硒化合物诱导高危型HPV亚型宫颈癌细胞凋亡

Selenium compound induces apoptosis of cervical carcinoma cells of high risk HPV subtypes

目的探讨二氧化硒(SeO_2)对高危型HPV亚型宫颈癌细胞凋亡的诱导作用及其分子途径。方法不同浓度SeO_2分别作用于高危型HPV亚型宫颈癌细胞He La(HPV18+)和Caski(HPV16+)24 h,光学显微镜下观察细胞形态;四甲基偶氮唑蓝(MTT)比色法检测细胞增殖及活力;流式细胞计量术(FCM)检测细胞凋亡率;Western blot测定细胞内caspase-3和p53蛋白表达;Stem-loop实时定量PCR检测凋亡相关miRNA LET-7a表达。结果 SeO_2作用后宫颈癌细胞变圆、皱缩;SeO_2呈量效依赖关系抑制宫颈癌细胞增殖,其中He La细胞在7.5~30μmol/L组抑制作用显著;Caski细胞在SeO_2低浓度组即有显著抑制作用(P<0.05)。宫颈癌细胞凋亡率亦随SeO_2浓度升高而升高,在Caski细胞上升更加明显。SeO_2可明显上调宫颈癌细胞系中caspase-3与p53蛋白水平,在He La细胞中二者均于7.5μmol/L处达到峰值。Caski细胞从7.5μmol/L组开始凋亡蛋白表达量显著性升高(P<0.05)。SeO_2还可显著上调两细胞系实验组细胞LET-7a表达水平,且均在7.5μmol/L处出现峰值。结论 SeO_2通过上调高危型HPV亚型宫颈癌细胞中凋亡相关蛋白p53蛋白及miRNA LET-7a的表达,经caspase-3途径诱导细胞凋亡。对于SeO_2诱导宫颈癌细胞凋亡,HPV16+型较HPV18+型宫颈癌细胞更为敏感。

Objective To investigate the inducing effects and related pathways of selenium dioxide（ SeO_2） on the apoptosis in 2 human cervical carcinoma cell lines of high risk HPV subtypes. Methods He La（ HPV-18-positive）and Caski（ HPV-16-positive） cells were incubated with different concentrations of SeO_2 for 24 h respectively. Morphological changes of He La and Caski cells were observed under inverted optical microscope; cell proliferation and activity were examined by MTT assay; flow cytometry was employed to detect the cell apoptosis; the expressions of apoptosis-related proteins caspase-3 and p53 in cervical carcinoma cell lines were determined by Western blot analysis; the effects of SeO_2 on apoptosis-related miRNA LET-7 a expression was detected by stem-loop reverse transcription-polymerase chain reaction（ RT-PCR）. Results Cell morphology was obviously changed in vitro. Cells became rounded and shrunken. SeO_2 markedly inhibited cell proliferation and viability in a dose-dependent mannerin both cell lines; In He La cells the inhibitory effects induced by 7. 5-30 μmol/L of SeO_2 were significant（ P〈0. 05）; The inhibitory effects on Caski were statistical significant（ P0. 05） even with low concentrations of SeO_2.The apoptosis induced by SeO_2 increased dose-dependently in cervical carcinoma cell lines,which were higher in Caski. SeO_2 up-regulated the apoptosis-related proteins in cervical carcinoma cell lines. The expressions of caspase-3 and p53 in He La cells both significantly increased as compared with control group（ P0. 05）,and peaked at the concentration of 7. 5 μmol/L. Treated with higher concentrations（ 7. 5-30 μmol/L） of SeO_2,the expression on Caski cells increased significantly（ P0. 05）. SeO_2 induced of expression of apoptosis-related miRNA LET-7 a both in He La cells and Caski cells with statistical meanings（ P0. 05）; the effects reached its peak at the concentration of 7. 5 μmol/L bothly. Conclusions SeO_2 shows anti-tumor properties via apoptosis pathway by up-regulating the expressions of apoptosis-related proteins caspase-3,the mechanisms of can be potentially explained by p53 and LET-7 a in cervical cancer cell lines. The apoptosis-inducing effect of SeO_2 is more sensitive in HPV16＋cell line compared with HPV18＋cell line.