本文選題：鼻咽癌 + 絲裂原和應(yīng)激激活的蛋白激酶�。� 參考：《醫(yī)學(xué)研究生學(xué)報(bào)》2017年04期

【摘要】：目的絲裂原和應(yīng)激激活的蛋白激酶1(MSK1)的異�；罨诙喾N腫瘤的發(fā)生中起著重要作用。采用小干擾RNA(siRNA)抑制MSK1的表達(dá),觀察其對人鼻咽癌細(xì)胞CNE2增殖的影響,并探討其可能機(jī)制。方法構(gòu)建靶向MSK1的siRNA真核表達(dá)質(zhì)粒,轉(zhuǎn)染CNE2細(xì)胞并篩選獲得穩(wěn)定表達(dá)細(xì)胞株。將細(xì)胞分為空白對照組、陰性對照組、實(shí)驗(yàn)組�？瞻讓φ战M:不轉(zhuǎn)染質(zhì)粒的CNE2細(xì)胞;陰性對照組:穩(wěn)定轉(zhuǎn)染陰性對照質(zhì)粒的CNE2細(xì)胞(si-mock);實(shí)驗(yàn)組:穩(wěn)定轉(zhuǎn)染MSK1 siRNA質(zhì)粒的CNE2細(xì)胞(si-MSK1)。實(shí)時(shí)熒光定量PCR和Western blot檢測細(xì)胞中MSK1 mRNA和蛋白表達(dá)的改變;CCK-8和平板克隆形成實(shí)驗(yàn)檢測細(xì)胞增殖能力的改變;流式細(xì)胞術(shù)檢測細(xì)胞周期的改變;Western blot檢測組蛋白H3Ser10磷酸化水平的改變;雙螢光素酶報(bào)告系統(tǒng)和Western blot檢測c-jun轉(zhuǎn)錄活性和蛋白表達(dá)的改變。結(jié)果與空白對照組相比,實(shí)驗(yàn)組在48、72和96 h的細(xì)胞增殖抑制率明顯增高(P0.05);與陰性對照組相比,實(shí)驗(yàn)組細(xì)胞在24 h后生長速度明顯減慢,其48、72和96h的細(xì)胞增殖抑制率均明顯增高(P0.05)。與空白對照組比較,實(shí)驗(yàn)組細(xì)胞的克隆形成能力明顯降低(P0.01);與陰性對照組相比,實(shí)驗(yàn)組細(xì)胞克隆形成能力的降低,其克隆形成數(shù)目明顯減少[(221.00±20.08)個(gè)/300個(gè)細(xì)胞vs(99.67±15.57)個(gè)/300個(gè)細(xì)胞,P0.01]。與陰性對照組相比,實(shí)驗(yàn)組細(xì)胞周期發(fā)生明顯改變,G0/G1期細(xì)胞比例增加,而S期細(xì)胞比例明顯減少(P0.01)。與空白對照組和陰性對照組相比,實(shí)驗(yàn)組能顯著降低組蛋白H3Ser10磷酸化水平(P0.01),而總組蛋白H3的表達(dá)則無明顯改變。與空白對照組相比,實(shí)驗(yàn)組c-jun蛋白表達(dá)量和轉(zhuǎn)錄活性明顯下降(P0.05);與陰性對照組相比,實(shí)驗(yàn)組CNE2細(xì)胞c-jun轉(zhuǎn)錄活性明顯降低[(100.00±0.00)%vs(48.77±10.71)%,P0.05]。結(jié)論采用siRNA干擾MSK1表達(dá)可有效抑制人鼻咽癌細(xì)胞的生長、增殖,其機(jī)制可能與抑制組蛋白H3 Ser10磷酸化,進(jìn)而下調(diào)c-jun轉(zhuǎn)錄活性有關(guān)。
[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 鹵20.08 / 300 vs(99.67 鹵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 鹵0.00)%vs(48.77 鹵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.
【作者單位】： 廣東醫(yī)科大學(xué)廣東省醫(yī)學(xué)分子診斷重點(diǎn)實(shí)驗(yàn)室;廣東醫(yī)科大學(xué)病理生理學(xué)教研室;
【基金】：國家自然科學(xué)基金(81502411) 廣東省科技計(jì)劃項(xiàng)目(2014A020212560)
【分類號】：R739.63

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