【摘要】：目的研究ADP核糖基化因子6(Arf6)對(duì)雄激素非依賴(lài)性前列腺癌PC-3細(xì)胞株增殖、遷移和侵襲能力的影響并初步探討其可能的分子作用機(jī)制。方法設(shè)計(jì)合成3條針對(duì)不同靶向區(qū)域的Arf6特異性si RNA序列,轉(zhuǎn)染細(xì)胞后通過(guò)real-time PCR和蛋白質(zhì)印跡法檢測(cè)其對(duì)Arf6的干擾效果,篩選出干擾效果最佳的si RNA序列;通過(guò)噻唑鹽(MTT)實(shí)驗(yàn)、劃痕實(shí)驗(yàn)、及transwell細(xì)胞遷移和侵襲實(shí)驗(yàn)觀察si RNA干擾Arf6表達(dá)對(duì)PC-3細(xì)胞增殖、遷移和侵襲的影響;蛋白質(zhì)印跡法檢測(cè)AKT、p-AKT、ERK1/2、p-ERK1/2和Rac1蛋白表達(dá)水平的變化。結(jié)果與空白對(duì)照組相比,轉(zhuǎn)染陰性對(duì)照序列對(duì)PC-3細(xì)胞內(nèi)源性Arf6的m RNA和蛋白表達(dá)水平無(wú)明顯影響,3條si RNA序列均能抑制Arf6的表達(dá),其中si RNA-3對(duì)PC-3細(xì)胞Arf6表達(dá)干擾效果最好,Arf6m RNA和蛋白抑制率分別為(91.88±3.13)%和(86.37±0.57)%。si RNA-3干擾Arf6表達(dá)抑制PC-3細(xì)胞的增殖,且PC-3細(xì)胞體外遷移距離和侵襲細(xì)胞數(shù)較空白和陰性對(duì)照組明顯減少(P0.05)。蛋白質(zhì)印跡法檢測(cè)發(fā)現(xiàn)轉(zhuǎn)染si RNA-3的PC-3細(xì)胞p-ERK1/2和Rac1表達(dá)水平明顯降低,而AKT、p-AKT和ERK1/2表達(dá)水平較對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義。結(jié)論 si RNA干擾Arf6表達(dá)可顯著抑制PC-3細(xì)胞的增殖、遷移和侵襲能力,其分子作用機(jī)制可能與p-ERK1/2和Rac1表達(dá)下調(diào)相關(guān)。
[Abstract]:Objective to investigate the effects of ADP ribosylation factor 6 (Arf6) on the proliferation, migration and invasion of androgen independent prostate cancer PC-3 cell line and to explore its possible molecular mechanism. Methods three Arf6 specific si RNA sequences targeting different target regions were designed and synthesized. After transfection, real-time PCR and Western blotting were used to detect the interference effect on Arf6. The effects of si RNA interfering Arf6 expression on the proliferation, migration and invasion of PC-3 cells were observed by scratch assay and transwell cell migration and invasion assay, and the changes of AKT,p-AKT,ERK1/2,p-ERK1/2 and Rac1 protein expression levels were detected by Western blot. Results compared with the blank control group, the expression of m RNA and protein of endogenous Arf6 in PC-3 cells was not significantly affected by the transfection of negative control sequence. All the three si RNA sequences could inhibit the expression of Arf6. The inhibitory rates of Arf6m RNA and protein on Arf6 expression in PC-3 cells were (91.88 鹵3.13)% and (86.37 鹵0.57)%, respectively. Si RNA-3 interfered with Arf6 expression inhibited the proliferation of PC-3 cells, and the migration distance and the number of invasive cells of PC-3 cells in vitro were significantly decreased compared with the blank and negative control groups (P0.05). Western blot analysis showed that the expression of p-ERK1/2 and Rac1 in PC-3 cells transfected with si RNA-3 was significantly decreased, while the expression of AKT,p-AKT and ERK1/2 was not significantly different from that of the control group. Conclusion si RNA interference with Arf6 expression can significantly inhibit the proliferation, migration and invasion of PC-3 cells, and its molecular mechanism may be related to the down-regulation of p-ERK1/2 and Rac1 expression.
【作者單位】： 南方醫(yī)科大學(xué)南方醫(yī)院泌尿外科;南方醫(yī)科大學(xué)藥學(xué)院;
【基金】：Supported by Natural Science Foundation of Guangdong Province(S2013010014537) by Science and Technology Planning Project of Guangdong Province(2012B031800263;2014A010107012;2014A020212260)~~
【分類(lèi)號(hào)】：R737.25

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