Research Of IRE1 With Cell Proliferation And Apoptosis During ERS

Objective To clone IRE1 gene and construct its six different structural fragments; construct target IRE1 mRNA interference plasmid pSUPER-IRE1. Study the relationship between IRE1 and cell proliferation , cell apoptosis with SMMC -7721 when ERS .Methods (1) Construct the RNA interference plasmids pS1-IRE1 and pS2-IRE1. Transfected them into HeLa and HepG2 cells, then mRNA level and protein expression were detected with RT-PCR and Western blot.(2) The six fragments were amplified from pCMV-IRE1 vector and inserted into eukaryotic expression vector pcDNA3.1(-) respectively by PCR. After the identification of double-enzyme digestion and sequencing, the recombinant plasmids were transfected into LO2 cells. Western blot assay was carried out to analyse the expression of each protein, and the online software SWISS-MODEL was used to predict their protein products.(3) The recombinant plasmids were transfected into SMMC-7721 cells with ERS, and the relationship between IRE1 and cell proliferation was observed by MTT and cell counting methods.(4) Detect cell cycle distribution and rate of cell apoptosis with flow cytometry.Results: (1) RNA interference plasmids pS1-IRE1 and pS2 were constructed successfully, after transfected into HeLa and HepG2 cells the mRNA and protein level were decreased comparing with the control(P<0.05).(2) Successfully constructed the eukaryotic expression vector pIRE1,pD-Kinase,pD-RNase,pNLD,pKinase,pR+K and pRNase . WB test the expression of the plasmids. Determine that IRE1 was locationed in cytoplasm by IF.(3) After transfected during ERS ,we saw pIRE1 promote cell proliferation and the other plasmids inhibit cell proliferation, the differences of cell proliferation and the cell number were statistical significance (P<0.05).(4) After transfected during ERS ,we saw pIRE1 inhibit cell apoptosis and the other plasmids promote cell apoptosis, the differences of cell proliferation and the cell number were statistical significance (P<0.05).Conclusion The recombinant plasmids of full-length,six fragments and RNA interference plasmids were constructed successfully : pIRE1, pD-Kinase, pD-RNase,pNLD, pKinase,pR+K ,pRNase,pS1 and pS2. During ERS, IRE1 will promote cell proliferation and inhibit cell apoptosis.