siRNA抑制MSK1表达对人鼻咽癌CNE2细胞增殖的影响及其机制

发布时间：2018-05-11 18:05

本文选题：鼻咽癌 + 丝裂原和应激激活的蛋白激酶　；参考：《医学研究生学报》2017年04期

【摘要】：目的丝裂原和应激激活的蛋白激酶1(MSK1)的异常活化在多种肿瘤的发生中起着重要作用。采用小干扰RNA(siRNA)抑制MSK1的表达,观察其对人鼻咽癌细胞CNE2增殖的影响,并探讨其可能机制。方法构建靶向MSK1的siRNA真核表达质粒,转染CNE2细胞并筛选获得稳定表达细胞株。将细胞分为空白对照组、阴性对照组、实验组。空白对照组:不转染质粒的CNE2细胞;阴性对照组:稳定转染阴性对照质粒的CNE2细胞(si-mock);实验组:稳定转染MSK1 siRNA质粒的CNE2细胞(si-MSK1)。实时荧光定量PCR和Western blot检测细胞中MSK1 mRNA和蛋白表达的改变;CCK-8和平板克隆形成实验检测细胞增殖能力的改变;流式细胞术检测细胞周期的改变;Western blot检测组蛋白H3Ser10磷酸化水平的改变;双萤光素酶报告系统和Western blot检测c-jun转录活性和蛋白表达的改变。结果与空白对照组相比,实验组在48、72和96 h的细胞增殖抑制率明显增高(P0.05);与阴性对照组相比,实验组细胞在24 h后生长速度明显减慢,其48、72和96h的细胞增殖抑制率均明显增高(P0.05)。与空白对照组比较,实验组细胞的克隆形成能力明显降低(P0.01);与阴性对照组相比,实验组细胞克隆形成能力的降低,其克隆形成数目明显减少[(221.00±20.08)个/300个细胞vs(99.67±15.57)个/300个细胞,P0.01]。与阴性对照组相比,实验组细胞周期发生明显改变,G0/G1期细胞比例增加,而S期细胞比例明显减少(P0.01)。与空白对照组和阴性对照组相比,实验组能显著降低组蛋白H3Ser10磷酸化水平(P0.01),而总组蛋白H3的表达则无明显改变。与空白对照组相比,实验组c-jun蛋白表达量和转录活性明显下降(P0.05);与阴性对照组相比,实验组CNE2细胞c-jun转录活性明显降低[(100.00±0.00)%vs(48.77±10.71)%,P0.05]。结论采用siRNA干扰MSK1表达可有效抑制人鼻咽癌细胞的生长、增殖,其机制可能与抑制组蛋白H3 Ser10磷酸化,进而下调c-jun转录活性有关。
[Abstract]:Objective the abnormal activation of mitogen and stress-activated protein kinase 1 (MSK1) plays an important role in the carcinogenesis of various tumors. Small interfering RNAs siRNAs were used to inhibit the expression of MSK1, to observe its effect on the proliferation of human nasopharyngeal carcinoma cell line CNE2, and to explore its possible mechanism. Methods siRNA eukaryotic expression plasmid targeting MSK1 was constructed and transfected into CNE2 cells and stable expression cell lines were obtained. The cells were divided into blank control group, negative control group and experimental group. Blank control group: untransfected CNE2 cells; negative control group: CNE2 cells stably transfected with negative control plasmids; experimental group: CNE2 cells stably transfected with MSK1 siRNA plasmids. The changes of MSK1 mRNA and protein expression in cells were detected by real-time fluorescence quantitative PCR and Western blot. The changes of cell proliferation were detected by CCK-8 and plate clone formation assay. The changes of cell cycle were detected by flow cytometry, the phosphorylation of histone H3Ser10 was detected by Western blot, the transcriptional activity and protein expression of c-jun were detected by double luciferase reporting system and Western blot. Results compared with the blank control group, the cell proliferation inhibition rate of the experimental group was significantly higher than that of the control group at 48, 72 and 96 hours, and the growth rate of the experimental group was significantly slower than that of the negative control group after 24 hours, and the cell proliferation inhibition rate of the experimental group at 48, 72 and 96 hours was significantly higher than that of the negative control group. Compared with the blank control group, the clone forming ability of the experimental group was significantly lower than that of the negative control group, and that of the experimental group was significantly lower than that of the negative control group [221.00 卤20.08 / 300 vs(99.67 卤15.57 / 300cell P 0.01]. Compared with the negative control group, the cell cycle in the experimental group increased significantly in G _ 0 / G _ 1 phase, while the cell proportion in S phase decreased significantly (P _ (0.01). Compared with the blank control group and the negative control group, the phosphorylation level of histone H3Ser10 was significantly decreased in the experimental group, but the expression of total histone H3 was not changed. Compared with the blank control group, the expression and transcription activity of c-jun protein in the experimental group decreased significantly (P 0.05), and the c-jun transcription activity of the CNE2 cells in the experimental group was significantly lower than that in the negative control group [100.00 卤0.00)%vs(48.77 卤10.71 P 0.05]. Conclusion siRNA interference with MSK1 expression can effectively inhibit the growth and proliferation of human nasopharyngeal carcinoma cells. The mechanism may be related to the inhibition of histone H3 Ser10 phosphorylation and down-regulation of c-jun transcription activity.
【作者单位】：广东医科大学广东省医学分子诊断重点实验室;广东医科大学病理生理学教研室;
【基金】：国家自然科学基金(81502411) 广东省科技计划项目(2014A020212560)
【分类号】：R739.63

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