RNA干扰G_α13对人卵巢癌HO-8910PM细胞侵袭转移的影响

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细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：RT-PCR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/G1期，且膜上Rho GTPases与GTP结合活性明显下降。结论：靶向Gα13的重组shRNA质粒能够有效抑制Gα13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 G_α13 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 G_α13 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 G_α13,and changes of protein expression of subunit of G_α13 were detected by western blot.The effects of G_α13 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 G_α13 was successively developed;RNAi could effectively down-regulate the mRNA and protein level of G_α13;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 G_α13 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.

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