| Objective: EMS1 gene is an oncogene, first cloned and known from breast cancer, head and neck squamous cell carcinoma. Our forward research found EMS1 is a gene related to gastric cancer, highly expressed in gastric cancer, and closely related to the metastasis. Studies have shown that EMS1 gene can be regulated by mi RNAs in tumorigenesis and development. Here we screen and identify the targeted mi RNAs of EMS1 gene by biological information science technology and experiment in vitro on post-transcription, and elucidate the molecular mechanism of the EMS1 gene come high-expression in gastric cancer. It will be helped to provide a theoretical basis for the treatment and prognosis of gastric cancer.Methods:1.Using bio-informatical software to predict the probable targeted mi RNAs of EMS1 gene, and taking literature retrieval in Pubmed, CNKI website to screen out the higher likelihood of the targeted mi RNAs which was not reported.2.Using the cationic liposome method transfected the screened mi RNA mimic/inhibitor instantaneous into MGC803 cells. Using Western blot technique to detect the expressed conditions of EMS1 gene of the blank group, the mi RNA mimic/inhibitor negative control group and mi RNA mimic/inhibitor experimental group after been transfected 48 h later.3.The effects of transient transfection of mimic/inhibitor mi RNA o n the proliferation, migration and invasion of MGC803 cells were detecte d by MTT assay, scratch test and in vitro migration and invasion assay.Result:1) Bioinformatics results showed that: eleven mi RNAs including mi R-501-3p, mi R-182, mi R-338-5p, mi R-183, mi R-502-3p, mi R-545, mi R-329, mi R-603, mi R-326, mi R-526 a,, mi R-548d-3p could be the targeted mi RNAs of EMS1.The literature searching results show that there are fi ve mi RNAs up-regulated in tumor tissues, such as mi R-182, mi R-501-3p, mi R-338-5p mi R-183 and mi R-502-3p. In addition there are five m i RNAs down regulated expression in tumor tissues, they are mi R-182, mi R-545, mi R-603, mi R-329, mi R-326. However the effects of mi R-526 a and mi R-548d-3p to tumor cells is still not reported. Among th e five down-regulated mi RNAs, mi R-326 and mi R-182-5p are targeted m i RNAs of EMS1 gene which have already been reported. The correlatio n between the other three mi RNAs and EMS1 gene correlation is not yet studied. The literature searching results found that mi R-545-3p, mi R-329-3p, and mi R-603 may be the targeted mi RNAs of EMS1 gene.Targeted binding site predicted results showed that mi R-545-3p's binding site of EMS1 3'UTR is 162-168, mi R-329-3p's binding sites of EMS1 3'UTR is 863-869 and 1184-1190, mi R-603's binding site of EMS1 3'UTR is 1042-1048.2) Western blot indicates that these three mi RNAs mimic transfect ed groups, as mi R-545-3p, mi R-329-3p and mi R-603 groups, compare d with the blank group and NC group, the EMS1 protein expression in mi R-545-3p group is decreased, p < 0.05, the difference has statisticall y significant. mi R-329-3p and mi R-603 group, compared with blank grou p and NC group, The level of EMS1 protein expression has no significa nt difference(p > 0.05). After respectively transient transfecting mi R-545-3p, mi R-329-3p and mi R-603 inhibitor to MGC803 Cells,these three tra nsfected groups compared with the blank group and NC group, the expre ssion of EMS1 protein in mi R-545-3p group is increased, p < 0.05, th e difference has statistically significant. mi R-329-3p and mi R-603 grou p, compared with blank group and NC group, the expression of EMS1 protein has no significant difference(p > 0.05).3) Results of MTT experiment: compared with the blank control gro up and NC group, MGC803 Cells of gastric cancer which were transien tly transfected with mi R-545-3p mimic, decreased the cell proliferation ability(p< 0.05). However when the cells transfected with mi R-545-3p inhibitor, their proliferation ability would be enhanced(p < 0.05). Th e experimental results show mi R-545-3p up-graduated expression can inh ibit the proliferation of human MGC803 cells of gastric cancer. Its inhi bition can enhance the MGC803 proliferation ability.4) Migration and invasion experiment: the migration assay result s showed MGC803 cells which were transiently transfected mi R-545-3p mimic, compared with blank group and mimic NC group cell, migratio n and invasion ability was decreased(p< 0.05). MGC803 cells transientl y transfected mi R-545-3p inhibitor, compared to blank group and inhibito r NC group, cell migration and invasion capability was improved(p< 0.05).Conclusion:Mi R-545-3p is a candidate mi RNA on targeting regulating EM S1 gene, a gastric cancer related gene, which can inhibit the prolife ration, migration and invasion of MGC803 cells probably by inhibit ing the expression of EMS1. |
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