B7-H1 Was Regulated By P53 Through MicroRNAs

Part.1 Establishment of p53-knock out Cell lineObjectives:Establishment of p53 null cell in A375 cells using CRISPR/CAS9 system.Method:Two gRNAs were designed and inserted into pBT-U6-Cas9-2A-GFP expression vectors. Then vector was transfected into K562 cells. Surveyor Mutation Detection Kits were used for determining the efficiency of both gRNA. And better one was used for knock out p53 in A375 cell line. Then vector was transfected into A375 and mutation was detected. The GFP positive cells were sorting by FCAS. To from colonies, selected cells were grown one cell per well in 96 wells plate. To select p53 knock-out colonies, lOGy X-ray was administrated to all colonies and then expression of p53 was tested. The p53 negative colonies were selected to further PCR and sequencing to confirm gene editing. Furthermore, the expression of p21 after X-ray stimulation was examined in p53 mull or mutant colonies by western blot.Results:Both gRNAs can edit target gene. The gRNA 1 show higher editing efficiency. Then choose gRNA 1 to transfect A375. After FACS sorting and colony formation,23 clones were achieved. And 3 of 23 clones showed absent of p53 expression by western blot. PCR showed size of p53 products changed in the 3 clones and sequencing results displayed insertion of deletion occurred in all negative clones target region of p53. And functional assay showed absent of p21 expression in p53 null clones.Conclusion:This research indicated that we successfully used CRISPR/CAS9 system to knock out p53 gene in A375 cell line.Part.2 p53 down regulate expression of B7-H1 via microRNA-34a and microRNA-200bObjectives:To explore the B7-H1 expression level after p53 knock out and the mechanism of p53 modulate B7-H1 expression.Method:The expression level of B7-H1 in A375, HCT116, A375?p53, HCT116Ap53 cell lines were evaluated by FACS. The mRNA level of B7-H1 was determined by RT-PCR. To explore underlying mechanism, different microRNA profiles between A375 and A375Ap53 cells was analyzed using microRNA array. And candidate microRNA was selected by prediction software. Then 8 candidate microRNAs mimics were transfected and Luciferase report vector was designed to identify microRNAs targeting the mRNA of B7-H1. Furthermore, the relationship between p53 and level of microRNA-34a and microRNA-200b were evaluated in p53+/-A375 and HCT116 cells. And the p53 and B7-H1 level was determined by western blot.Results:Expression of B7-H1 in A375 and HCT116 was lower compared to in its p53 knock out cell lines. The level of B7-H1 mRNA showed no differences between A375 and A375Ap53 or HCT116, HCT116?p53 cells. The microRNA-34a and microRNA-200b mimics reduced the B7-H1 level in A375Ap53 cells. And it was confirmed that microRNA-34a and microRNA-200b can bind with B7-H1 mRNA 3'UTR. Loss of p53 decreased the expression of microRNA-34a and microRNA-200b and increased expression of B7-H1 protein.Conclusion:p53 promoted transcription of microRNA-34a and microRNA-200b, which can target 3'UTR of B7-H1 and inhibit its expression.Part.3 microRNA-34a and microRNA-200b modulate proliferation and function of Jurkat cellsObjectives:to explore effect of microRNA-34a and microRNA-200b to Jurkat cells via modulation of B7-H1.Mothod:The PD-1 expression in Jurkat cells was induced by TPA and evaluated by western blot. The activated Jurkat cells was co-cultured with A375?p53 with or without anti-B7-H1 antibody. Then PCNA level of Jurkat cells was analyzed. A375?p53 was transfected with microRNA-34a and microRNA-200b mimics and inhibitors and then co-cultured with activated Jurkat cells. After 48h, Jurcat cells were isolated and PCNA was evaluated. The supernatant of co-culture system was isolated then the level of IL-2 and IFN-gamma was analyzed.Results:level of PD-1 increased after incubated with TPA. The proliferation of activated Jurkat cells significantly inhibited after co-culture with A375?p53. And such inhibition effect was removed on the condition of anti-B7-H1 antibody administration. A375Ap53 showed stronger ability in inhibiting proliferation of Jurkat cells than A375 p53 positive cells. However, this inhibition in Jurkat by A375?p53 was eliminated under transfection of microRNA-34a and microRNA-200b mimics. The level of IL-2 and IFN-gamma in co-culture supernatant increased after transfecting microRNA-34a and microRNA-200b mimics.Conclusion:Activation of B7-H1/PD-1 pathway inhibit proliferation of Jurkat cells, which can be inhibited by administration of B7-H1 antibody. MicroRNA-34a and microRNA-200b can affect function of T cells through B7-H1/PD-1 pathway.