| Background and ObjectiveWith advances in medical technology, the early diagnosis of cancer has made considerable progress. Hepatocellular carcinoma(HCC) as having a high incidence of cancer mortality in Asia and Africa, early diagnosis is still relatively difficult. The vast majority of patients at the time of diagnosis are at advanced disease, but patients with delayed diagnosis led to miss the timing of surgery, and thus lead to high mortality rates of hepatocellular carcinoma. At present, the main means confined to early HCC screening serum alpha-fetoprotein (AFP) and imaging, and its application has made relatively good effect of early diagnosis, but both sensitivity and specificity are relatively low, in the application there are many defects. Research has shown that there is currently another microRNA involved in the occurrence and development of HCC. Therefore it is necessary to evaluate the diagnostic value of microRNA in hepatocellular carcinoma.HCC is one of the most common malignant tumors, liver cancer development and progression is a multi-factor and a multi-step process. microRNA is a small non-coding RNA fragments. Studies showed that microRNA can involved in growth, metabolism, proliferation and apoptosis of the cell, and plays an important role in regulating the expression of the gene. Since its discovery in 1993, more and more evidence of their involvement in the development and progression of various cancers. Therefore it is essential to study the specific mechanism of microRNA in cancer and its role of downstream genes regulation in cancer is valuable for the cancer diagnosis, treatment and prognosis. In tumors, the role of microRNA is heterogeneity and can be expressed as oncogenes or tumor suppressor genes, and studies have also indicated that microRNA regulatory mechanism is complicated, even exists the self-regulation phenomenon, and therefore illustrate the specific microRNA can benefit the clinical practice.microRNA is a small non-coding RNA fragment, there is a certain level of expression in serum. Its main role through targeted binding fragment and thus participate in the regulation of gene mRNA transcription or protein translation. Studies have shown that microRNA involved in a number of physiological and pathological processes, including organ development, inflammation and tumorigenesis development. Studies have shown that microRNA involved in the occurrence and development of HCC, have potential diagnostic value.To date, more than 2500 individuals have been recorded in the category miRBase database V20, whereas with the further study of their number is rapidly increasing. Taking into account one microRNA can regulate a variety of target mRNA transcripts or one mRNA transcript can be targeted by a number of microRNA regulation phenomenon, it can be roughly estimated that about 10-40% above mRNA sequences targeted by microRNA regulation. Therefore, it is necessary to focus on microRNA target genes for further study. microRNA in tissues associated with the development process and can occur either the expression level of differentiation may also be a transient period of differential expression. By labeling microRNA can accurately distinguish cancerous tissue from normal tissue. Further studies have shown that microRNA present in succession as a diagnostic and prognostic biomarkers for cancer. microRNA genes typically located in the intron RNA and clustering of the RNA polymerase Ⅱ and a length of several kb miRNA transcribed genomic primary microRNA (pri-microRNA). Then the primary microRNA genes occur in some specific parts of the nucleus generate shear precursor microRNA (pre-microRNA) by the Drosha RNA cleavage to generate the mature microRNA (mature-microRNA) in the cytoplasm by Dicer RNA cleavage. Then the mature microRNA-induced silencing complex under (miRISC) to effect binding Argonaute 2 (AG02) activation. In fact targeted mRNA sequence 3’untranslated region through 2-8 nucleotides in mature microRNA 5’complementary sequence binding, mRNA if fully complementary between the mature microRNA and its target mRNA and often leads to degradation of deadenylated without improving the complementarity cause translational inhibition. Mature microRNA may also regulate gene expression by binding to the 5’UTR or a coding region of the target gene mRNA (CDS). CDS-site-specific binding tend to be more of a target gene mRNA translational inhibition, and 3’UTR sites tended to promote mRNA degradation. There are a number of studies have shown that microRNA variety of cancer-related genes have regulatory role, in fact, played a role in oncogenes or tumor suppressor genes. And a considerable number of studies have confirmed their participation in the microRNA by the development of cancer among the epithelial-mesenchymal transition process.microRNA-301 SKA2 is located in intron one microRNA, which can lead to malignant cell proliferation regulation, enhance the invasiveness and angiogenesis increase, and downregulation can inhibit malignant cell proliferation, migration and tumor growth. And microRNA-301 in human pluripotent liver cell self-renewal may be further adjusted by targeting the immune and proteomic diversity in SFRS2 and MDB2a. Recent studies have indicated that the phenomenon microRNA-301 upregulated in the presence of prostate cancer and pancreatic cancer, but the phenomenon appears downregulated in cholangiocarcinoma. And studies have shown that microRNA-301 expression in HCC was significantly higher than the adjacent tissues.Nevertheless microRNA-301 in the diagnosis of hepatocellular carcinoma needs further research value, thus the main purpose of this study was to explore microRNA-301 diagnosis value in hepatocellular carcinoma and its mechanism.Material and method1. Patients and cell lineThis study was collected in January 2014 to 2015 before July,52 cases of hepatocellular carcinoma in patients undergoing surgery serum samples,38 cases of control serum samples (38 cases of healthy people),36 patients with liver cancer in men, with an average age of 53.50 years,16 women patients with an average age of 47.19 years. All were pathologically diagnosed as hepatocellular carcinoma, excluding associated with other malignancies, serious infections and other serious systemic disease, all patients has No treatment of chemotherapy or other biological immunotherapy.13 patients with tumor diameter= 5cm, and tumor diameter> 5cm in 39 patients, with portal vein tumor thrombus in 20 cases, AFP> 400μg/L in 36 patients with AFP≤400μg/L in 16 patients, HBsAg (+) in 39 patients, HBsAg (-) in 13 patients, Barcelona early stage cancer patients (A period) in 7 cases, medium (B stage) in 25 cases and advanced (C stage and above) in 20 cases. All samples of each patient were informed in advance and obtain permission from the Ethics Committee of the experiment to obtain permission Hospital. All specimens were 15 minutes away from the body into the inactivated enzyme RNA cryopreservation tubes and stored at -80℃ refrigerator.52 patients were collected tumor tissue, paraneoplastic tissue and relative normal tissue. Normal liver cell line LO2 and hepatoma cell line MHCC-97H, SMMC-7721, HepG2, Hep3B, Huh-7 were bought form Chinese Academy of Science in shanghai for the study of microRNA-301 mechanism.2. Contents of the studyPortion 1microRNA-301 diagnostic significance in hepatocellular carcinoma:52 cases of hepatocellular carcinoma patients for the study,38 cases of controls (38 cases of healthy people). Quantitative PCR was observed by measuring the serum microRNA-301 expression levels in patients with control group and experimental group, the application of independent samples t-test analysis of experimental group and serum microRNA-301 expression level of patients in the control group differences, and ROC curves explore the diagnostic cutoff value, the expression level of microRNA-301 for diagnostic assessment of the effectiveness, then apply the univariate analysis to evluate the relationship between the expression level in clinical parameters of gender, age, tumor size and HBsAg and analysis the correlation microRNA-301 expression level in patients grouped by Barcelona HCC staging and the the serum relative expression levels of microRNA-301 in early, medium and advanced Barcelona stages patients.Portion 2mechanisms of microRNA-301 in hepatocellular carcinoma:52 cases of hepatocellular carcinoma patients, paraneoplastic relatively normal tissue for the study, evaluation and analysis microRNA-301 tissues in the case of the above; and a variety of hepatocellular carcinoma cell line based detecting expression levels of microRNA-301 in hepatoma cells, and filter out the obvious differences between the two cell lines for the study. By transfection of microRNA-301 detection of tumor cell proliferation, migration and invasion, and to detect the expression FoxF2, BBC3, AKT, PTEN, MMP13, E-cadherin, N-cadherin and other proteins.3. MethodsPart1Quantitative PCR was observed by measuring the expression levels of microRNA-301 in patients of experimental group and a control group, ROC curves and explore its diagnostic value and analyze patient sex, age, stage and HBsAg Barcelona correlation between the level of expression.Part 2Quantitative PCR assay 52 cases were observed in patients with hepatocellular carcinoma, paraneoplastic relatively normal tissue, evaluation and analysis microRNA-301 in the case of the above-mentioned tissues; quantitative PCR method for determination of microRNA-301 was observed in normal liver cells and liver LO2 cell line MHCC-97H, SMMC-7721, HepG2, Hep3B, Huh-7 expression. After verification microRNA-301 differentially expressed in cells and tissues, selected differentially expressed cell lines the most obvious test. Good selection of hepatocellular carcinoma cell lines were used microRNA-301 transfection, by quantitative PCR to verify transfection efficiency, while the cells transfected cell proliferation, migration and invasion experiments microRNA-301 overexpression and to express after detection expression FoxF2, BBC3, AKT, PTEN, MMP13, E-cadherin, N-cadherin and other proteins, initially identified the mechanism in the development of hepatocellular carcinoma.4.Statistical methodsAll data was analysis by statistical software Spss 20.0 computer processing. Normal distribution of measurement data is record as mean±standard deviation, and non-normal distribution is record as digits mean, setting P<0.05 was statistically significant.Results1.Experimental group (52 patients with HCC) and control group (n= 38) relative expression levels were measured in serum microRNA-301 by mean t test, with homogeneity of variance between the two groups can be further comparison, mean equation t test results suggest that P< 0.001, microRNA-301 relative expression level difference between the two groups was statistically significant, the results suggested that serum microRNA-301 relative expression level was higher in patients with hepatocellular carcinoma, P<0.001. The diagnosis of hepatocellular carcinoma serum microRNA-301 first expression level cutoff value 0.203850, sensitivity 86%, specificity 63.2%, area under the curve 0.77, P<0.001.2.52 patients in the experimental group and 38 in the control group did ROC curve for diagnosis predictive analysis, according to Youden index diagnostic cutoff value prediction, Youden index Youden index (J)= sensitivity+specificity-1, when Youden index maximum concentration values corresponding to the best cutoff value. In this experiment the best cutoff value Cutoff= 0.203850, Youden index= 0.865 (sensitivity)-0.368 (1-specificity) at, microRNA-301 relative expression amount of time that is greater than 0.203850 diagnosis of hepatocellular carcinoma, according to the diagnostic cutoff value, sensitivity 86.5%, specificity was 63.2%. Figurel-3 and Table 1-3 and the area under the ROC curve of 0.77 prompt> 0.5 and P<0.01, the diagnostic ROC curve results suggest the diagnosis of a statistically significant effect.3. These results suggest that microRNA-301 relative expression level and age, gender and tumor size was no significant correlation, P values were 0.415,0.399,0.056; and with the Barcelona stage, whether PVTT, AFP level and whether HbsAg positive correlation, P value were 0.004,0.035,0.023,0.001 respectively.4. Barcelona installments early, mid and late to microRNA-301 relative expression levels of single factor analysis of variance, early in 7 cases,25 cases of the mid-late 20 cases, suggesting that the packet by Tablel-5 tests found heterogeneity of variance, so the use Tamhane test, test prompted the early and middle microRNA-301 relative expression levels had no significant difference, P= 0.545; and early and middle and late, respectively, the relative expression levels have microRNA-301 difference, P values were 0.006 and<0.001.. microRNA-301 expression level of correlation analysis showed that both the Barcelona stage Pearson correlation coefficient 0.942, P<0.001.5. microRNA-301 expression level of Barcelona staging Pearson correlation coefficient analysis showed that both of 0.825, P<0.001.6.52 cases of hepatocellular carcinoma in patients with advanced carcinoma microRNA-301 relative expression levels higher than in patients with early.7. In this study, patients were divided into stages by Barcelona early, middle and late three groups, the variance of the three groups of patients microRNA-301 relative expression of single factor analysis, three groups of Tablel-5 Tips variance still missing, so the line Tamhane test, test results suggest that early, middle and late three groups were significantly different (P value less than 0.001), and the relative expression levels were microRNA-301 ladder-like changes, late> mid> early.8.52 cases of hepatocellular carcinoma tissues, adjacent tissues and normal liver tissue paired opposite each paired comparison showed there were significant differences between groups, P values were less than 0.001, statistically significant. The microRNA-301 relative expression levels is cancer tissue> adjacent tissues> relatively normal liver tissue.9. After transfection of these cells lines, microRNA-301 expression of hepatoma cell lines in each group, The expression levels as follows:SMMC-7721> Hep3B> MHCC-97H> HepG2> Huh-7> L02. The microRNA-301 expression level between SMMC-7721 and Huh-7 cell line is most obvious (Huh-7<SMMC-7721).10. microRNA-301 significant effect the proliferation, migration and invasion of hepatoma cell line SMMC-7721 and Huh-711. After overexpression and noexpresssion of microRNA-301, expression levels of FoxF2, MMP13, E-cadherin and N-cadherin proteins and other changes in SMMC-7721 cell lines.Conclusions1. microRNA-301 upregulated in serum of patients with HCC, cancer tissue and cell lines for oncogene microRNA.2. The relative expression levels of serum microRNA-301 has a certain diagnostic value of hepatocellular carcinoma.3.The expression levels of serum microRNA-301 in patients correlate to hepatocellular carcinoma Barcelona stage, portal vein thrombosis, AFP and HBsAg.4. By changing the expression level of microRNA-301 can significantly affect the impact of hepatoma cell line proliferation, migration and invasion.5. microRNA-301 expression level by changing the expression levels of FoxF2, MMP13, E-cadherin and N-cadherin and other proteins involved in the development and progression of HCC. |
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