人口腔鳞状细胞癌SCC9细胞miR-146a-5p表达及对细胞增殖和凋亡的影响

Expression of miR-146a-5p in human oral squamous cell carcinoma SCC9 cells and its influence on the cell proliferation and apoptosis

目的探讨miR-146a-5p在人口腔鳞状细胞癌SCC9细胞系中的表达及对SCC9细胞增殖、凋亡的影响。方法取对数生长期人口腔鳞状细胞癌SCC9细胞和人正常口腔角质HOK细胞,采用实时荧光定量PCR法检测miR-146a-5p mRNA、肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor associated factor 6, TRAF6)mRNA相对表达量。取对数生长期人口腔鳞状细胞癌SCC9细胞,随机分为观察组(转染miR-146a-5p mimics)和空白对照组(转染等量ddH2O),转染24、48 h,采用CCK-8法检测2组细胞增殖,FITC-PI荧光双染法检测细胞凋亡;转染24 h,采用实时荧光定量PCR法检测PI3K mRNA、Akt mRNA、mTOR mRNA相对表达量,Western blot法检测PI3K、Akt、mTOR蛋白相对表达量。结果 SCC9细胞miR-146a-5p mRNA相对表达量(8.33±0.58)高于HOK细胞(1.04±0.01),TRAF6 mRNA相对表达量(0.05±0.01)低于HOK细胞(1.05±0.01)(P<0.05)。转染24、48 h,观察组SCC9细胞增殖吸光度值(1.65±0.02、1.96±0.04)高于空白对照组(1.33±0.01、1.57±0.02)(P<0.05),SCC9细胞凋亡率[(0.89±0.01)%、(1.23±0.01)%]低于空白对照组[(2.77±0.01)%、(4.23±0.01)%](P<0.05)。转染24 h,观察组Scc细胞PI3K mRNA、Akt mRNA、mTOR mRNA相对表达量(2.98±0.05、8.20±0.06、5.34±0.03)高于空白对照组(1.05±0.01、1.03±0.01、1.07±0.01),PI3K、Akt、mTOR蛋白相对表达量(2.32±0.01、10.23±0.02、5.25±0.01)高于空白对照组(0.62±0.01、4.58±0.01、0.09±0.01)(P<0.05)。结论 SCC9细胞miR-146a-5p表达上调,TRAF6表达下调;miR-146a-5p可促进SCC9细胞增殖,抑制其凋亡,其机制可能是增强PI3K/Akt/mTOR信号通路表达。

Objective To investigate the expression of miR-146 a-5 p in human oral squamous cell carcinoma SCC9 cell line and its influence on the proliferation and apoptosis of SCC9 cells. Methods The human oral squamous carcinoma SCC9 cell line and human normal oral keratinocytes(HOK) in logarithmic growth phase were detected the relative expressions of miR-146 a-5 p and tumor necrosis factor receptor-associated factor 6(TRAF6) mRNAs by real-time fluorescence quantitative PCR. The SCC9 cells in logarithmic growth phase were randomly divided into highly-expressed miR-146 a-5 p group(transfected with miR-146 a-5 p mimics) and blank control group(transfected with ddH2O). The cell proliferation was detected by CCK-8 method, and the apoptosis was detected by FITC-PI fluorescent double staining method after 24 and 48 h of transfection;the relative expressions of PI3 K, Akt and mTOR mRNAs were detected by real-time fluorescence quantitative PCR, and the relative expressions of PI3 K, Akt and mTOR proteins were detected by Western blot after 24 h of transfection. Results The relative expression of miR-146 a-5 p mRNA was higher in SCC9 cells(8.33±0.58) than that in HOK(1.04±0.01), while the relative expression of TRAF6 mRNA was lower in SCC9 cells(0.05±0.01) than that in HOK(1.05±0.01)(P<0.05). After 24 and 48 h of transfection, the optical densities were higher in observation group(1.65±0.02, 1.96±0.04) than those in blank control group(1.33±0.01, 1.57±0.02)(P<0.05), and the cell apoptotic rates of SCC9 cells were lower in observation group((0.89±0.01)%,(1.23%±0.01)%) than those in blank control group((2.77±0.01)%,(4.23±0.01)%)(P<0.05). After 24 h of transfection, the relative expressions of PI3 K, Akt and mTOR mRNAs as well proteins were higher in observation group(2.98±0.05, 8.20±0.06,5.34±0.03;2.32±0.01,10.23±0.02,5.25±0.01)than those in blank control group(1.05±0.01,1.03±0.01,1.07±0.01;0.62±0.01,4.58±0.01,0.09±0.01)(P<0.05).Conclusion miR-146 a-5 p is up-regulated and TRAF6 is down-regulated in SCC9 cells.miR-146 a-5 p could promote SCC9 proliferation and inhibit its apoptosis probably by improving PI3 K-Akt-mTOR signaling pathway.