The Expression And Clinical Prognostic Significance Of Micro RNA-32 In Hepatocellular Carcinoma

ABSTRACTObjective: Primary hepatic carcinoma(PHC) is the seventh malignant carcinoma worldwide and the second common cause of death from cancer, hepatocellylar carcinoma(HCC) accounts for 90.0% of primary liver cancer. The pathogenesis of HCC is complicated and poorly understood,and current therapeutic strategies for HCC are less effective,the patients with hepatocellular carcinoma are proned to relapse and metastasis after surgery, its five-year survival rate is only 15.0%-45.7%. Despite with the progress of the molecular biology, virology and genetics,more HCC related markers were founded and used in clinical, such as:alpha-fetoprotein(AFP),phosphatidylinositol(GPC3), hepatocyte growth factor(HGF), AFP m RNA, GGT m RNA and so on, there is lack of ideal biomarkers for HCC diagnosis and therapy. Micro RNAs(mi RNAs) are a class of non-coding endogenous small(22nt)RNAs,having very important regulating role in cell.mi RNA is involved in regulating cell differentiation, cell invasion, apoptosis and other cell functions,closely related with cancer occurrence and development.Each mi RNA can have multiple target genes, and several mi RNAs can also adjust the same gene. This complex regulatory network either by a mi RNA to regulate the expression of multiple genes,or through a combination of several mi RNAs to regulate the expression of a gene.mi R-32 is located on human chromosome 9q31.3,mi R-32 is upregulated in a variety of tumors, playing an important role in the pathogenesis of cancer-causing genes in prostate cancer,colon cancer,pancreatic cancer, kidney cancer,intestinal type gastric cancer,multiple myeloma and so on.But the mechanistic and prognostic significance of aberrant expression of mi R-32 in human hepatocellular carcinoma are far from clear.In this research,we detect the expression of mi R-32 in hepatocellular carcinoma tissue,to invistigate the relationship between the gene and the clinical data,pathological characteristics and lifetime of the data analysis of the gene in hepatocellular carcinomathe significance of the progress and prognosis,in order to find the mechanistic and prognostic significance of mi R-32 in human hepatocellular carcinoma.To provide theoretical support for mi R-32.Methods:1 Subjects and specimens: 50 patients with hepatocellular carcinoma were enrolled in our study who were taken surgical resection at the Fourth Hospital of Hebei Medical University Hepatobiliary Surgery form March 2010 to June 2014. 50 cases were 46 male and 4 females, aged 25 to 73 years and the average age of 52.8±10.5 years. Preoperative determine hepatitis type, tumor marker levels by serology, based on laboratory tests one week before surgery,Child-Pugh grading criteria to assess liver function, imaging and intraoperative findings to assess the presence or absence of liver cirrhosis and the tumor gross features.Tumor diameter range from 2cm to 15 cm. 50 cases of liver function Child-Pugh A grade forty-seven, B grade three cases. Clinical stage of the 50 cases were different, there were stage Ⅰand stage Ⅱ 29 cases, stage Ⅲ and stage Ⅳ 21 case. This group of patients with hepatitis B surface antigen(HBs Ag) positive 40 cases, negative 10 cases; hepatitis C antibody(HCV-Ab) were negative.All patients before and after surgery were not received any adjuvant therapy.All specimens were 100 cases, including fresh 50 cases of hepatocellular carcinoma tissue specimens and corresponding adjacent 50 cases of cirrhosis tissues.Specimens after resection stored in liquid nitrogen immediately, stored at-80 ℃ refrigerator for spare.Specimens embedded in paraffin HE staining,two pathologist for histological diagnosis.Collecting patients smoking history, drinking history, history of hepatitis, family history and other medical history, registrating patients blood routine, clotting function, liver function, AFP, history of hepatitis B and other laboratory data, recording tumor size, number, and whether a merger of metastasis and other pathological data.2 Detected the expression of mi R-32 in HCC tissues and corresponding adjacent tissues:RNA was extracted using Trizol method, reversed transcription c DNA after determination of the purity and integrity of RNA samples,using SYBR Green real–time fluorescent quantitative PCR technology to detect 50 pairs of hepatocellular carcinoma tissue and the corresponding pericarcinomatous tissue the expression of mi R-32. The specific stem-loop reverse transcription primers of mi R-32:5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATA CGACTGCAACTT-3’.The specific stem-loop reverse transcription primers of U6:5’-AACGCTTCACGAATTTGCGT-3’.The real-time fluorescent quantitative PCR primer of mi R-32:5’-GTGCAGGGTCCGAGGTATT-3’(forward)/5’-GCCGCTATTGCACATTACTAAGTT-3’(reverse).The real- time fluorescent quantitative PCR primer of U6: 5’-CTCGCTTCGGCAGCA CA-3’(forward)/ 5’-AACGCTTCACGA ATTTGCGT-3’(reverse).3 Statistical analysis was performed using SPSS software version 17.0(SPSS Inc,USA),using Kolmogorov-Smimov test method for normal test data. Non-normal data indicate the median, using the Wilcoxon test for paired samples between the two groups. Count data using Chi-square test or Fisher’s Exact Test, survival analysis using the Kaplan-Meier method, univariate analysis using the Log-rank test, multivariate analysis using COX model.P<0.05 was considered as statistical significantance.Results:1 Comparing the expession of mi R-32 in hepatocellular carcinoma andadjacent tissuesIn this study,50 cases of hepatocellular carcinoma and correspondingadjacent tissues,33 cases(66.0%) the expression of mi R-32 relativeincreased.The different expression of mi R-32 between hepatocellularcarcinoma and corresponding adjacent tissues has statisticalsignificance(Z=-2.495,P=0.013);showed that mi R-32 expression inhepatocellular carcinoma was significantly higher than the adjacent tissues.2 Comparing the expression of mi R-32 with differentclinicopathological features in hepatocellular patientsThe relative expression level was correlated with metastasis(P <0.05), The relative expression of mi R-32 in HCC was higher in patients with metastasis(85.0%)than in patients without metastasis(53.3%)(P=0.021).However, no significant associations were found between mi R-32 in HCC with age(P=0.118), sex(P=1), tumor size(P=0.321), AFP level(P=0.924), tumor number(P=0.858),Child-Pugh grading of liver function(P=1),HBs Ag status(P=0.117),the clinical stage(P=0.058).(P>0.05)3 Relationship between the expression status of mi R-32 and the survival time in hepatocellular carcinoma patients.Kaplan-Meier analysis of the expression of mi R-32 positive patients showing 1-,2-,3-year survival rate were 61.1%、44.1%、27.2%, the median survival time of 16.0 months, the average survival time was 23.7±3.1 months; negative patients 1-,2-,3-year survival rate were 76.5%、58.8%、41.2%,the median survival time of 31.0 months, the average survival time was 32.2 ± 4.7 months,the difference was not statistically significant(P=0.109). Tumor diameter>5cm patients showing 1-,2-,3-year survival rate were 61.6%、39.2%、22.4%, the median survival time of 14.0 months, the average survival time was 22.4 ± 2.7;tumor diameter <5cm patients 1-,2-,3-year survival rate were 82.5%、73.3%、64.2%, the median survival time of 46.0 months, the average survival time was 40.2±5.2 months,the difference was statistically significant(P=0.01).Child-Pugh graded A patients 1-,2-,3-year survival rate were 69.3%、51.4%、33.6%, the median survival time of 25.0 months, the average survival time was 27.7±2.8 months;Child-Pugh graded B patients 1-,2-,3-year survival rate were 0%, the median survival time of 8.0 months, the average survival time was 8.5±0.5months,the difference was statistically significant(P=0.035). The metastasis patients with 1-,2-,3-year survival rate were 48.0%、32.0%、21.3%, the median survival time of 12 months, the average survival time was 18.9±3.4 months;no metastasis patients with 1-,2-,3-year survival rate were 79.0%、61.0%、39.5%, the median survival time of 33.0 months, the average survival time was 32.1±3.5 months,the difference was statisticallysignificant(P =0.018).In order to analyze the independent factors relating to the total survival time of hepatocellular carcinoma patients,3 variables were selected to conduct the COX Analysis.The variables are Child level, tumor size, tumor metastasis. The analysis demonstrated that tumor size(P=0.043)is an independent prognostic factor of hepatocellular carcinoma.Conclusions:1 The up-regulation of mi R-32 may play a significant role in the initiation and progression of hepatocellular carcinoma.2 Mi R-32 expression state associated with the presence of metastasis for patients with hepatocellular carcinoma, indicating that abnormal expression of mi R-32 may reflect the condition of patients with hepatocellular carcinoma in a certain extent.3 Still can not believe that mi R-32 expression status as a prognostic indicator in patients with hepatocellular carcinoma, tumor size can be used as an independent factor of hepatocellular carcinoma.