胶原三股螺旋重复蛋白1基因真核表达载体的构建及其与microRNA-30b关系初探

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

目的 构建pCTHRC1-IRES2-EGFP真核表达载体,初步探讨胶原三股螺旋重复蛋白1基因(CTHRC1)与microRNA(简写为miR)-30b的关系。方法 设计特异性引物,通过PCR扩增合成含有CTHRC1 CDS+3′UTR区的序列,并克隆至pIRES2-EGFP真核表达载体,得到pCTHRC1-IRES2-EGFP真核表达载体。采用NheⅠ/XhoⅠ双酶切电泳和测序法进行鉴定;通过脂质体转染法转染Huh7及HepG2细胞评价转染效率;将miR-30b mimic、control、pCTHRC1-IRES2-EGFP质粒和pIRES2-EGFP质粒转染至293T细胞中,分别采用Western Blot和实时荧光定量PCR法检测转染48 h后CTHRC1蛋白的表达水平及miR-30b的表达水平,初步探讨CTHRC1基因与miR-30b的关系。两组间比较采用独立样本t检验。结果 采用NheⅠ/XhoⅠ双酶切pCTHRC1-IRES2-EGFP真核表达载体,得到5300 bp和1100 bp两条片段,与预期结果一致,测序结果也显示序列与GeneBank公布序列完全一致,表明pCTHRC1-IRES2-EGFP真核表达载体构建成功。2 μg和4 μg的pCTHRC1-IRES2-EGFP质粒转染Huh7及HepG2细胞,转染效率分别可达90%、89%和85%、83%。在293T细胞中,miR-30b组CTHRC1表达下降(P<0.05)。而过表达CTHRC1组中miR-30b的表达下降(P<0.05)。结论 成功构建了pCTHRC1-IRES2-EGFP真核表达载体;在293T细胞中,CTHRC1基因表达与miR-30b的表达水平相反。

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-30b. 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 pCTHRC1-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-30b mimic, control, pCTHRC1-IRES2-EGFP plasmid, and pIRES2-EGFP plasmid were transfected into 293T cells, Western Blot and quantitative real-time PCR were used to measure the protein expression of CTHRC1 and the expression of miR-30b at 48 hours after transfection. The association between CTHRC1 gene and miR-30b 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 pCTHRC1-IRES2-EGFP eukaryotic expression vector, and two fragments with a length of 5300bp and 1100bp 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 pCTHRC1-IRES2-EGFP plasmid had a transfection efficiency of 90% and 89%, respectively, while 4 μg pCTHRC1-IRES2-EGFP plasmid had a transfection efficiency of 85% and 83%, respectively. In 293T cells, the expression of CTHRC1 decreased to the miR-30b group(P<0.05). The expression of miR-30b 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-30b in 293T cells.