论文部分内容阅读
目的研究RNA干扰沉默人乳腺癌细胞VPAC1受体对VIP跨位激活EGFR/HER2和VEGF的分泌的影响。方法使用小干扰RNA(siRNA)探讨血管活性肠肽(VIP)对人乳腺癌细胞的作用机制。向雌激素依赖的(T47D)和雌激素非依赖的(MDA-MB-468)乳腺癌细胞转染VPAC1受体的siRNA完全消除VIP刺激分泌促进的血管生成因子(血管内皮生长因子,VEGF)的作用,以及跨位激活表皮生长因子受体(EGFR/HER2)。结果 VPAC1受体阻断导致T47D细胞增殖率下降至10%,MDA-MB-468细胞增殖下降至25%;采用ELISA试剂盒测定VPAC1受体沉默对乳腺癌细胞胞外血管内皮生长因子VEGF165分泌的影响,无论有无VIP诱导时,VEGF165分泌均被完全抑制(P<0.05);VIP可以导致HER2/EGFR的跨位激活造成磷酸化,沉默VPAC1受体后,其磷酸化消失(P<0.001);采用酪氨酸激酶抑制剂AG-825和AG-1478(10μmol/L)可以抑制VIP诱导的VEGF分泌活性,敲除VPAC1受体也完全阻断了VIP诱导的VEGF165分泌。结论实验结果阐明了人乳腺癌细胞中VIP-VPAC1受体信号通路机制,同时揭示了采用VPAC1受体拮抗剂联合靶向治疗乳腺癌的潜在用途及采用RNA干扰VPAC1受体分子疗法的潜在意义。
Objective To investigate the effect of RNA interference (RNAi) silencing VPAC1 receptor on human breast cancer cells on transactivation of VIP by activating EGFR/HER2 and VEGF secretion. Methods Small interfering RNA (siRNA) was used to investigate the mechanism of action of vasoactive intestinal peptide (VIP) on human breast cancer cells. siRNA transfection of VPAC1 receptors to estrogen-dependent (T47D) and estrogen-independent (MDA-MB-468) breast cancer cells completely abolished VIP-stimulated secretion-promoted angiogenic factors (vascular endothelial growth factor, VEGF) Role, as well as transposition activation of epidermal growth factor receptor (EGFR/HER2). Results VPAC1 receptor blockade resulted in the decrease of the proliferation rate of T47D cells to 10%, and the proliferation of MDA-MB-468 cells decreased to 25%; ELISA kit was used to determine the secretion of VEGFR1 receptor-expressed extracellular vascular endothelial growth factor VEGF165 in breast cancer cells. The effect of VEGF165 secretion was completely inhibited with or without VIP induction (P<0.05); VIP could lead to phosphorylation of HER2/EGFR transposition activation and phosphorylation of VPAC1 receptor after silencing (P<0.001). The use of tyrosine kinase inhibitors AG-825 and AG-1478 (10 μmol/L) can inhibit VIP-induced VEGF secretion activity, knocking out VPAC1 receptor also completely blocks VIP-induced VEGF165 secretion. Conclusion The experimental results elucidate the mechanism of VIP-VPAC1 receptor signaling pathway in human breast cancer cells, and reveal the potential use of VPAC1 receptor antagonists in combination with targeted therapy for breast cancer and the potential significance of RNA interference VPAC1 receptor molecular therapy.