双干扰质粒共转染对胃癌BAG-1基因亚型表达的抑制作用

doi:10.3971/j.issn.1000-8578.2013.04.003

双干扰质粒共转染对胃癌BAG-1基因亚型表达的抑制作用

Inhibitory Effects of Cotransfection of Two Short Hairpin RNA on Expression of BAG-1 Subtypes in Gastric Carcinoma

摘要

摘要: 目的构建针对人BAG-1（Bcl-2-associated athanogene 1）基因的短发卡RNA（short hairpin RNA，shRNA）真核表达质粒，探讨其对人胃癌SGC-7901细胞BAG-1主要亚型表达的抑制作用及对细胞生长的影响。方法以人BAG-1 mRNA编码区为RNA干扰靶点，设计2条不同的位于BAG-1基因P29、P33、P46、 P50亚型的两个通用外显子上64nt寡核苷酸干扰片段A、B及1条阴性对照片段S，将其定向克隆到带有卡那霉素抗性和增强绿色荧光蛋白的真核表达载体pGenesil-1上，将其转染至SGC-7901细胞中，设空白对照组（未转染质粒SGC-7901-Un）、阴性对照组（转染阴性对照质粒pGensil-Neg）、干扰组（转染干扰质粒pGensil-BAG1-A和pGensil-BAG1-B），用RT-PCR、Western blot检测BAG-1基因的干扰效果。MTT法检测细胞生长情况并绘制生长曲线。结果酶切及测序结果证实，pGensil-BAG1-A和pGensil-BAG1-B干扰质粒及阴性对照质粒pGensil-S构建成功。质粒在SGC-7901细胞中的转染效率达40%~50%。阴性对照组细胞BAG-1基因表达与空白对照组无明显差异，干扰组细胞BAG-1基因表达较空白对照组显著下降（P<0.01），表达抑制率在mRNA水平为(63±9.8)%，在蛋白水平对BAG1-P46、BAG1-P32亚型的抑制率分别为(94.3±1.08)%、(66.1±6.5)%。生长曲线表明细胞生长受到了抑制。结论针对人BAG-1基因的shRNA真核表达质粒pGensil-BAG1-A和pGensil-BAG1-B共转染能高效特异的抑制胃癌SGC-7901细胞中BAG-1主要亚型的表达，并抑制人胃癌SGC-7901细胞的生长。

Abstract: Objective To construct the eukaryotic expression plasmids of short hairpin RNA(shRNA) specific for Bcl-2 associated athanogene 1(BAG-1);and to observe their inhibitory effects on the main subtypes expression of BAG-1 gene in human gastric carcinoma SGC-7901 cells. Methods Two distinct interference segment A and B of 64nt oligonucleotides which were located on two common exons of BAG-1 gene P29, P33, P46 subtypes and one negative control segment S, were designed and directionally cloned into eukaryotic expression plasmids pGenesil-1 containing kanamycin resistance gene and GFP to target the human BAG-1 mRNA coding region. Lipofectamin TM 2000 was used to cotransfect plasmids into SGC-7901 cells. Negative plasmid-transfected, untransfected SGC-7901 cells and transfected interference plasmids pGenesil-BAG-A and pGenesil-BAG-B served as negative, blank, and interference control respectively. Interference effects were measured by RT-PCR and Western blot. Cell proliferation was detected by MTT assay and cell growth curve was drawn to analyze the inhibitory effects. Results The clone was verified through restriction enzyme digestion and sequence analysis of hairpin-siRNA expression DNA segment and adjacent vector sequence. The transfection rate in SGC-7901 cells was 40%-50%. Compared with the untransfected control, the negative control cell BAG-1's gene expression showed no obvious difference. However, the mRNA and protein of BAG-1 in SGC-7901 cells was significantly inhibited by BAG-1 shRNA (both P<0.01). The inhibition rate of mRNA was (63 ±9.8)%. The inhibition rate of P46 and P32 BAG-1 proteins were (94.3±1.08)% and (66.1±6.5)%, respectively. The proliferation of cells was inhibited significantly compared with the control group. Conclusion The vectors containing hairpin-siRNA expression DNA segment are successfully constructed, and cotransfection plasmids of pGensil-BAG1-A and pGensil-BAG1-B can significantly inhibit BAG-1gene's main subtype expression and cell proliferation of human gastric carcinoma SGC-7901 cells.

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