Aberrant Expression Of MiRNAs And Biological Function Study Of MiR-574-3p In Gastirc Cancer

Gsatric Cancer (GC) remains a major challenge to human health, especially indeveloping countries. Nearly one million new cases are diagnosed worldwide each year.Early detection and therapy of gastric cancer could improve patients suvival and life quality,while traditional cancer detections are always lack of efficiency in diagnosis of patients inearly stage. Rencently, researches focuses on the gene-based detection and therapy of cancer.As newly gene-based detection and treatment approaches, mcroRNAs (miRNAs) in cancerespecially in early stage cancer have been investigated widely.The discovery of miRNA provides a novel tool for studying the tumorigenesis,diagnosis and treatments of cancers. MiRNAs approximately19-24nucleotides in length, areevolutionarily conserved small single-stranded non-coding RNA molecules. They workmainly at post-transcriptional level by binding to the sequences in the3' untranslated regions(3'UTR) of their targeted mRNAs resulting in translational repression and gene silencing.Aberrant expressions of miRNAs between cancer and normal tissues have been studied inseveral types of human cancers. They may play roles in the development and progression ofcancers similar to those played by oncogenes or tumor suppressor genes. Gastric cancer (GC)is the second leading cause of death in cancer worldwide. For the diagnosis and treatment ofGC, it's necessary to explore highly sensitive and specific tumor markers and noveltherapeutic targets. Studies on the aberrantly expressed microRNAs in gastric cancerhighlighted the viewpoint that some microRNAs can potentially be considered as tumorbiomarker in GC patients.In order to gain more information on this issue, we employed AFFX miRNA expressionchips to study the aberrant expressions of miRNAs and quantitative real-time PCR toidentify and confirm miRNAs with aberrant expressions in gastric cancer. We also searchedthe potential targets for miR-574-3p through bioinformatics analysis combined with ourprevious screening results of gene expression profiles, and validated them by transfectingmiR-574-3p mimics into SGC7901cell (a gastric cell line). In addition, we study the effectof miR-574-3p transfection on cell viability, migration, invasion activity in SCG7901cell.1. Aberrant expression of miRNAs in gastric cancer tissuesTo obtain the aberrant expressions of miRNAs between gastric cancer and normal tissues, we employed AFFX miRNA expression chips to study the differences of miRNAexpression profiles in13pairs of gastric cancer tissues and matched normal tissues adjacentto the tumor.16miRNAs were found to be aberrantly expressed, of which14weredown-regulated and2up-regulated in gastric cancer tissues comparing with their adjacentnormal tissues. Among these miRNAs, up-regulation of miR-455-3p and down-regulation ofmiR-574-3p, miR-1207-5p, miR-486-5p and miR-768-5p in gastric cancer have not beenreported to the best of our knowledge.To confirm the accuracy of our chip results, based on the maximum expressiondifference, we chose miR-31, miR-574-3p, miR-455-3p and miR-768-5p for furtherexperimental validation using quantitative real-time PCR. The results of the real-time PCRshowed that miR-31, miR-574-3p and miR-768-5p expressed at significantly lower levels ingastric cancer tissues compared to their matching references(P<0.01), and miR-455-3pexpressed at a higher level in gastric cancer tissues (P<0.01), which were consistent with themicroarray data.Interestingly, we noted that the expression level of miR-574-3p was significantlyreduced in cancer tissues with higher levels of differentiation (P <0.05); and a similarobservation is made on early stages of the cancer (P <0.05). In addition, the expression levelof miR-574-3p in gastric cancer was not affected by the gender and the age of the patients.Since the aberrant expression of miR-574-3p has not been reported before, to gainfurther insights about it, we studied mRNA targets and functions of miR-574-3p in furtherexperiments.2. Prediction and preliminary validation of the potential targets of miR-574-3pMiRNAs modulate gene expression by interacting with their target mRNAs resulting inmRNA degradation and/or translational repression. To find out the potential targets ofmiR-574-3p, we performed bioinformatics prediction analysis, based on our previouspublished data. The prediction of the target genes for miR-574-3p was performed usingTargetScan and miRBD. Among the31predicted targets of miR-574-3p, RXRA and CUL2showed high potentials since both RXRA and CUL2were predicted by both programs. Inaddition, we also noted from our160sets of gastric cancer tissue mRNA microarrays thatonly RXRA and CUL2out of the807up-regulated mRNAs were predicted to be the targetsof miR-574-3p. We validated the results of the prediction and the microarray assays byperforming real-time PCR. The results showed that expression levels of RXRA and CUL2 were indeed increased in gastric cancer tissues.To provide the direct evidence to validate the RXRA and CUL2gene are the targets ofmiR-574-3p, we transfected miR-574-3p mimics into SGC7901cell. Then, we detected theexpression of RXRA and CUL2mRNA expression in24h and48h later by real-time PCR,respectively. The PCR results showed that CUL2mRNA in miR-574-3p-transfectedexpressed at a lower level compared with Mock. While we didn't found the change of RXRAmRNA level after the miR-574-3p mimics transfection. The results indicated that only CUL2gene could be the target of miR-574-3p.3. Effect of miR-574-3p transfection on cell viability, migration and invasionactivityTo study the biological role of miR-574-3p in gastric cancer, we performedgain-of-function studies using miR-574-3p-transfected SGC7901cell lines. The CCK8assaydemonstrated that cell proliferation was reduced to74.5%of Mock inmiR-574-3p-transfected. We further analysed the effects of miR-574-3p on the migratoryand invasive behaviour of gastric cell lines. The migration assays showed that the SGC7901cells migration activity of miR-574-3p-transfected was specifically reduced byapproximately37.9%. Moreover, the invasion assay also indicated that miR-574-3p mimicsreduced the invasion of SGC7901cells by63.7%. These findings suggest that miR-574-3plevel seems to be closely associated with the invasion and migration of gastric cancer celllines.In summary, miR-574-3p was down-regulated in gastric cancer tissues were identifiedby microarray and real-time PCR. We found that CUL2could be the target of miR-574-3p ingastric cancer cell. Further, we also found that decreased cell proliferation, migration, andinvasive activities in miR-574-3p transfectants, suggesting that miR-574-3p is a candidatetumor suppressive miRNA in human gastric cancer.