Expression,Clinical Significance And Biological Function Of EVI5 In Hepatocellular Carcinoma

Objective: Malignant tumor is a major social and public health problem.Hepatocellular carcinoma(HCC)is a common malignant tumor worldwide.We use characteristics of tumor cell proliferation and migration of hepatocellular carcinoma as the breakthrough point,screen the differential m RNA by high throughput chip technique,obtain the target gene related to cell proliferation and metastasis via literature search and analysis,explore the relationship between the gene and clinical pathological characteristics and prognosis.Moreover,we explored the biological function of cells by cell experiments and analyzed the effect of the gene on the proliferation and migration of hepatoma cells.We hope to find characteristic biomarkers to provide reference for clinical diagnosis and treatment,and provide alternative target for biological target therapy of HCC.Methods:(1)5 pairs of samples of cancer tissues and paracancerous tissues were examined and analyzed in HCC.Total RNAs was quantified by the Nano Drop ND-2000 and the RNAs integrity was assessed using agarosegel electrophoresis.The sample labeling,microarray hybridization and washing were performed based on the manufacturer’s standard protocols.First of all,total RNAs were transcribed to double strand c DNAs and then synthesized c RNAs.Next,2nd cycle c DNAs were synthesized from c RNAs.Followed fragmentation and biotin labeling,the 2nd cycle c DNAs were hybridized onto the microarray.After washing and staining,the arrays were scanned by the Affymetrix Scanner 3000.Arraysignal intensities were analyzed with Expression Console Software.Raw data probes were normalized using robust multiarray analysis(RMA)for the background correction and quantile algorithm.Then,to define the differential expression profiles and alternative splicing events within the different variants,a one-way Anova was performed in the Transcriptome Analysis Console Software.The GO analysis were carried.We analyzed the differentially expressed genes by Pub Med,and obtained the target gene(EVI5)related to cell proliferation and metastasis.(2)The clinical and pathological data of 205 patients with hepatocellular carcinoma treated with radicalhepatectomy were analyzed retrospectively.Real-time quantitative polymerase chain reaction(RT-PCR)and Western blotting were used to detect the expression of EVI5 in HCC.(3)The expression and distribution of EVI5 in paraffin embedded specimens were detected by immunohistochemistry(IHC).(4)Using SPSS16.0 software package for statistical analysis.Pearson’s chi-squared tested and analyzed the relationship between EVI5 expression and clinicopathological features.(5)Univariate analysis of clinicopathological factors that may affect the prognosis of patients with hepatocellular carcinoma was performed using the log-rank test.Based on the Cox regression analysis model,multiple factors analysis was performed to screen the significantsingle factors,and finally the independent risk factors affecting prognosis were determined.Kaplan-Meier method and log-rank test were used to analyze the influence of EVI5 on the prognosis of patients under different factors.(6)RT-PCR and Western blotting were used to detect the expression of EVI5 in multiple HCC cell lines.(7)Si RNA interference fragments transiently were transfected into hepatoma cells,knocking down the expression levels of EVI5 in HCC cells.The effect of EVI5 on the biological phenotype of human HCC cells was revealed by comparing the proliferation,clonal formation and migration phenotype of HCC cells.Results:(1)Using P < 0.05 and log2|FC|≥1 as screening threshold,438 differentially expressed m RNAs were screened,among which 204 up-regulated genes and234 down-regulated genes.Through Pub Med analysis of differential genes,we confirmed that EVI5 is one of the genes related to cell proliferation and metastasis.(2)The levels of EVI5 in m RNA and protein were significantly up-regulated in HCC tissues.(3)The expression of EVI5 protein was closely related to clinicopathological features,including liver function(P=0.013),vascular invasion(P=0.015),and TNM staging(P=0.014).(4)Single factor log-rank test results showed that the serum alpha fetoprotein level(P=0.034)and postoperative pathological grade(P=0.027),tumor number(P=0.011),tumor size(P=0.018),vascular invasion(P<0.001),TNM stage(P=0.001),EVI5 expression level(P=0.001)all have effects on the overall survival time;serum alpha fetoprotein level(P=0.003)and postoperative pathologicalgrade(P=0.003),tumor number(P=0.008),tumor size(P=0.004),vascular invasion(P < 0.001),TNM stage(P=0.028),EVI5 expression level(P=0.002)have effects on the recurrence free survival time.According to Cox regression analysis model,we analyzed 7 significant factors analysis of the impact on overall survival and recurrence free survival time of single factors by multivariate analysis,and ultimately determined the tumor number(P=0.046),EVI5 expression level(P<0.001)were independent prognostic factors affecting overall survival;serum alpha fetoproteinlevel(P=0.023),tumor number(P=0.036)and EVI5 expression level(P <0.001)were independent prognostic factors for recurrence free survival time.Kaplan-Meiersurvival analysis and log-rank test were used to analyze the effect of different expression levels of EVI5 on the relapse free survival time of HCC patients.The results showed that the high expression of EVI5 significantly reduced the recurrence free survival time.(5)Compared with the normal liver cell line LO2,the m RNA and protein levels of EVI5 in HCC cells were significantly up-regulated.(6)Knockdown of endogenous EVI5 by si RNA interference fragments could decrease the proliferation activity,colony forming ability and migration rate of HCC cells,and compared with the control group,the difference was statistically significant(P<0.05).Conclusions:(1)High throughput microarray analysis showed differential expression of EVI5 in tumor and adjacent non-tumor liver tissues in HCC.(2)EVI5 is up-regulated in hepatocellular carcinoma compared with non tumorous liver tissue adjacent to HCC.(3)EVI5 was up-regulated in HCC cells compared with normal hepatocytes.(4)EVI5 mainly showed cytoplasm staining in immunohistochemical staining.(5)EVI5 may play the role of oncogene in primary hepatocellular carcinoma.The expression levels of EVI5 were inversely related to the prognosis of patients with primary liver cancersurgery,and patients with high EVI5 expression exhibited worse prognosis and shorter relapse free survival time.(6)EVI5 can be an independent biological marker related to prognosis.(7)EVI5 is related to the proliferation and migration ability of HCC cells.Knockdown of EVI5 expression in hepatoma cell lines can reduce tumor proliferation activity,colony forming ability and migration ability.