RNA干扰G_(α13)对人卵巢癌HO-8910PM细胞侵袭转移的影响  

Effect of RNA interference of G_(α13) on invasion and metastasis of human ovarian cancer HO-8910PM cells

作　　者：符伟玉[1] 曾振飞[1] 兰柳波[1] 吴科锋[2] 

机构地区：[1]广东医学院生物化学与分子生物学研究所,湛江524023 [2]广东天然药物研究与开发重点实验室,湛江524023

出　　处：《药物分析杂志》2017年第1期90-96,共7页Chinese Journal of Pharmaceutical Analysis

基　　金：广东省科技计划项目(No 2011B031800343);湛江市财政资金科技专项竞争性分配项目(No.2013A01008)

摘　　要：目的:利用小干扰RNA技术沉默G_(α13)基因的表达,探讨其对人卵巢癌HO-8910PM细胞生长与转移的影响。方法:以Shuttle Vector 1.0-CMV为载体,构建针对G_(α13)的shRNA重组表达质粒;转染人卵巢癌细胞HO-8910PM,利用RT-PCR和Western Blot分别检测G_(α13)基因mRNA和蛋白的表达水平。体外通过MTT法、Transwell小室、Matrigel胶模型、流式细胞术,观察G_(α13)被沉默后对人卵巢癌HO-8910PM細胞生长与侵袭转移的影响;放射自显影方法分析Rho GTPases活性。结果:RTPCR和Western Blot结果显示,Shuttle Vector 1.0-CMV G_(α13)B siRNA转染组G_(α13)基因的表达水平被有效抑制。与对照组相比,Shuttle Vector 1.0-CMV G_(α13)B siRNA转染组的细胞侵袭、迁移力明显下降(P<0.01),细胞被阻滞在G_0/G_1期,且膜上Rho GT Pases与GTP结合活性明显下降。结论:靶向G_(α13)的重组shRNA质粒能够有效抑制G_(α13)基因在HO-8910PM细胞中的表达,并能降低HO-8910PM细胞侵袭迁移的能力,其机制可能与降低细胞膜上Rho GTPases活性有关,可为卵巢癌的靶向治疗提供参考。Objective： To investigate the inhibitory effect of Gα13 gene silencing on the growth and metastasis of human ovarian carcinoma HO-8910PM cell line by RNAi system. Methods： A RNAi system expressing shRNAs targeting Gα13 was developed based on the Shuttle Vector 1.0-CMV. After transfection, RT-PCR assay was used to determine the changes of mRNA expression of subunit of Gα13, and changes of protein expression of subunit of Gα13 were detected by western blot. The effects of Gα13 knockdown on HO-8910PM cells growth and metastasis were analyzed by MTT assay, Transwell migration assay and Matrigel invasion assay.GTP-binding activity of Rho GTPases was detected by autoradiography. Results： A series of RNAi system expressing shRNA targeting Gα13 was successively developed; RNAi could effectively down-regulate the mRNA and protein level of Gα13; RNAi can inhibit growth and metastasis of HO-8910PM cell （ P〈0.01 ）, induce cell cycle to pause at G0/G1 phase, and decrease the binding abilities of Rho GTPases and GTP. Conclusion： Recombinant plasmid expressing shRNA targeting Gα13 has successively performed the RNAi effects in HO-8910PM; the RNAi system can inhibit growth and metastasis of HO-8910PM cells via decreasing the activation of Rho GTPases, which can provide a reference for targeted therapy in ovarian cancer.

关 键 词：溶血磷脂酸 RNA干扰技术 侵袭迁移 基因表达 卵巢癌 

分 类 号：R917[医药卫生—药物分析学]