| Objective: primary hepatic carcinoma(PHC)is one of the most common malignant tumors with high incidence and mortality.About 90% of PHC is hepatocellular carcinoma.About 383 thousand people die of hepatocellular carcinoma(accounted for 1/2 of the total number deaths of hepatocellular carcinom)in China each year.Although the knowledge of hepatocellular carcinoma has been deepened,its pathogenic is not completely clear.With the progress of multidisciplinary research,the number of markers related to hepatoma liver cancer is being discovered by researchers,and the clinical application is becoming more and more popular.The clinical practice shows that the high sensitivity and specificity of biological markers are still scarce,and the prominent advantage of such markers is obviously beneficial to the diagnosis,treatment and prognosis of hepatocellular carcinoma.MicroRNA(miRNA)can effectively regulate several kinds of life behaviors in living cells,and the endogenous miRNA length is about 20-24 nucleotides.MiRNA is involved in the development,growth,metabolism,apoptosis and tumor formation of the organism,which will obviously affect the formation and development of a variety of malignant tumors.Some analyses also show that over 50% of miRNA is located in the tumor related regions of the genome,suggesting that microRNA may be closely related to cancer.MiR-378 is located in the human chromosome 5q32,which is down regulated in the diseases such as gastric cancer,colorectal cancer and bone marrow dysplasia,but in breast cancer and lung cancer,the expression is up.MiR-532 is located in the human chromosome Xp11.23,and its up-regulation and downregulation are not certain.In different diseases,the expression is different,such as: expression up regulation: chronic lymphocytic leukemia;down regulation of expression: lung cancer.From the aspect of HCC,these two kinds of microRNA have not been studied yet.In this experiment,the fresh hepatocellular carcinoma specimens and paracancerous specimens were used as the research object to detect the expression of microRNAs in these specimens.At the same time,the relationship between the pathogenesis of hepatocellular carcinoma and the clinical data of the patients was analyzed,and the relationship between the expression of the two microRNAs and the prognosis of the patients was determined by the results of analysis.To provide theoretical support for the clinical application of miR-378 and miR-532.Method:1 The research objects and specimens: The specimens were collected from 90 cases of Hepatocellular carcinoma in fourth hospital of Hebei Medical University in 2010~2016 years.None of the patients received any radiotherapy or chemotherapy before operation.There were 75 men,15 women,the age range is 25-78 years old and the median age is 56 years old.Based on the Child-Pugh grading standard,the liver function was evaluated,the type of hepatitis virus infection and the level of serum tumor markers were determined,the liver cirrhosis was assessed by the intraoperative and imaging findings,and the degree of liver cirrhosis was judged,to evaluate the characteristics of the tumor.The diameter of the tumor was 2-15 cm.87 cases of liver function were grade Child-PughA,and 3 cases were B.All patients underwent AJCC clinical staging.There were 64 patients in stage I and stage II,26 in stage III and IV.Among the specimens,74 patients were positive for hepatitis B surface antigen(HBsAg)and 16 were negative.All the patients in the study were negative for hepatitis C antibody(HCV-Ab).No preoperative adjuvant treatment was performed in all patients.The specimens were immediately stored in liquid nitrogen and then stored in-80 C refrigerator.All the specimens obtained were routinely stained with HE.Finally,the tumor and its adjacent normal tissues were diagnosed by the senior doctors.The individual history of patients,such as whether the patient has a history of smoking,whether there is a history of drinking,whether the hepatitis B has been infected,or whether there is a certain genetic history in the family,records the patient's detailed laboratory indicators,and registers the tumor related clinical pathological data of the patients.All patients were followed up by mobile phone or letter sending.The follow-up period was until October 2016.2 Chip hybridization technology of miRNA :From the specimens of hepatocellular carcinoma,5 samples were selected.The primers Poly(A)tail was retroactively transcribed to the standard RNA,and miRNA chips were hybridized,washed,scanned,and generated data.The application of Internet miRNA target gene prediction software(Targetscan,MiRanda)by bioinformatics analysis and summary,the predicted target gene output the differential expression of miRNA.(We finished this part of the experiment with the help of the Shanghai Ming biological information company).3 Detection of the expression of miR-378 and miR-532 in hepatocellular carcinoma tissue:Extraction of total RNA by Trizol,the extracted RNA samples were measured the purity and integrity.After the test,the standard RNA was reverse transcribed to cDNA.Then the SYBR Green real-time fluorescent quantitative PCR technique was used to detect the expression of miR-378 and miR-532 in the pathological and paracancerous tissues of the liver cancer patients(90 cases).The miR-378 Specific stem loop primers:5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATA CGACGCCTTCTG-3',The U6 specific stem ring reverse transcriptional primers is 5 '-AACGCTTCACGAATTTGCGT-3'.The specific stem ring primers for miR-532 is 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACT GCAAGCC-3',MiR-378 real-time fluorescent quantitative PCR up stream primer sare:up stream primers:5'-GTGCAGGGTCCGAGGTATT-3',down stream primers: 5 '-GCCGCACTGGACTTGGAGTCAGAA-3'.The up stream primers for U6 real-time fluorescence quantitative PCR were 5 '-CTCGCTTCGGCAGCACA-3,and the down stream primers was 5-AACGCTTCACGAATTTGCGT-3'.The upstream primers of miR-532 real-time fluorescent quantitative PCR are: upstream primers: 5 '-GTGCAGGGTCCGAGGTATT-3' down stream primers: 5 '-GCCGCCCTCCCACACCCAAGGCTT-3'.4 Statistical analysis:Using SPSS21.0 software for analysis,use the Kolmogorov-Smimov test method to test all the data in normal distribution.Using the X2 test or Fisher's exact test to the count data.All non normal data were analyzed using paired samples Wilcoxon test;Kaplan-Meier analysis method was used to analyze the survival of patient,single factor analysis using Log-rank test,multivariate analysis using COX model.P<0.05 has statistical significance for the difference.Result:1.Analysis of the expression of miRNA in hepatocellular carcinomaThe selected 5 pairs of hepatocellular carcinoma tissues and corresponding para cancerous tissues were selected with high differential expression(at least 2 times).The total number of miRNA is 148,there are 102 down-regulated expression of miRNA in HCC tissues than in adjacent tissues,while the other 46 are up-regulated miRNA.2.Relative expression of miR-378 and miR-532 in the tissues of hepatocellular carcinoma and the corresponding para cancerous tissuesIn 90 cases of hepatocellular carcinoma tissues and corresponding normal tissues,the expression of the miR-378 in normal tissues decreased in 64 cases than in cancer tissues(71.11%),the difference was statistically significant(Z=-2.529,P=0.011),indicating that the expression of miR-378 in hepatocellular carcinoma tissues was significantly lower than that of adjacent tissues.The expression level of miR-532 in hepatocellular carcinoma tissues was higher than that in adjacent tissues.There were 52 cases(57.77%),and the difference was statistically significant(Z=-1.986,P=0.047).This result indicates that the expression of miR-532 in hepatocellular carcinoma tissues is significantly higher than that in adjacent tissues.The results are in agreement with the results of the miRNA chip.3.The relationship between the expression of miR-378,miR-532 and the clinicopathological features in patients with hepatocellular carcinomaThe abnormal expression was significantly correlated with tumor size and clinical stages in patients with hepatocellular carcinoma in miR-378(P<0.05),the tumor diameter < 5 cm of the patients with the positive expression rate of miR-378 was(57.14%),the positive rate for the expression of diameter larger than 5 cm of the patients of miR-378 was(75.36%),the difference was statistically significant(P=0.047).The positive rate of down-regulation of miR-378 in clinical stage I and II patients was(67.18%).The positive rate of miR-378 down regulated in patients with III stage and IV stage was(80.76%),and the difference was statistically significant(P=0.005),but the different expression of miR-378 has no significantly with age(P=0.136),gender(P=0.238),AFP value(P=0.593),tumor number(P=0.692),Child-Pugh grade(P=0.406),hepatitis B surface antigen(P=0.502)and tumor metastasis(P=0.673).(P > 0.05).Abnormal expression of miR-532 with tumor size and AFP value and clinical stages were significantly correlated(P<0.05),miR-532 is up-regulated in tumor diameter < 5 cm of the patients The positive rate was(90.47%),the positive rate increased with diameter larger than 5 cm with the expression of miR-532 was(47.82%),the difference was statistically significant(P=0.003);the positive rate of miR-532 expression increased in AFP is less than or equal to 400 of patients was(68.75%),the positive rate of AFP is more than 400 of patients was(42.86%),the difference was statistically significant(P=0.024);The positive rate of miR-532 expression in clinical stage I and II patients was(68.5%).The positive rate of miR-532 expression in clinical stage III and IV stage patients was(30.77%),the difference was statistically significant(P=0.001).But the abnormal expression of miR-532 has no connection with age(P=0.097)? gender(P=0.849)?tumor number(P=0.946),Child-Pugh grade(P=0.132),hepatitis B surface antigen(P=0.07)and tumor metastasis(P=0.849),there were no statistically significant differences between the different clinicopathological characteristics(P greater than 0.05).4.The relationship between the expression of miR-378,miR-532 and the survival prognosis of patients with hepatocellular carcinomaKaplan-Meier Analysis showed that miR-378 downregulated the expression of hepatocellular carcinoma in 1 year and 3 year survival rates were 73.7% and 55.1%,the average survival time was 20.61±2.38 months;miR-378 expression in 1,3 year survival rates were 82.8% and 44.5%,the average survival time was 24.23±3.85 months,the difference shows no statistical significance(P=0.918).The expression of miR-532 is upregulated in hepatocellular carcinoma patients with 1 and 3 year survival rates were 66.8%,37.1%,the average survival time was 20.12 ±2.52 months;the down expression with 1 year and 3 year survival rates were 90.4% and 67.7%,the average survival time was 23.67±3.31 months,the difference was statistically significant(P= 0.029).Independent prognostic factors for overall survival in patients with hepatocellular carcinoma and analysis of the various single factor,we analysis the multi factor with COX analysis,the results show that for miR-378,the tumor metastasis is independent prognostic factors predicting survival of patients after liver transplantation(P=0.025),the rest of the parameters P values were greater than 0.05,was not independent prognostic factor for the prediction of survival of patients with hepatocellular carcinoma after hepatectomy.For miR-532,miR-532 expression(P=0.000)and tumor size(P=0.01),AFP(P=0.029)were independent prognostic factors predicting survival of patients after liver transplantation.The parameters of the P values are greater than 0.05,was not an independent prognostic factors predicting survival of patients with hepatocellular carcinoma after hepatectomy.Conclusions:1.There were differentially expressed of two microRNAs in hepatocellular carcinoma,the expression of miR-378 in hepatocellular carcinoma tissues than in paracancerous tissues reduced,the expression of miR-532 in hepatocellular carcinoma compared with paracancerous tissues is higher,suggesting that miR-378 and miR-532 play a certain role in hepatocellular carcinoma.2.The differential expression of miR-378 in hepatocellular carcinoma is relate to tumor size and clinical stages,and miR-532 expression of tumor patients is relate to the tumor size and AFP and clinical stages,the results show that differential expression of these two microRNAs may reflect the disease condition of patients with hepatocellular carcinoma in a certain extent.3.The low expression of miR-378 is not as a temporary state of patients with hepatocellular carcinoma prognosis,but the high expression of miR-532 can be used as the condition of patients with hepatocellular carcinoma after judgment index;Tumor metastasis can be used as independent predictors of survival in patients,at the same time the size of tumor and the high expression of miR-532,AFP can be used as independent predictors of survival in patients with hepatocellular carcinoma after operation. |
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