Construction And Identification Of Eukaryotic Expression Plasmids Expressing Shrna Sections Targeting Human MKRN1 Gene And Its Biological Effects To HEK293 Cells

Objective To construct eukaryotic expression plasmids expressing short hairpin RNA(shRNA)sections targeting human MKRN1 gene and determine their effects on MKRN1 gene expression and the bionomics in HEK293 cell line. For further study MKRN1 lay the foundation for the function of the gene.Methods According to the sequence of human MKRN1 gene,the oligonucleotides of shRNA were designed and synthesized and directionally cloned into plasmid pGenesil-1 with enhancing green fluorescence protein(EGFP) gene and Kan gene. The recombinant plasmids were confirmed by enzyme digestion identified and DNA sequencing.The recombinant plasmids were transfected into HEK293 cell line by LipofectamineTM 2000,the effects on MKRN1 gene at mRNA and protein levels were observed.Select the most effective interference sequence, transfected into HEK293 cells. The effect of the recombinant plasmid(pGenesil-1-MKRN1-shRNA1) to HEK293 cells was detected by MTT assay;Cell cycle distribution was detected by FCM;the effects on hTERT gene at mRNA and protein levels were detected by RT-PCR and Western blot.Results Three shRNA expressing recombinants and the corresponding negative control plasmid were constructed and transfected into HEK293 cell successfully.MKRN1 transcript was reduced by about 26.2 -52.4% ,the protein of MKRN1 was reduced by about 27.9-69.1% in three transfectants respectively . With transfected plasmid pGenesil-1-MKRN1-shRNAhk and non-transfected cells compared, MTT testing showed that transfection of recombinant plasmid pGenesil-1-MKRN1-shRNA1 can significantly promote the proliferation of HEK293 cells. FCM detection found that transfection of recombinant plasmid pGenesil-1-MKRN1-shRNA1 group G2 phase and S phase cells increased, indicating that pGenesil-1-MKRN1-shRNA1 can promote the proliferation of HEK293. RT-PCR, Western blot detection showed that recombinant expression plasmid transfectionpGenesil-1-MKRN1-shRNA1 of HEK293 cells, hTERT gene mRNA expression level and protein level was significantly increased. Conclusion Eukaryotic expression plasmids expressing shRNA sections targeting human MKRN1 gene can inhibit HEK293 cell-specific gene expression in human MKRN1 to increase in the expression of hTERT gene and promote cell proliferation.