| Objective: Hepatocellular carcinoma(HCC)is a common malignant tumor all around the world,and the mortality rate of HCC ranks top 3 among all tumors.Albeit the life expectancy of HCC patients have been prolonged by5 years with the help of comprehensive treatment that gives priority to operative treatment,the rate of HCC patients who could live 5 years longer is only from 15.0% to 45.7% due to very complicated pathogenesis of HCC,biological markers with lower specificity and susceptibility.Therefore,the research emphasis of HCC lies in delving into molecular biological mechanisms of HCC,seeking for special diagnosis and treatment targets of HCC patients.MicroRNA(miRNA)is a kind of non-coding and small RNA fragments,which widely existing in eukaryotic.It can widely participate in body's physiological and pathological process through regulating the expression of target genes.MiR-187-3p belongs to miR-187 and it can locate the chromosome 18q12.2.It has been reported that miR-187-3p shows low level expression in colorectal cancer,prostatic cancer,non-small cell lung cancer,renal cell carcinoma and adenocarcinoma of esophagus,demonstrating that miR-187-3p plays the role of tumor suppressor gene.However,some other experiments verify that miR-187-3p shows high level expression in peripheral T-cell lymphoma.Besides,the above-mentioned experiments also demonstrate that miR-187-3p can increase the invasion ability of breast cancer cell and that miR-187-3p is related to the prognosis of breast cancer patients.Hence,miR-187-3p can play different roles in varied tumors.Nevertheless,the expression status and effects of miR-187-3p in hepatocellular carcinoma tissues have never been reported.This paper aims to filtrate the differential expression of miRNA existing in fresh hepatocellular carcinoma tissues via Gene Chip Hybridization Technology,test the expression status of miR-187-3p with the help of Real-time PCR,and analyze the development and progression of miR-187-3p in hepatocellular carcinoma,its clinical diagnosis and prognostic value combined with clinical data of patients,survival data and pathological features,providing theoretical foundation for the clinical application of miR-187-3p.Methods:1 Subjects and specimens: Participants and Specimen: 90 patients with HCC who had been operated in Department of Hepatobiliary Surgery,Fourth Hospital of Hebei Medical University from April 2010 to August 2016 were selected.75 male patients and 15 female patients were included and the ages of them were between 25 and 75.Liver functions,types of hepatitis and tumor marker levels were evaluated according to laboratory indexes a week before the operation and Child-pugh grading standards.The status of liver cirrhosis and features of tumors were also estimated via imageological examination and intraoperative findings.The diameter of tumors was found 2-25 cm.There were 87 cases of Child A liver function and 3 cases of Child B liver function.Also among the chosen 90 patients,there were 39 Stage I patients,29 Stage II patients and 22 Stage III patients.Meanwhile,74 patients were(HBsAg)positive and the others were(HBsAg)negative.Please note that all the 90 patients did not accept chemotherapy,radiotherapy or other antitumor therapies.There were 180 specimens in total,90 fresh hepatocellular carcinoma tissue specimens and 90 corresponding liver tissues adjacent to carcinoma included.All the specimens were immediately preserved in liquid nitrogen after sampling and then were placed in-80? deep freezer for further use.In the meanwhile,all the specimens were dealt with common paraffin embedding and HE staining.Afterwards,the histodiagnosis of all specimens were finished by two pathologists.Patients' viral hepatitis background,history of alcohol intake,smoking history and family history were collected;liver function of patients,AFP,blood routine examination,coagulation function,hepatitis A,B and C and other laboratory indexes of patients were recorded;types of tumorpathology,sizes and numbers of tumors,whether the portal vein and its branches tumor suppository or not and other pathology materials were registered.Additionally,the patients were interviewed by telephone or in the form of letters.The start dates of follow-up visits were usually the dates of operations;the deadline of follow-up visits was October 11,2016.2 MicroRNA chip hybridization technology:5 patients were chosen from90 patients with HCC,using primer Poly(A)Tailing reverse transcription conform to the standard of total RNA.And then gene chip hybridization,washing and scanning were adopted to generate data.With the help of bioinformatics analysis and online service sites of miRNA target gene prediction software(Targetscan,MiRanda),miRNA target gene prediction featuring differential expression was outputted and the intersection of 2prediction target genes were extracted.(The experiment is assisted to complete by the QiMing Biological information company of Shanghai)3 Test for expression status of miR-187-3p in hepatocellular carcinoma tissue and corresponding liver tissues adjacent to carcinoma: RNA was extracted by virtue of Trizol method.The purity and integrality of RNA was also tested.And then the qualified DNA was reserved as cDNA.Finally,the expression status of miR-187-3p in 90 pairs of hepatocellular carcinoma tissues and corresponding liver tissues adjacent to carcinoma was examined via SYBR Green real-time PCR analysis technique.As a result,the specific stem-loop reverse transcription primer of miR-187-3p was 5'-GTCGTATC CAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCGGCTGC-3';the specific stem-loop reverse transcription primer of U6 is 5'-AACGCTT CACGAATTTGCGT-3'.Meanwhile,the sense primer of miR-187-3p Realtime PCR was 5'-GTGCAGGGTCCGAGGTATT-3' and the reverse primer was 5'-GCCGCTCGTGTCTTGTGTTGCAGC-3'.The sense primer of U6Real-time PCR was 5'-CTCGCTTCGGCAGCACA-3' and the reverse primer was 5'-AACGCTTCACGAATTTGCGT-3'.4 Statistical analysis: statistical analysis was performed through statistical software SPSS 21.0 and normal distribution test was also used to deal withexperimental data relying on Kolmogorov-Smimov.The non-normal data was expressed in median and paired samples between the two groups were tested by Wilcoxon.Enumeration data were examined by Chi-squared test or Fisher's Exact Test;survival analysis was calculated by Kaplan-Meier;univariate analysis was scrutinized by Log-rank and multivariate survival analysis by COX regression model.P<0.05 was considered that difference was statistical significance.Results:1 The expression profiles analysis of HCC: Use of microRNA chip hybridization technology,148 kinds of microRNAs with more than 2 times different expression in hepatocellular carcinoma were found,of them 102 kinds of microRNA in hepatocellular carcinoma tissue kept lower expression than that in paracancerous tissue and the remaining 46 kinds of microRNA were completely opposite.2 Comparing the expession of miR-187-3p in hepatocellular carcinoma and adjacent tissuesIn this study,90 cases of hepatocellular carcinoma and corresponding adjacent tissues,63 cases(70.0%)the expression of miR-187-3p relative decreased.The different expression of miR-187-3p between hepatocellular carcinoma and corresponding adjacent tissues has statistical significance(Z=-2.891,P=0.004);showed that miR-187-3p expression in hepatocellular carcinoma was significantly lower than the adjacent tissues,consistenting with the results of microRNA chip.3 Comparing the expression of miR-187-3p with different clinicopathological features in hepatocellular patientsThe relative expression level was correlated with tumor size and clinical stages(P<0.05),The relative expression of miR-187-3p in HCC was lower in patients with Tumor diameter ?5cm(75.0%)than in patients with Tumor diameter<5cm(59.1%)(P=0.017).The positive rate of expression in stage ?of HCC is 69.2%,in stage ? is 79.3% and in stage ? is 59.1%,The differe-nce is statistically significant(P=0.002).However,no significant associations werefound between miR-187-3p in HCC with sex(P=0.119),age(P=0.976),the portal vein and its branches tumor suppository or not(P=0.392),HBs Ag status(P=0.342),tumor number(P=0.867),AFP level(P=0.198).(P>0.05)4 Relationship between the expression status of miR-187-3p and the survival time in hepatocellular carcinoma patients.Kaplan-Meier analysis of the expression of miR-187-3p negative patients showing 1-,3-,5-year survival rate was 87.8%,56.2%,24.7%,the average survival time was 40.470±4.407 months;positive patients 1 year survival rate was 90%,the average survival time was 64.600±4.996 months,the difference was statistically significant(P=0.011).The patients of HCC have tumor suppository in the portal vein and its branches with 1 year survival rate was50.9%,the average survival time was 17.568±2.326 months,the median survival time was 19 months;no tumor suppository patients with 1-,3-,5-year survival rate was 93.8%,68.4%,59.3%,the average survival time was51.266±4.164 months,the median survival time was 70 months,the difference was statistically significant(P=0.005).Tumor diameter <5cm patients 1 year survival rate was 82.9%,the average survival time was 58.802±5.791 months,the median survival time was 70 months;tumor diameter?5cm patients showing 1-,3-,5-year survival rate was 95%,59.0%,37.2%,the average survival time was 41.713±4.606 months,the median survival time was 26 months,the difference was statistically significant(P=0.032).Stage?patients with 1-,3-year survival rate was 86.4%,47.8%,the average survival time was58.629±5.203 months.Stage?patients with 1 year survival rate was 77.6%,the average survival time was 30.593±6.290 months.Stage ? patients with 1year survival rate was 56.8%,the average survival time was 20.729±1.914 months,the difference was statistically significant(P=0.001).Child-pugh graded A patients 1-,3-,5-year survival rate was 97.5%,71.2%,63.3%,the average survival time was 47.109±3.984 months,the median survival time was 63 months;Child-pugh graded B patients 1-,3-,5-year survival rate was0%,the average survival time was 19.667±6.006 months,the median survival time was 26 months,the difference was no statistically significant(P=0.437).Age <60 patients with 1-,3 year survival rate were 88.9%,62.3%,the average survival time was 49.916±4.785 months,Age ?60patients with 1 year survival rate was 78.5%,the average survival time was 39.836±6.172 months,the difference was no statistically significant(P=0.441).In order to analyze the independent factors relating to the total survival time of hepatocellular carcinoma patients,some variables were selected to conduct the COX Analysis.The variables are miR-187-3p abnormal expression,clinical stage,tumor size and so on.The analysis demonstrated that miR-187-3p abnormal expression,clinical stage and tumor numbers are independent prognostic factor of hepatocellular carcinoma(P<0.05).Conclusions:1 HCC tissues have differentially expressed miRNAs,and miR-187-3p in HCC tissues was lower from corresponding adjacent carcinoma tissues,explaining miR-187-3p may participate in the occurrence and development of HCC.2 MiR-187-3p expression state associated with tumor size and clinical staging for patients with hepatocellular carcinoma,indicating that abnormal expression of miR-187-3p may reflect the condition of patients with hepatocellular carcinoma in a certain extent.3 MiR-187-3p expression status,clinical stage and tumor numbers can be used as independent factors,predicting postoperative survival outcomes for patients with hepatocellular carcinoma. |
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