稳定干扰鸟苷酸环化酶C基因表达的人胃腺癌细胞株的筛选和建立

Screening and Establishment of Human Gastric Adenocarcinoma Cell Line with Stable Interference of Guanylyl Cyclase C Gene Expression

背景:正常胃黏膜组织不表达肠上皮细胞特异性受体鸟苷酸环化酶C(GC-C),但肠化生、异型增生和胃腺癌组织存在GC-C异位表达。目的:筛选并建立GC-C特异性短发夹RNA(shRNA)稳定转染的胃腺癌细胞株。方法:构建靶向GC-C基因的shRNA表达载体,以脂质体法转染胃腺癌细胞株SGC-7901,实时荧光定量RT-PCR和蛋白质印迹法分别检测GC-C mRNA和蛋白表达。G418筛选阳性克隆并建立GC-C shRNA稳定转染的SGC-7901细胞株,并行RT-PCR鉴定。结果:GC-C shRNA可抑制SGC-7901细胞株的GC-C基因表达,以GC-C sh3的抑制效果最佳,转染48 h后的GC-CmRNA和蛋白沉默率分别为77.0%和62.2%。有效筛选并建立了GC-C shRNA稳定转染的SGC-7901细胞株,GC-CmRNA沉默率为65.3%。结论:成功建立了GC-C shRNA稳定转染的SGC-7901细胞株,为下一步研究奠定了基础。

Background： Guanylyl cyclase C （GC-C）, a specific receptor of intestinal epithelial cell, is not expressed in normal gastric mucosa, but is ectopically expressed in intestinal metaplasia, dysplasia and gastric adenocarcinoma tissue. Aims： To screen and establish the gastric adenocarcinoma cell line with stable interference of GC-C gene by short hairpin RNA （shRNA）. Methods： shRNA expression vector targeting GC-C was constructed and transfected into gastric adenocarcinoma cell line SGC-7901 by LipofectamineTM 2000. Real-time fluorescent quantitative RT-PCR and Western blotting were conducted to assess the mRNA and protein expressions of GC-C, respectively. The positive cell clone was screened by G418, and the stable GC-C shRNA transfected cell line SGC-7901 was established and verified by RT-PCR. Results： GC-C shRNA could successfully inhibit the expression of GC-C in cell line SGC-7901, and the inhibitory effect was most prominent in GC- C sh3. The inhibition rates of GC-C mRNA and protein after 48 hours of transfection were 77.0% and 62.2%, respectively. The stable GC-C shRNA transfected SGC-7901 cell line effectively screened had a GC-C mRNA inhibition rate of 65.3%. Conclusions： A stable GC-C shRNA transfected cell line SGC-7901 is successfully established, providing a basis for further studies.