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目的研究利用小分子干扰技术(RNAi),构建Grp78靶向RNA干扰质粒载体,观察其对人类卵巢癌细胞SKOV3Grp78基因表达的抑制作用。初步探讨RNA干扰技术抑制Grp78的表达作为卵巢癌基因治疗的可行性。方法从GeneBank中选取人类卵巢癌细胞Grp78基因序列,采用siRNA Target Designer-Version 1.51设计软件设计Grp78基因发卡寡核苷酸,序列符合siRNA表达载体psiSTRIKETMU6的要求并与psiSTRIKETM质粒载体连接,采用脂质体转染法将含有特异性小分子干扰Grp78 mRNA的重组载体psiSTRIKETM/Grp78导入卵巢癌细胞系SKOV3中,分别在转染前、转染后24h、48h和72h通过RT-PCR和Western blot检测Grp78 mRNA及蛋白水平的表达情况,用流式细胞仪检测细胞凋亡。四甲基偶氮唑蓝(MTT)比色法测定细胞经梯度浓度紫杉醇处理后的细胞存活率。结果成功构建RNA干扰质粒载体,靶向Grp78 RNA干扰质粒载体命名为psiSTRIKETM/Grp78。将上述质粒转染到卵巢癌细胞后,观察到psiSTRIKETM/Grp78能够有效的抑制Grp78 mRNA及蛋白表达。转染psiSTRIKETM/Grp78基因后的卵巢癌细胞增殖受到抑制,出现凋亡征象,且对化疗药物的敏感性增加。结论构建的RNA干扰真核表达载体psiSTRIKETM/Grp78能明显抑制Grp78 mRNA及蛋白的表达,增加卵巢癌细胞的化疗敏感性。RNAi技术抑制Grp78表达为卵巢癌的基因治疗开辟了新的思路。
Objective To study the inhibitory effect of Grp78 targeting RNA interference plasmid vector on the expression of human ovarian cancer cell line SKOV3Grp78 by using small interfering RNA (RNAi) technology. Preliminary study of RNAi inhibition of Grp78 expression as ovarian cancer gene therapy feasibility. Methods The Grp78 gene sequence of human ovarian cancer cells was selected from GeneBank. The Grp78 gene hairpin oligonucleotide was designed by using siRNA Target Designer-Version 1.51 design software. The sequence was in accordance with the siRNA expression vector psiSTRIKETMU6 and ligated to psiSTRIKETM plasmid vector. Liposome The recombinant vector psiSTRIKETM / Grp78 containing specific small interfering Grp78 mRNA was transfected into ovarian cancer cell line SKOV3 by transfection method. Grp78 mRNA was detected by RT-PCR and Western blot before transfection, 24h, 48h and 72h after transfection And protein level of expression, using flow cytometry to detect apoptosis. MTT assay was used to determine cell viability after gradient paclitaxel treatment. Results The RNA interference plasmid vector was successfully constructed and the targeting Grp78 RNA interference plasmid vector was named psiSTRIKETM / Grp78. After transfection of these plasmids into ovarian cancer cells, psiSTRIKETM / Grp78 was observed to inhibit Grp78 mRNA and protein expression. Proliferation of ovarian cancer cells transfected with psiSTRIKETM / Grp78 gene is inhibited, signs of apoptosis appear, and chemosensitivity increases. Conclusion The RNA interference eukaryotic expression vector psiSTRIKETM / Grp78 can significantly inhibit the expression of Grp78 mRNA and protein and increase the chemosensitivity of ovarian cancer cells. RNAi technology inhibits the expression of Grp78 gene therapy for ovarian cancer has opened up new ideas.