目的

探讨乳腺癌细胞MDA-MB-231 中癌基因RhoA 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RhoA 过表达质粒pcDNA3.0-V14RhoA、对照质粒pcDNA3.0、RhoA 沉默质粒pcDNA3.0-shRhoA 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RhoA 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 表示,采用方差分析。

结果

MDA-MB-231 细胞中上调RhoA 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RhoA 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RhoA 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RhoA 表达的变化可以引起胞内外VEGF 水平的变化,并且RhoA 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 pcDNA3.0 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 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.