Different MicroRNAs Expression Profile Of Gastric Cancer With Potential Metastasis And Verified The Function Of Target Gene

【Background】Malignant tumor is the main disease to threaten human’s health and liferecently, which is a intractable problem all over the world, everyone is afraid ofthe cancer when talking about it. The prevalence of gastric cancer takes the secondplace among all kinds of malignant tumor, which is only next to Lung Cancer.There are nearly600thousands people dieing from gastric cancer every year, themain reason is that the early diagnosis of gastric cancer is very difficulty but thetreatment rates is low. About2/3patients have been found with long-distancemetastasis before identify their gastric cancer or peritoneal metastasis, whichcould lead to losing the opportunity of operation and declining life quality ofpatients.The5-years’ survival rate of the gastric cancer patients with metastasis inthe middle stages or latter stages are less than15%.The process of gastric cancer peritoneal metastasis comprises a series ofindependent events taking place early or late, and is the outcome of differentaction of multi-molecule in different ways after multiple steps. With the progressand development of science and technology, the important effect of the regulationof gastric cancer peritoneal metastasis genes in the process of their occurrence anddevelopment has been paid attention by researchers. The high-speed developmentof gene technology bring hope to identify the molecule principle of all kinds oftumor. However, the study of single molecule can not identify the passageway ofgastric cancer peritoneal metastasis and find out the way among different molecules. It may help to find out the principle of gastric cancer peritonealmetastasis even all other malignant tumor to discover the genes which havedifferent expression in the process of gastric cancer peritoneal metastasis and totest and verify their function in the process of gastric cancer peritoneal metastasis,further research reciprocal function among massive molecules. High throughputselection brings up new opportunity to understand the mechanism of peritonealmetastasis, at the present time, high throughput selection to research tumormetastasis fundamentally divide into genes different expression and the selectionof depend phenotype by tactics, the most typical selection of genes difference isgene chips technology, which has found many different expression genes inprimary foci and metastases of tumor, but how these genes adjust the process ofmetastasis need further studing. In the recent years, microRNA is very close to theregulation mechanism of happen, development, metastasis and invasion of tumor.At present time, it is very important to put high throughput selection of tumormetastasis into the study of peritoneal metastasis.【Objects】1. Select out the miRNAs with different expression in gastric cancer cellsGC9811and gastric cancer peritoneal high metastasis cells GC9811-p,and testdifference multiple, select out the genes that may relate to peritonealmetastasis as objective molecules to study;2. Do functional experiments of objective molecule miRNA-21-5p andmiRNA-146-5p, explore their function in the process of gastric cancer andgastric cancer peritoneal metastasis.【Methods】1. cells culture, total RNA extracting: selected the cells GC9811and GC9811-pcells coming from at the same time and fostered at the same condition, then picked up total RNA after48hour logarithmic growth, send it to companywith dry ice freezed to select out the miRNA with different expression in cellGC9811and GC9811-p miRNA array when the total RNA’s OD is between1.8and2.0; analysised the miRNA by SPSS and drawn out its different expressionatlas；2. test and verified the different expression of foal gene in cell9811and9811-pby RT-PCR, count relative value by2-△△CT, selected U6as internal referencecontrast;3. virus packaged and transfection: take virus packaged for negative contrastvirus and objective gene down-regμLated virus, selected the parental cells lineGC9811to do objective gene expression virus transfection obstructed;4. tested and verified the expression of negative contrast and objective moleculesmiRNA-21-5p and miRNA-146a-5p after transfection by down-regulatedvirus by means of RT-PCR and immunofluorescence；5. migration experiment and invasion assay proved the transform ability ofobjective gene;6. tested and verified the target genes affected by objective gene pass throughbioinformatics.【Results】1. Through the selection of miRNA Microarray Service, statistics testing andcluster analysis: there are155miRNA with the difference between GC9811and GC9811-p, among GC9811-p,79miRNA are down-regulated,76areup-regμLated and8miRNA’s signal strength is more than500and8’s Pvalue is less than0.01,42’s is less than0.05,19’s is less than0.10.2. Further we tested and verified the8molecules whose signal strength are morethan500and P is less than0.01by the experimental way of RT-PCR, whose different expression are the same with the selection outcome of miRNAMicroarray Service.3. Transfect negative contrast virus, miRNA-21-5p down-regulated virus andmiRNA-146a-5P down-regulated virus into cell9811, respectively marked toGC9811-NC, GC9811-21-IH and GC9811-146a-IN, test the three virus aresuccessfully transfected by immunofluorescence.4. Experiment RT-PCR verified the expression of GC9811-21-IH andGC9811-NC actually declined than their miRNA-21-5p and GC9811-146a-IHand GC9811-NC were down-regulated than their miRNA-146a-5p;5. The experiment of invasion and transfer in vitro verified the transform numberof GC9811-21-IH and GC9811-146a-IH was more than GC9811-NCtransfected negative contrast virus after the aim molecules miRNA-21-5p andmiRNA-146a-5p down-regulated expression gene and negative contrast genetransfection gastric cancer cell9811【Conclusion】1. Select out153miRNA related to gastric cancer peritoneal metastasis, amongwhich, there are79miRNA are down-regulated and74are up-regulated in cellGC9811-P.2. miRNA-21-5p and miRNA-146a-5p are inhibit gastric cancer cell GC9811invasion and migration by different target genes, different signalingpathways.