| Background:The death rate of pancreatic cancer is very high. Currently, surgery is the best choice for the treatment of pancreatic cancer, but when diagnosed, resectable only 80% of cases, most have been diagnosed with advanced, non-surgical opportunities, and the five-year survival rate is less than 5%. Therefore, an urgent need to investigate its molecular pathogenesis. Gene mutation, transcription and translation of non-normal, abnormal methylation can lead to aberrant miRNA expression in tumors, most of the miRNA can be used as tumor suppressor gene or oncogene.It plays a role in inhibiting cancer.It may regulate the cell cycle process, cell differrantiation or cell death.And it may affect the tumor cell proliferation.Objective:1.We investigated the hypermethylation of miRNA in the pancreatic cancer promoter. We found the hypermethylation miRNA expression in pancreatic cancer cells. We found the miRNA expression with the influence of DNA methylation.We detected the expression of miR-615-5p in 25 pairs of pancreatic carcinoma and adjacent tissues.2.With miR-615-5p’s In situ hybridization of tissue microarray, we investigated the miR-615-5p expression in pancreatic cancer and we studied the relationship between the clinical pathology, the prognosis and the expression of the miRNA.3.We studied the downstream target genes of miR-615-5p and signaling pathways.And we explored the related functions of miR-615-5p in pancreatic carcinoma cells.Methods:1.We try to find the miRNA with the methylated peak differences with the application of DNA methylation chip and MeDIP in pancreatic cancer cells and normal pancreatic tissue.We studied the different expression of miR-615-5p in normal pancreatic tissue and pancreatic carcinoma cell lines using quantitative PCR.With the application of demethylation drug treatment for pancreatic cancer cell line, we investigated the miR-615-5p differences before and after drug using with quantitative PCR. To detect the miR-615-5p expression in 25 pairs of pancreatic cancer and adjacent tissues using quantitative PCR.2.102 pairs of paraffin were used to create the tissue microarray with tissue microarray instrument (1 pair including one case of pancreatic tissue and the corresponding one cases of adjacent tissues).We detected the expression of miR-615-5p in 102 pairs of adjacent pancreatic tissue and cancer tissue with in situ hybridization.According to the results of miR-615-5p in situ hybridization in tissue microarray, we described the relationship between miR-615-5p expression and clinical data of the patients.The further analysis of the ralationship between miR-615-5p expression in pancreatic tissue and its prognosis was done.Comparative analysis of miR-615-5p in patients with pancreatic cancer and survival time was done with Kaplan-meier method.Correlation analysis of survival time factor was done using Cox regression.3.We created a stable over-expression of miR-615-5p pancreatic cancer cell lines.And we respectively investigated the vitality, invasion, movement, cell cycle and apoptosis of the pancreatic cancer cells with the over-expression of miR-615-5p using EdU, MTT, Transwell invasion assay, wound healing assay, flow cytometry in vitro.We analysed the mRNA expression microarray sequencing results of pancreatic cancer cell line with overexpressed miR-615-5p.We compared the low expressing miR target genes and miR:615-5p regulatory genes of gene analysis software system’predicting (TargetScan, etc.).And we obtained the intersection of genes and downstream target genes.And we analysed the gene regulation of miR-615-5p relevant networks and signaling pathways.Results:l.With DNA methylation chip and methylated DNA immunoprecipitation Tip, in normal pancreatic tissue and PANC-1 cell line,we found a total of eight miRNA with methylated peak:miRNA-615, miRNA-663b, miRNA-574, miRNA-1826, miRNA-1247, miRNA-675, miRNA-483, miRNA-663. Using BSP+TA cloning, we found the results that the methylation rate of miRNA-615 in PANC-1 was remarkblely higher than in the normal pancreatic tissue. With quantitative PCR,we detected the miR-615-5p expression in the pancreatic carcinoma cell lines was noteblely higher than in the normal pancreatic tissue. Comparing with the demethylation drug-treated pancreatic cancer cell lines,the expression of miR-615-5p raised in the normal pancreatic carcinoma cell lines.Using normal pancreatic tissue as a reference, in comparison with the adjacent normal tissue,the expression of miR-615-5p in 21 pairs of human pancreatic tumor was dramatically lower.2. According to the aim of our design,we made two pieces of tissue microarray, with dot matrix arranged in neat rows, with no shift phenomenon, where three sites are missing, no lack of meaningful organization on site.We obtained a meaningful 102 pairs of samples (102 cases of pancreatic cancer and 102 cases of corresponding adjacent tissues, tumor pathology by AJCC 7th grade, I grade 10 cases, ii grade 80 cases, III grade 12 cases,).The observed rate of morphological tissue site was greater than 80%. There was no slice off,ectopic wrinkle phenomenon in HE staining.And in situ hybridization staining position was clear. Background was clean too.In situ hybridization compared with pancreatic carcinoma, miR-615-5p was significantly higher in the adjacent tissue of the tumor (P<0.05).There was no noteble association between miR-615-5p expression and the patient age, gender, lymph node metastasis and nerve violations (P<0.05). There is a noteble association between the expression level of miR-615-5p and tumor differentiation, tumor size, grade, presence or absence of vascular invasion. The survival time significantly increased in miR-615-5p staining positive patients.With the miR-615-5p substitution model made by clinical parameters showed that miR-615-5p is one of the relatively independent factors associated with the survival time of patients with pancreatic cancer.3.EdU staining results were that pancreatic cancer cell’s proliferation decreased with the miR-615-5p. MTT results suggested that miR-615-5p inhibit the activity of SW1990 cells.Transwell experiments suggested that miR-615-5p reduced the invasiveness of SW1990 cells.Draw marks experiments suggest that miR-615-5p reduced the migration of SW1990 cells.MiR-615-5p microarray sequencing results showed that there were 319 down-regulated genes in miR-615-5p stable transfected cell lines SW1990 compared with GFP control group.The comprehensive comparison of the down-regulated genes in miR-615-5p stable transfected cell line SW1990 compared with control group and the miRNA target prediction system’ results,we obtained the intersection of 14 genes.Bioinformatics software analysis pointed out that 14 differentially expressed genes related signaling pathways including PI3K signaling pathway, RAP1 signaling pathway,Cancer pathways, neuroactive ligand-receptor interaction and the gap connections.The gap junctions are significantly enriched signaling pathways.Conclusion:1.The level of miR-615-5p in pancreatic carcinoma cell lines was strikingly lower than in the normal pancreatic tissue.DNA hypermethylation may be an important mechanism of the low expression of miR-615-5p.2.The application of miR-615-5p in situ hybridization on tissue microarrays and semi-quantitative can help us find the expression of miRNA in tissuessimultaneously qualitative. Expression of miR-615-5p in situ hybridization has high accuracy, specificity and positive predictive value in finding the differences between pancreatic cancer and normal pancreatic tissue.The miR-615-5p is important in the development of the clinical prediction in pancreatic cancer.3.The miR-615-5p can inhibit the cell proliferation, viability, invasion and movement of the pancreatic cacinoma cells.Using High-throughput mRNA expression microarray based sequencing, we established a diversified networks of genes related to the regulation of miR-615-5p in pancreatic cancer screening with bioinformatics methods.It provided convenience for the later study of extensive mechanism in pancreatic cancer. |
