Research Of Transcription Factor XBP1s With Cell Proliferation And Apoptosis

Objective:X-box binding protein 1(XBP1) is an important transcription factor of endoplasmic reticulum signaling pathway,with construct XBP1s eukaryotic expression vector pcDNA3.1(-)-XBP1s and the target XBP1s mRNA interference plasmid pSUPER-XBP1s, use normal liver cell L02, hepatoma cells HepG-2 and HeLa cells to study the relationship between XBPls and cell proliferation, cell apoptosis.Methods:(1) Trizol method to extract total RNA from HepG-2 cells, RT-PCR amplified fragment of XBP1s gene. XBP1s was inserted into the pGEM-T vector by TA cloning technology to construct plasmid pGEM-T-XBP1s. Construct the prokaryotic expression plasmid pcDNA3.1(-)-XBP1s by molecular cloning technology.(2) Synthetic two pairs of XBPls siRNA oligos, inserted into the empty plasmid pSUPER to construct the RNA interference plasmid pSUPER-XBP1s.(3) The two expression plasmids, pcDNA3.1(-)-XBP1s and pSUPER-XBP1s, were transfected into HepG-2 cells and L02 cells, then mRNA level and protein expression were detected with RT-PCR and Western blotting after cells transfected empty vector and pSUPER-XBPls.(4) The two recombinant plasmids, pcDNA3.1(-)-XBP1s and pSUPER-XBP1s, were transfected into L02 cells and HepG-2 cells, And the relationship between XBP1s and cell proliferation was observed by MTT and cell counting methods.(5) Firstly, establish cell model of endoplasmic reticulum stress(ERS) with the ER stress inducer tunicamycin(Tm) and thapsigargin (Tg),Then transfect above-mentioned recombinant plasmids into HepG2 cells, L02 cells and Hela cells,And detect cell cycle distribution and cell apoptosis rate with flow cytometry,observe the morphology of apoptotic cells with immunohistochemistry and electron microscopy.Results:(1) Successfully constructed the eukaryotic expression vector pcDNA3.1 (-)-XBP1s and RNA interference plasmid pSUPER-XBPls.(2) After transfected eukaryotic expression vector pcDNA3.1(-)-XBP1s to HepG-2 cells, the mRNA and protein level were increased comparing with the control, the difference is statistical significance(P<0.05).While pSUPER-XBP1s can significant inhibit XBP1s gene transcription and protein expression in HepG-2 cells, the difference is statistical significance(P<0.05).(3) After the two recombinant plasmids transfected into L02 cells and HepG-2 cells, comparing with the control, the differences of cell proliferation and the cell number were also statistical significance(P<0.05). (4) Immunohistochemistry and HE staining results showed that, XBP1s expression will be decreased and cells morphology will be changed after transfected into pSUPER-XBP1s comparing with the control during ERS.(5) Cell cycle distribution results show that, the S phase significant reduced after transfected into pSUPER-XBP1s comparing with the control, however, the S phase clearly increased after transfected into pcDNA3.1 (-)-XBP1s, the differences were statistical significance(P<0.05).Cell apoptosis rate results show that, compare with the control, ERS leads to cell apoptosis, XBP1s expression will be increased in ERS, and XBP1s will relief the ERS-mediated apoptosis.The apoptotic bodies will be observed in ERS-mediated apoptosis by Electron microscopy.Conclusion:Successful construction the plasmids of eukaryotic expression vector pcDNA3.1 (-)-XBP1s and RNA interference vector pSUPER-XBPls. XBP1s will advance cell proliferation. And during ERS, the inhibition of XBP1s will increase cell apoptosis.