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目的探讨miR-101与宫颈癌干细胞对5-氟尿嘧啶敏感性的关系并研究其机制。方法流式细胞术分选Hela细胞系的肿瘤干细胞。荧光定量PCR检测Hela肿瘤干细胞中miR-101的表达水平。MTT法检测Hela肿瘤干细胞在5-氟尿嘧啶和miR-101处理下的细胞活力。Western blot试验检测5-氟尿嘧啶和miR-101对Hela肿瘤干细胞c-met表达水平,PI3K、AKT磷酸化水平及caspases活化水平的影响。流式细胞术检测Hela肿瘤干细胞在5-氟尿嘧啶和miR-101处理下的凋亡率。结果 Hela干细胞中miR-101的表达水平相较Hela非干细胞细胞显著下降。Hela干细胞转染miR-101后,其对5-氟尿嘧啶的敏感性显著上升。Western blot试验显示miR-101能显著降低Hela肿瘤干细胞的c-met蛋白表达水平,表明c-met是miR-101的靶点。Western blot流式细胞实验结果表明在Hela肿瘤干细胞中,miR-101通过下调c-met的表达抑制PI3K和AKT的磷酸化,从而提高Hela肿瘤干细胞对5-氟尿嘧啶依赖的凋亡信号的敏感性,促进caspase-9和caspase-3的活化。结论 miR-101通过下调c-met的表达提高宫颈癌干细胞对5-氟尿嘧啶的敏感性。
Objective To investigate the relationship between miR-101 and cervical cancer stem cell sensitivity to 5-fluorouracil and its mechanism. Methods Flow cytometry was used to screen tumor stem cells of Hela cell line. Fluorescent quantitative PCR was used to detect the expression of miR-101 in Hela tumor stem cells. Cell viability of Hela tumor stem cells treated with 5-Fluorouracil and miR-101 was detected by MTT assay. The effect of 5-fluorouracil and miR-101 on the expression of c-met, PI3K, AKT phosphorylation and activation of caspases in Hela tumor stem cells were detected by Western blot. Flow cytometry was used to detect the apoptosis rate of Hela tumor stem cells treated with 5-Fluorouracil and miR-101. Results The expression level of miR-101 in Hela stem cells was significantly lower than that in Hela non-stem cells. The sensitivity of Hela stem cells to 5-fluorouracil increased significantly after transfected with miR-101. Western blot showed that miR-101 significantly reduced c-met protein expression in Hela tumor stem cells, indicating that c-met is the target of miR-101. The results of Western blot showed that miR-101 could inhibit the phosphorylation of PI3K and AKT by down-regulating the expression of c-met in Hela tumor stem cells, and thus enhance the sensitivity of Hela tumor stem cells to 5-fluorouracil-dependent apoptotic signals, Promote the activation of caspase-9 and caspase-3. Conclusion miR-101 enhances the sensitivity of cervical cancer stem cells to 5-fluorouracil by down-regulating the expression of c-met.