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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 silence the expression of G_ (α13) gene by small interfering RNA (RNAi) technology and investigate its effect on the growth and metastasis of human ovarian cancer HO-8910PM cells. METHODS: Shuttle Vector 1.0-CMV was used as a vector to construct shRNA recombinant expression vector targeting G_ (α13). Human ovarian cancer cell line HO-8910PM was transfected. The mRNA and protein of G_ (α13) were detected by RT- The level of expression. The effects of G13 (α13) silencing on the growth and invasion of human ovarian cancer HO-8910PM cells were observed by MTT assay, Transwell chamber, Matrigel gel electrophoresis and flow cytometry. The activity of Rho GTPases was analyzed by autoradiography. Results: The results of RTPCR and Western Blot showed that the expression level of G_ (α13) gene in Shuttle Vector 1.0-CMV G_ (α13) B siRNA group was effectively inhibited. Compared with the control group, the cell invasion and migration of Shuttle Vector 1.0-CMV G_ (α13) B siRNA group were significantly decreased (P <0.01), cells were arrested in G_0 / G_1 phase, and Rho GT Pases GTP binding activity decreased significantly. CONCLUSION: Recombinant shRNA targeting G_ (α13) can effectively inhibit the expression of G_ (α13) gene in HO-8910PM cells and reduce the invasion and migration of HO-8910PM cells. The mechanism may be related to the decrease of Rho GTPases Activity, which may provide a reference for the targeted therapy of ovarian cancer.