| Background and purpose:In our country, gallbladder stone is an extremely common significant digestive disease that affects some 10-15% of the global population. Gallbladder stone is also the main cause of biliary pancreatitis, and closely related to iatrogenic lesions of the biliary tract. It also is the factor of gallbladder carcinoma development. The molecular mechanisms involved in gallstone formation are far from clear. Currently, it is believed as the combined effect of genetics and environment. In recent years, the studies to explore the occurrence of gallstones mechanisms, which eventually formed three consistent basic consensus, has become a classic of gallstone disease hypothesis:a, Supersaturated cholesterol in bile, which is necessary for gallstone formation; b, ubalance of pro- and anti- nucleus components, results in the formation of cholesterol single crystal water; c, aberration of dynamics in bliliary tract, which involed in promoting the occurrence of the gallstone. microRNA (miRNA) has been shown to regulate many diseases, but no reports in the gallstone. Therefore, we analyze the miRNA and mRNA expression profile of gallbladder stone and gallbladder polyp by using a high-throughput sequencing technology, and explored the roles of miRNA and mRNA in the development of gallstone and their molecular mechanism in the occurrence and development of gallbladder stone.Method:1. The total RNA from the gallbladder epithelium cells of gallbladder stone and gallbladder polyp were detected by the high-throughput deep sequencing to achieve the mRNA gene expression profile. Then the differentially expressed genes with more than2-fold changing were screened out and annotated by the gene ontology and KEGG signalingpathway with the tools and databases integrated by the website DAVID. By the aboveanalysis, the main functions and signaling pathways enriched with differentially expressedgenes were found out.2. the same total RNA was detected by the method of Solexa miRNA High-throughputsequencing to achieve the mRNA gene expression profile. The differentially expressedmiRNA with more than 2-fold changing were screened out and used to achieve the targetgenes based on the prediction database of website TARGETSCAN. Then the target geneswere annotated by the gene ontology and KEGG signaling pathway with the tools anddatabases integrated by the website DAVID. The analysis results of mRNA and target geneswere compared to find the clues for the follow-up research.3. Gene regulatory network with differentially expressed miRNA and mRNA was contructed using the methods of bioinformatics (difference expression of miRNA are defined as:a Fold-change> 1 or a Fold-change value≤1, and p-value<0.01; difference expression of mRNA is defined as:FDR≤0.001 with 2 times or more flod change).4. The rest were used for qRT-PCR confirmation and fulfillmiRNAfunctional study.5. Differential expressed miRNA and mRNA were screened out and validated through the method of dual luciferase report gene, and the mechanism was further comfirmed in vitro cell model to by the western blot experiments.Results: 1. Comparative analysis of mRNA between gallbladder stone and gallbladder polypIn the study of mRNA gene expression profiles, we found 525 differentially expressedgenes with more than 2-fold changing and 334 genes within them researched before. Thegene ontology analysis suggested that these genes enriched in the process of developmentalprocess, biological regulation, cellular process and biological adhesion. The KEGG pathwayanalysis suggested ion transfort and Insulin-related pathway pathway were significantlyregulated (P<0.05).2. Comparative analysis of miRNA between gallbladder stone and gallbladder polyp In the study of miRNA gene expression profiles, we found 17 differentially expressedgenes with more than 2-fold changing. Based on the database of websiteTARGETSCAN, we achieved 1962 target genes. In the following GO analysis, we found these genes enriched in the biological processes:developmental process, biological regulation, cellular process,cellular component organization, growth and et al. In the KEGG signaling pathway analysis,we found that 29 pathway were regulated. Excepting the pathway relating to tumor, therewere 16 signaling pathway left, most of which were proved to relating toion transport. In the 16 pathways, the ion transport signaling pathway was significantly regulated by the miRNA.By Miranda v3.3 software, significant difference expression of target genes of 17 miRNAs were predicted. Although almost each miRNA targetting to a multitude of mRNA, we finally choose the predicted target energy minimum 10 gene constructs the miRNA and its target gene regulation networks.Further analysis found there were only one differential expression of miRNA and its predicted target genes also has a significant difference (miR-210 and ATP 11a).3. RT-qPCRThe results of Real-time PCR experiments showed that the expression of ATP11A, TRDN,IFI27, MYL3, RPS4Y1, USP9Y, AMH, SLC28A and miR-192, miR-133a, miR-210, miR-200c, miR-194, miR-891a was consistent with the results of high-throught sequencing, which suggested the data is credible to further study.4. The roles of miRNA and target genes in HGBECThe relationship between miR-210 and its target gene ATP11A was verified by Dual luciferase reporter experiments. With treatment with miR-210 mimics, ATP11a expression in HGBEC decreased obviously. In the gallstone sample, the expression of miR-210 was negatively associated with the expression of ATP11A.Conelusion:We found 17 differentially expressed micRNA and 525 differentially expressed mRNA in the 10 samples with gallbladder stone, which enriched in cell adhesion, growth, apoptosis, etc., and related to the ion transport, insulin pathway, immune system. The integrating analysis showed differentially expressed 17 miRNAand 114 mRNA. Eventually miR-210 and its target genes ATP11a were identified as significant difference expression. |
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