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目的:通过RNA干扰乳腺癌MCF-7细胞组蛋白去乙酰化酶1(HDAC1)基因,观察对其细胞周期及细胞生长的影响。方法:将乳腺癌MCF-7细胞分成3个组:空白对照组(不予任何处理)、阴性对照组(予以阴性si RNA+脂质体)、si RNA组(HDAC1si RNA+脂质体)。人工合成针对HDAC1基因的小分子干扰RNA构建HDAC1-si RNA重组质粒,通过脂质体转入乳腺癌MCF-7细胞。用Real Time-PCR法和Western-blot法分别检测HDAC1基因的m RNA及蛋白表达情况;MTT法检测细胞增殖情况;流式细胞术检测细胞周期变化。结果:转染24h后人乳腺癌MCF-7细胞中HDAC1m RNA表达量在si RNA组为(0.15±0.02)、空白对照组为(1.00±0.03)、阴性对照组(0.83±0.04)。与空白对照组和阴性对照组相比,si RNA组细胞中HDAC1m RNA表达量下降明显(P<0.05)。Western blot显示HDAC1蛋白表达量亦明显显示:si RNA组表达量比空白和阴性对照组低(P<0.05)。MTT法检测显示3组细胞中,si RNA组的细胞生长速度较其他两组慢(P<0.05)。流式细胞术检测显示3组乳腺癌细胞细胞周期分布显示si RNA组细胞周期S期明显减少(P<0.05),G1期比例增加(P<0.05)。结论:通过RNA干扰能有效的抑制人乳腺癌MCF-7细胞HDAC1的表达,同时抑制细胞增殖、引起细胞周期停滞。
OBJECTIVE: To investigate the effect of HDAC1 gene on the cell cycle and cell growth of MCF-7 breast cancer cells by RNA interference. Methods: Breast cancer MCF-7 cells were divided into three groups: blank control group (without any treatment), negative control group (negative si RNA + liposomes) and si RNA group (HDAC1siRNA + liposomes). Synthetic HDAC1-si RNA recombinant plasmids were synthesized by lipofectamine into human breast cancer MCF-7 cells. The expression of m RNA and protein of HDAC1 gene were detected by Real Time-PCR and Western-blot respectively. The cell proliferation was detected by MTT assay. The cell cycle was detected by flow cytometry. Results: The expression of HDAC1mRNA in human breast cancer MCF-7 cells transfected for 24 h was (0.15 ± 0.02) in the siRNA group, (1.00 ± 0.03) in the blank control group and (0.83 ± 0.04) in the negative control group. Compared with the blank control group and negative control group, the expression of HDAC1mRNA in si RNA group decreased significantly (P <0.05). Western blot showed that the expression of HDAC1 protein was also significantly lower than that of the blank and negative control groups (P <0.05). MTT assay showed that the cell growth rate of si RNA group was slower than that of the other two groups (P <0.05). Flow Cytometry showed that the cell cycle distribution of the 3 groups of breast cancer cells showed that the cell cycle of si RNA group was significantly decreased (P <0.05), and the proportion of G1 phase was increased (P <0.05). Conclusion: The RNA interference can effectively inhibit the expression of HDAC1 in human breast cancer MCF-7 cells, inhibit cell proliferation and cause cell cycle arrest.