Containing P53 Binding Sites Conserved MicroRNA Expression And Purification Of Vector And Its Application & MicroRNA Related Proteins And Mutant Ku80 Expression

Objective: It is reported that p53signaling pathways could cross talkwith microRNA (miRNA) regulation pathway. More and more studiesrevealed that miRNAs were involved in p53signaling pathway and p53could also regulate miRNA biosynthesis. To promote the expression ofmiRNAs under the transcriptional regulation activity of p53, weconstructed miRNA expression vector containing p53consensussequence (p53CON).With this vector, we could enhance miRNAsexpression in cells with wild-type p53，and also detect whether p53hastranscriptional regulation activity in converse.Methods: Modified commercial microRNA expression vectorpCMV-miR was constructed by inserting p53CON before the polyclonalsites.To confirm the efficiency of the modified vector,we inserted theprecursor sequences of miR-138, miR-34a and miR-21, which werealready known as p53-regulated miRNAs, into pCMV-miR separatelyand constructed successfully the vectors of pCMV/p53-miR-138,pCMV/p53-miR-34a and pCMV/p53-miR-21. Then the pCMV/p53-miR-138, pCMV/p53-miR-34a and pCMV/p53-miR-21expression vectors were transfected into HeLa cells which highly expressing wildtype p53and H1299cells which lack expression of p53individually,anddetected the expression of the microRNAs and the mRNA levels of theirrelated target genes by fluorescence quantitative PCR. The protein levelsof the target genes of miR-138, miR-34a and miR-21, that is Cyclin D3,CDK2and PTEN respectively, were also detected by Western blot assay.Results: The expression levels of miR-138, miR-34a and miR-21weresignificantly increased in HeLa cells transfected with modified miRNAexpression vectors, and the mRNA levels as well as protein levels ofCyclin D3, CDK2and PTEN were significantly decreased. Our resultsconfirmed the regulation of miR-138, miR-34a and miR-21by p53andthe efficiency of the constructed vectors.Conclusion: Under the regulation of p53, the expression level ofmiRNAs could be effectively increased. The constructed vectorpCMV-miR could be used not only to promote the expression of miRNAswith p53conserved binding sites, but also to detect whether p53existtranscriptional regulation activity in converse. Objective: Ku protein is an evolutionarily conserved DNA bindingprotein. Human Ku is a heterodimer of two polypeptides, Ku70(XRCC6)and Ku80(XRCC5), so named because the molecular weight of thehuman Ku protein is around70kDa and80kDa.The Ku80gene locatedin2q33-34, encoding732amino acids, containing an αβ domain, DNAbinding domain, Ku70binding domain and DNA-PKcs binding domain.It is recently reported that Ku80can bind with RNA. Studies have shownthat the Ku protein is capable of binding yeast telomerase RNA andinternal ribosome entry site sequence. Previous work in our laboratoryconfirmed that Ku80might be involved in miRNA processing. Toinvestigate the mechanism of Ku protein in the regulation of miRNAmature processing, the eukaryotic and prokaryotic expression vectors ofKu80and its mutants were constructed. The eukaryotic expression vectorwas transfected into HeLa cells and the expression of Ku80and its mutants were detected. The prokaryotic expression vectors of Ku80andits mutants were expressed in E.coli, and the Ku80and its mutant proteinswere purified.Methods: The cDNAs of Ku80gene and its mutants (deletion of αβdomain or deletion of DNA binding domains) were PCR amplified.Vector of pcDNA6was used to construct the eukaryotic expressionvectors, and pET-28a (+) was used to construct the prokaryotic expressionvectors. After the construction of all the vectors, the vectors wereconfirmed by sequencing. Then the eukaryotic expression vectors wastransfected into HeLa cells, Western blotting was used to detect theexpression of Ku80and its mutants. The prokaryotic expression plasmidswere transformed into Escherichia coli BL21, and the proteins wereinduced by IPTG and purified with Ni column chromatography.Results: Western blotting results showed that the Ku80and its mutantscould be expressed in HeLa cells with their eukaryotic expression vectors.SDS-PAGE and analysis Western blotting results showed that the Ku80and its mutants were expressed in E.coli and could be purified by Nicolumn.Conclusion: Recombinant Ku80and its mutants were expressed both ineukaryotic or prokaryotic systems. The purified Ku80and its mutantprotein provides the materials for further study of the relationship ofKu80and miRNAs.