| Objective 1. To investigate the expression of P115, MIF and their roles in the tumorigenesis and progression of gastric carcinoma. 2.To access the effect of P115 on the growth of gastric carcinoma cell lines BGC-823 , MKN-28. 3. To explore the possible underlying moleculer mechanism involved in the proliferation of gastric carcinoma cell influenced by P115.Methods 1. mRNA and protein experessions of P115 and MIF in human normal gastric mucosa, gastric carcinoma tissues, and in three cell lines(normal gastric epithelial cell line GES-1, differentiation of gastric carcinoma cell lines MKN-28, BGC-823)were detected by RT-PCR, SP method and Western-blot . 2. To construct the specific small hairpin RNA (shRNA) expressing vectors against the P115 and the eukaryotic Full-length expression plasmid of p115(pEGFP-N1-P115), the recombinants were transfected into gastric carcinoma cell lines BGC-823 and MKN-28, respectively; then transfection efficiency were observed by Fluorescence microscopy and flow cytometry. Real-time-PCR and Western-blot were performed to detect the expression levels of P115 mRNA and protein. 3. MTT assay , flow cytometry were also used to detect the proliferation effect of P115-shRNA on BGC-823 ; the expression levels of cyclinD1, mcm2, PCNA mRNA and protein before and after tansfection were examined with Real-time-PCR and Western-blot respectively. 4. MTT assay, flow cytometry were also used to detect the proliferation effect of pEGFP-N1-P115 on MKN-28;the expression levels of cyclinD1, mcm2, PCNA mRNA and protein before and after tansfection were examined with Real-time-PCR and Western-blot respectively. 5.The interaction between P115 and MIF in BGC-823 and MKN-28 cells was detected by co-immunoprecipitation; Real-time-PCR, Western-blot, ELISA were performed to detect the expression levels of MIF mRNA and protein, and the secretion level of MIF;the expression level of pERK1/2 were detected by Western-blot.Results1. P115 is over-expressed in gastric cancer tissues and cell lines. The positive rates of P115 and MIF were 73.3%,80%,respectively, in gastric carcinoma ; and 40%,46.7%,respectively in the normal gastric mucosa. The positive rates of P115 and MIF in the gastric carcinoma were higher in gastric carcinoma than in the normal gastric mucosa(P<0.01). Moreover, the expression of P115 was positively correlated with the expression of MIF(r =0.433). The mRNA and protein expression levels of p115,MIF were higher in the gastric carcinoma tissues than in the normal gastric mucosa tissues(P<0.01). RT-PCR, Immunocytochemistry , Western blot analysis showed that the mRNA and protein expression levels of P115 , MIF were higher in gastric carcinoma cell lines (MKN-28 and BGC-823) than in the normal gastric epithelial cell line GES-1(P<0.01).The expression levels of P115, MIF in poorly-differentiated cell line were higher than in the well-differentiated cell line(P <0.05). 2. P115-shRNA can reverse the proliferation of gastric cancer cell The recombinant vectors, P115-shRNA, were verified by sequencing. P115-shRNA expression plasmids can inhibit the expression of P115 gene ,P115-shRNA2 showed the highest inhibitory effection , the P115 mRNA expression was inhibited by 76.8% and protein expression was inhibited by 70.97%.; MTT assay showed the proliferation of P115-shRNA2 cells was inhibited significantly compared with that of untreated BGC-823 cells, Flow cytometry assay showed a lower distribution in S phase in P115-shRNA2, compared with untreated BGC-823 cells,the relative levels of cyclinD1,mcm2,PCNA mRNA and protein were significantly inhibited in P115-shRNA2 cells compared with untreated BGC-823 cells(P﹤0.01).3. Forced ectopic expression of P115 can increase the proliferation of gastric cancer cell. The recombinant vector pEGFP-N1-P115 was verified by sequencing. The relative levels of p115 mRNA and protein were significantly enhanced in pEGFP-N1-P115 cells compared with untreated MKN-28 cells(P<0.01), MTT assay showed the proliferation of pEGFP-N1-P115 cells was enhanced significantly compared with that of untreated MKN-28 cells, Flow cytometry assay showed a higher distribution in S phase in pEGFP-N1-P115 cells, compared with untreated MKN-28 cells; the relative levels of cyclinD1,mcm2,PCNA mRNA and protein were significantly enhanced in pEGFP-N1-P115 cells compared with untreated MKN-28 cells(P﹤0.01).4. P115 could up-regulate the secretion and expression of MIF,and up-regulate the phosphorylation level of ERK1/2 When MIF was immunoprecipitated by anti-MIF polyclonal antibody, P115 was identified from immunoprecipitated complex. The relative levels of MIF mRNA and protein, and the concentration of MIF in the supernatant were significantly inhibited in P115-shRNA2 cells compared with untreated BGC-823 cells(P﹤0.01),the relative level of pERK1/2 protein was significantly inhibited in P115-shRNA2 cells compared with untreated BGC-823 cells. However, the relative levels of MIF mRNA and protein, and the concentration of MIF in the supernatant were significantly enhanced in pEGFP-N1-P115 cells compared with untreated MKN-28 cells(P﹤0.01),the relative level of pERK1/2 protein was significantly enhanced in pEGFP-N1-P115 cells compared with untreated MKN-28 cells.Conclusion P115 is over-expressed in gastric cancer tissues and cell lines; P115 can increase the proliferation of gastric cancer cell; Our results strongly indicated that the growth increase effect of P115 could be explained in part through P115-mediated elevation of the expression and secretion levels of MIF, and then modulation ERK signal transduction pathway; These data suggested P115 may play a potential role in the progression of neoplasia,that have been considered as a potential target.
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