Study On The Role Of Runx3 Gene In The Immunology Regulation And The Relation Between Runx3 And Gastric Carcinoma

Objective To confirm the role of Runx3 on the levels of transcription factors in patients with gastric carcinoma,the expression of Runx3 in patients with gastric carcinoma were detected,and the relationship between the expression of Runx3 and Th1 type cytokines in peripheral blood mononuclear cells(PBMC) were studied.To clone and detect human Runx3 gene;To express Runx3 protein in E.coli system;To transfect SGC7901 cells with pIRES₂-Runx3(Runx3 was constructed into pIRES2-eGFP plasmid);and To research the function transform of SGC-7901;To investigate the feasibility of transcription factor Runx3 in biotherapy of gastric carcinoma.Methods(1) The gene expression of transcription factors Runx3,T-bet and cytokines IFN-γwere determined by RT-PCR and realtime-PCR from 100 patients with gastric cancer and 50 healthy person.(2)Runx3 gene was obtained through mRNA isolated from PBMC of health people and transcribed into cDNA by PCR with specific primers.The Runx3 gene was constructed into pMD19-T vector and detected by single and double restriction enzyme as well as sequencing.(3)Runx3 gene was constructed into pQE30 vector and expressed in E.coli M15 to get Runx3 fusion protein with His tag;The fusion protein was purified by IMAC column of Bio-Rad company;The purified protein was tested with anti-His by Western-blot.(4) Runx3 gene was constructed into pIRES₂-eGFP,and the recombined plasmid was transfected into SGC-7901 cells with liposome(pIRES₂-eGFP as negative control); After transfection,the eGFP was observated with fluorescence microscope,and the mRNA of Runx3,T-bet or IFN-γwas detected individually with RT-PCR in order to understand the role of Runx3 in SGC-7901.Results(1) The expression rates of Runx3,T-bet and IFN-γin patients were obviously lower than control groups,and there was a positive correlation between the expression of Runx3 and T-bet and IFN-γin patient(p＜0.01),but no relationship in healthy person.(2) The full length of Runx3 gene with open reading frame was cloned successfully from human PBMC and the purified protein was obtained.(3) The recombinant eukaryotic expression plasmids pIRES₂- Runx3 was produced, which was used to transfect SGC-7901 cells and the function to inhibit SGC-7901 cells was observed.ConclusionsIn short,the expression rates of Runx3 in patients with gastric cancer were obviously lower than control groups and there was a positive correlation between the expression of Runx3 and T-bet or IFN-γin patient,which indicated that Runx3 may be related to the immunology regulation of gastric cancer patients.Runx3 gene was cloned and expressed in E.coli system effectively;The purified protein was obtained;The gene-transfection methods were used to transfect Runx3 into SGC-7901 cells,which secret IFN-γ.Runx3 function towards inhibition of SGC-7901 cells was studied.It will be the basis for studying cancerimmune therapy of transferring gene Runx3.