2-ΔCT.For the convenience of analysis,the average relative expression of miRNA-4651(2-ΔCT=2)was used as the basis for classification.The expression of miRNA-4651 was divided into two groups:low expression(2-ΔCT≤2)and high expression(2-ΔCT>2);（4）The differences between the TACE group and the non-TACE group were tested using the Pearson chi-squared test.Logistic regression model was used to analyze the effect of miRNA-4651 expression level on the clinicopathological features of HCC.The correlation between miRNA-4651 expression level and tumor differentiation degrees or microvessel density（MVD）was tested using Spearman’s rank correlation method.The Kaplan-Meier survival model and the Cox regression model were used to analyze the effect of miRNA-4651 expression level on the prognosis of HCC and the efficacy of pa-TACE treatment.（5）Using HCC cell line SMMC-7721 as the research object,the sensitivity of tumor cells to doxorubicin（an anticancer drug used in pa-TACE procedure）was analyzed by the half-maximal inhibitory concentration（IC50）using terminal deoxynucleotidyl transferase-catalyzed DNA nick-end labeling（TUNEL）assay and flow cytometry assay.（6）High-throughput sequencing technology was used to analyze the possible target genes regulated by miRNA-4651,and the possible target genes obtained by screening were further verified by real-time quantitative PCR and Western blot analysis.Results:（1）There were no significant differences in the distribution of clinicopathological features such as age,gender,and tumor grade between the pa-TACE treatment group and the control group（P>0.05）;（2）Compared with the control group,pa-TACE treatment can effectively improve the overall survival（OS）of patients with HCC（median overall survival time:11.00 vs 24.00 months,P<0.05）and recurrence-free survival（RFS）（median recurrence-free survival time:8.00 vs 20.00 months）,P<0.05);（3）In tumor tissues,miRNA-4651 expression levels affected serum alpha-fetoprotein（AFP）levels[odd ratio（OR）=2.07,P<0.05],tumor grade（OR=2.46,P<0.05）,and tumor MVD（OR）=3.20,P<0.05),but did not affect other clinicopathological features（P>0.05）;（4）The results of Spearman’s correlation analysis showed that the expression level of miRNA-4651 in tumor tissues was positively correlated with tumor differentiation and MVD（correlation coefficients were 0.22 and 0.28,respectively,P<0.05）;（5）Compared with the low miRNA-4651expression group,high miRNA-4651 expression statistically shortened the OS of HCC patients（median overall survival time:29.00 vs 10.00 months,P<0.05）and RFS（median recurrence-free survival time:24.00 vs 7.00 months,P<0.05）;（6）Both single-factor and multi-factor Cox regression model analysis showed that increased miRNA-4651 expression increased the risk of death in HCC patients[hazard ratio（HR）were 2.80 and 2.79,respectively,P<0.05]and risk of tumor recurrence（HR were 2.39 and 2.30,respectively）.P<0.05);（7）Based on the stratified analysis of miRNA-4651 expression,pa-TACE treatment can improve the prognosis of patients with high miRNA-4651 expression,but not for patients with low miRNA-4651 expression;（8）Results in vitro cytology showed that miRNA-4651expression increased the sensitivity of cancer cell SMMC-7721 on anticancer drug doxorubicin;（9）High-throughput sequencing results showed that miRNA-4651 expression could down-regulate CYP2W1 expression,and this finding was further confirmed by real-time quantitative PCR and Western blot analysis.Conclusion:The current findings suggest that pa-TACE treatment can improve the prognosis of patients with HCC and his improvement in prognosis is more pronounced in patients with high miRNA-4651 expression.The possible mechanism might be that miRNA-4651 expression increases the sensitivity of HCC cells to anticancer drugs by down-regulating CYP2W1 expression in tumor cells.These results suggest that miRNA-4651 can be used as a valuable potential biomarker for patients with HCC whether receiving pa-TACE treatment or not. |
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