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目的探讨RNA干扰(RNA interference,RNAi)技术沉默AnnexinⅡ基因表达对人乳腺癌细胞MDA-MB-435S生物学特性的影响。方法体外化学合成AnnexinⅡ序列特异性双链小干扰RNA(siRNA),脂质体介导转染人乳腺癌细胞株MDA-MB-435S,采用荧光显微镜观察转染效率。收集siRNA干扰的细胞,RT-PCR、蛋白印迹和免疫细胞化学法分别检测AnnexinⅡmRNA和蛋白的抑制水平,并采用MTT法、流式细胞术测定细胞增殖能力和细胞周期的变化,Millicell小室测定细胞侵袭、迁移能力的变化。结果AnnexinⅡ-siRNA能有效抑制AnnexinⅡmRNA和蛋白的表达(P<0.05);经siRNA干扰后的细胞,细胞增殖能力显著减低(P<0.05);G0/G1期细胞明显增加,S期细胞明显减少(P<0.05);细胞侵袭、迁移能力明显降低(P<0.05)。结论采用siRNA干扰AnnexinⅡmRNA和蛋白表达后,能有效逆转乳腺癌细胞的某些恶性生物学特点,表明AnnexinⅡ与乳腺癌的发生、发展及转移有一定的关系。
Objective To investigate the effect of RNA interference (RNAi) technology on silencing AnnexinⅡ gene expression in human breast cancer cell line MDA-MB-435S. Methods Annexin Ⅱ sequence-specific double-stranded small interfering RNA (siRNA) was chemically synthesized in vitro and transfected into human breast cancer cell line MDA-MB-435S by liposome. The transfection efficiency was observed by fluorescence microscopy. The interference of siRNA was detected by RT-PCR, Western blot and immunocytochemistry respectively. The inhibitory effect of AnnexinⅡmRNA and protein was detected by MTT method. Cell proliferation and cell cycle were determined by flow cytometry. , Changes in migration ability. Results AnnexinⅡ-siRNA could effectively inhibit the expression of AnnexinⅡmRNA and protein (P <0.05). The proliferation of cells after siRNA interference was significantly reduced (P <0.05); the cells in G0 / G1 phase were significantly increased and the cells in S phase were significantly decreased P <0.05). The ability of cell invasion and migration was significantly decreased (P <0.05). CONCLUSIONS: siRNA targeting AnnexinⅡmRNA and protein expression can effectively reverse some malignant biological features of breast cancer cells, indicating that AnnexinⅡis closely related to the occurrence, development and metastasis of breast cancer.