The Screening Of Metastatic-related Genes In MiR-199a Regulated Melanoma Cells

Objective:By screening the tumor metastatic-related genes in miR-199a-regulated B16F10 melanoma cells with tumor metastatic PCR array, we mean to find more about the metastatic mechanism of melanoma and provide theoretic foundation for future research.Methods:To Construct miR-199a over-expressed plasmid and inhibitor, then transfer them to highly metastatic melanoma cells (B16F10) by gene transfer technology. As a result, miR-199a is highly or lowly expressed. After extracting RNA from the melanoma cells, we analyze the RNA samples with ultraviolet spectrophotometer and denatured agarose gel electrophoresis. Then we sent the RNA samples for tumor metastatic PCR array for screening the differently expressed metastatic genes (accomplished by Kangcheng biological industrial corporation in Shanghai). Then we pick out 2 or 3 differently expressed genes to do the confirmatory experiment through RT-PCR and agarose gel electrophoresis.Results:1. The RNA data shows the A260/A280 ratio is among 1.8 and 2.1,which means the RNA purity is qualified for the following experiment. The electrophoresis diaphragm also confirmed the purity of the RNA sample.2.Analyze the RNA sample with tumor metastatic PCR array: compare the inhibitor group to the overexpressed group,12 genes are up-regulated:Cd44,Cdh1,Cxcr4,Etv4,Fxyd5,Rpsa,Mmp3,Myc,Rb1,Tcf20,Hprt1,Actb1,3 genes are down-regulated:Ctsk,Itga7,Tnfsf10;Compare the inhibitor group to the blank group,14 genes are up-regulated:Cxc4,Ctbp1,Rpsa,Myc11,Mmp3,Mmp13,Kiss1r,Hgf,Htatip2,â…¡18,â…¡8rb,Cd82,Kiss1,Timp4, no genes are down-regulated significantly; compare the overexpressed group to the blank group,17genes up regulated:Cxcr4,Ctsk,Csf1,Ela2,Mmp3, Mmp13,P2ry5,Kiss1r,Hgf,Hras1,Htatip2,â…¡18,â…¡8rb,Kiss1,Timp4,Itga7,Tnfsf10,7 genes down regulated:Cdh1,Etv4,Fxyd5,Myc,Nme2,Rb1,Hprt1.3. Confirm the related gene expression with RT-PCR and agarose gel electrophoresis:Choose Myc and Tnfsf10 to do the confirmatory experiment among the differently expressed genes according to the PCR array data. The electrophoresis diaphragm is supportive of the previous experiment.Conclusions:Among the miR-199a-regulated melanoma genes, the potential genes related to up-regulation of melanoma metastasis are: Cd44,Cdh1,Ctbp1,Etv4,Fxyd5,Rpsa,Myc11,Myc,Nme2,Cd82,Rb1,Tcf20,Hprt1,Actb1; the potential genes related to down-regulation of melanoma metastasis are:Ctsk,Csf1,Ela2,P2ry5,Hras1,Ctsk,Itga7,Tnfsf10. According to our RT-PCR results, Myc is negatively regulated by miR-199a; while TnfsflO is positively regulated by miR-199a.