| BackgroundIn the genome all the transcripts can be divided into two categories: coding and non-protein coding RNA. For many years non-protein coding RNAs were thought to be as junk DNA and non-functional, but it is now believed that these non-coding sequences also play an important rule in regulation of genes expressions. Non-protein coding transcripts can be divided into two categories: household RNA and regulation of RNA, housekeeping transcript constitutively expressed in the RNA species are essential for normal cell function. The miRNA discussed in this article belong to the regulatory RNA, miRNAs regulate the expression of specific target gene at the post-transcriptional level, which involved a series of important life processes in the multiple organisms including cancer.Many experimental studies and clinical investigations have found that miRNAs acts as oncogene and/or anti-oncogene, thereby promoting or restraining the development of cancers. The initial reports of this effects were come from the study of the chronic lymphocytic leukemia (CLL). Accumulating evidence provided by worldwide scientis suggests miRNA expression is disregulated in many kinds of cancer, which involved with cancer initiation, development, and varied phenotypies. At present, however, few previous reports on the the miRNAs expressing in gastric have been published in the literature. Using Illumina miRNAs microarray platform, our previous work had shown that 6 miRNAs in 7 gastric adenocarcinoma tissues were up-regulated as compared to non-cancerous tissues. The tissues from gastric adenocarcinoma confirmed histologically and 7 normal gastric tissues which were away from the tumor′s margine over 5cm and without cancer cell histologically were collected duing surgical intervention for patients. The miRNAs expression profiling was performend on Illumina microarray platform for miRNAs, which include following steps: purified total RNA from the tissues, RNA polyadenylation, cDNA synthesis, miRNA-specific primer extension, PCR with fluoresenctly labeled universal primers, hybridization, array readout, and normalization of data.Purpose:Our previous study has observed the differences of miRNA expression profile in gastric carcinoma, this study was to validate the results from microarray in expanding the sample size using quantitative RT-PCR(qRT-PCR) to measure miRNA expression profiling of gastric cancer. The target gene and their associated KEGG pathway were predicted with bioinformatics tools in order to further study the relationship between the miRNA target genes and gastric cancer development and prognosis.Method:1,Pair-wise tissues pathologically confirmed from 50 cases of gastric adenocarcinoma and corresponding to their non-cancer gastric tissue which were away from the tumors over 5cm were collected during operations. Other 25 patients with chronic superficial gastritis were endoscopically biopsed as normal control. According to the nucleotide sequences deposited in miRNA database, primers were designed for 4 miRNAs. Quantitative real-time PCR analysis were performed using locked nucleic acids(LNAs) linear primers and SYBR GreenⅠto detect the expressions of miR-181a,miR-27a,miR-584 and miR-93 amony cancer tissues, non-cancer gastric tissue and normal gastric tissues.2,In order to investigate the relationship between the up-reglated miRNAs and the clinicopathologic factors in patients with gastric cancer, the correlation analyses were performed in variables including miR-181a and miR-27a expression levels, patient age, gender, pathological type, and lymph node metastasis.3,TargetscanS and miRanda computational approaches were used to predicts miR-181a and miR-27a target genes. The biological processes associated with predict targets were evaluated using KEGG pathway online resource.Result:1,When compared to adjacent non-cancerous tissues, the over expressions of miR-181a, miR-27a, miR-584 and miR-93 were seen in 44(88%),33(66%), 29(58%) and 29 patients(58%), respectively by fluorescence qRT-PCR . Wilcoxon signed rank paired sample test showed that, relative to adjacent non-cancerous tissues, miR-181a and miR-27a in gastric carcinoma tissues were statistically significant higher xpression (p <0.05). At the same time using two independent samples of Mann- Whitney test showed, compared with 25 chronic superficial gastritis, miR-181a and miR-27a in gastric cancer tissues were statistically significant higher expression (p = 0.05) .2,Both miR-181a and miR-27a expression levels did not statistically found associated with patient age, gender, pathological type, and lymph node metastasis.3,Predicted miR-181a and miR-27a corresponding to the target gene were 61 and 45, miR-181a and miR-27a target genes were involved in 11 and 15 KEGG pathways, including the human metabolic pathways, metabolism, genetic information processing Environmental information processing, cellular processes and organic systems such as molecular interactions and reaction networks.Conclusion:1,Over-expression of miR-181a and miR-27a in gastric carcinoma tissues are verified using quantitative real-time PCR method.2,Both miR-181a and miR-27a expression levels do not correlated with the patient age, gender, pathological type, and lymph node metastasis.3,Predicted miR-181a and miR-27a of the miRNAs target genes and KEGG pathways. |
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