Construction of the eukaryotic expression vector of collagen triple helix repeat containing 1 gene and its association with microRNA-30b

Objective To construct pCTHRC1-IRES2-EGFP eukaryotic expression vector,and to investigate the association between collagen triple helix repeat containing 1( CTHRC1) gene and miR-30 b. Methods Specific primers were designed and PCR was used for the amplification and synthesis of the sequence of CTHRC1 CDS + 3 'UTR region,which was then cloned into the pIRES2-EGFP eukaryotic expression vector to obtain the p CTHRC1-IRES2-EGFP eukaryotic expression vector. Nhe I/Xho I double enzyme electrophoresis and sequencing were used for identification,and the lipofection method was used to evaluate the transfection efficiency of Huh7 and HepG2 cells.After miR-30 b mimic,control,p CTHRC1-IRES2-EGFP plasmid,and pIRES2-EGFP plasmid were transfected into 293 T cells,Western Blot and quantitative real-time PCR were used to measure the protein expression of CTHRC1 and the expression of miR-30 b at 48 hours after transfection. The association between CTHRC1 gene and miR-30 b was analyzed. The independent samples t-test was used for comparison between two groups. Results Nhe I/Xho I double enzyme digestion was performed for the p CTHRC1-IRES2-EGFP eukaryotic expression vector,and two fragments with a length of 5300 bp and 1100 bp were obtained,which was consistent with the expected results;the sequencing results also showed that the sequence was exactly the same as that published in GeneBank,suggesting that the pCTHRC1-IRES2-EGFP eukaryotic expression vector was constructed successfully. In the transfection of Huh7 and HepG2 cells,2 μg p CTHRC1-IRES2-EGFP plasmid had a transfection efficiency of 90% and 89%,respectively,while 4 μg p CTHRC1-IRES2-EGFP plasmid had a transfection efficiency of 85% and 83%,respectively. In 293 T cells,the expression of CTHRC1 decreased to the miR-30 b group( P <0. 05). The expression of miR-30 b decreased in the group with overexpressed CTHRC1( P < 0. 05). Conclusion The eukaryotic expression vector of pCTHRC1-IRES2-EGFP is successfully constructed,and the expression of CTHRC1 is negatively correlated with the expression of miR-30 b in 293 T cells.