癌基因RhoA对乳腺癌细胞血管内皮生长因子表达的调控机制研究

Regulatory mechanism of oncogene Rho A on the expression of vascular epithelial growth factor in breast cancer cells

目的探讨乳腺癌细胞MDA-MB-231中癌基因Rho A对VEGF蛋白表达和胞外分泌的影响及可能的分子机制。方法将Rho A过表达质粒pc DNA3.0-V14Rho A、对照质粒pc DNA3.0、Rho A沉默质粒pc DNA3.0-shRho A转染到MDA-MB-231细胞中,通过Western blot和ELISA实验分别检测细胞内外VEGF蛋白的表达,通过Western blot和实时PCR方法分别检测Rho A对p53表达的影响及对VEGF的调控作用。均数比较用t检验;计量资料用x珋±s表示,采用方差分析。结果 MDA-MB-231细胞中上调Rho A表达后,胞内VEGF的蛋白表达水平增加;胞外分泌水平显著增加,与对照组相比差异具有统计学意义(F=4.020,P=0.032);MDA-MB-231细胞中沉默Rho A表达后,胞内VEGF的蛋白表达水平降低;胞外分泌水平显著降低,与对照组相比差异具有统计学意义(F=5.131,P=0.001);并且与0 h相比,在MDA-MB-231细胞中上调Rho A可以抑制p53的表达(48 h:F=3.231,P=0.043),而p53表达的降低可以增加VEGF的表达水平(48 h:F=3.226,P=0.015),均差异具有统计学意义。结论乳腺癌细胞MDA-MB-231中Rho A表达的变化可以引起胞内外VEGF水平的变化,并且Rho A可能是通过抑制p53的表达从而增加VEGF表达。

Objective To explore the effects of oncogene RhoA on protein expression of vascular epithelial growth factor（VEGF） and exocytosis in breast cancer cell line MDA-MB-231 and its potential molecular mechanism. Methods RhoA-overexpressed plasmid pcDNA3.0-V14RhoA, control plasmid peDNA3.0 and RhoA-silencing plasmid pcDNA3.0-shRhoA were transfected into MDA-MB-231 cells. After that, VEGF protein expression was detected by Western blot and ELISA assay. The effects of RhoA on p53 expression and on VEGF regulation were examined by Western blot or real-time PCR. The means were compared using t text, and the measurement data were expressed as x^- ± s and processed using analysis of variance. Results After up-regulating RhoA in MDA-MB-231 cells, intracellular VEGF protein level and extracellular secretion level were significantly increased compared with the control group （F = 4. 020, P = 0.032）, while after down-regulating RhoA in MDA-MB-231 cells, intracellular VEGF protein level and extracellular secretion level were significantly decreased compared with the control group （F=5. 131, P = 0. 001 ）. Compared with the control group, the upregulation of RhoA could inhibit p53 expression （ 48 h ： F = 3. 231 ,P=0. 043）and the decrease of p53 expression could promote VEGF expression（48 h：F=3. 226,P= 0. 015）. Conclusion In breast cancer MDA-MB-231 cells, the change of RhoA expression can cause the change of intracellular and extracellular VEGF expression, and RhoA may promote VEGF expression through inhibiting p53 expression.