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.%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G,C-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-CmRNA和蛋白表达.G418筛选阳性克隆并建立GC-CshRNA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-Csh3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-CshRNA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.
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