抑癌基因WWOX促进卵巢癌干细胞的凋亡及机制

发布时间：2018-08-31 10:37

【摘要】：背景:研究表明,WWOX能够影响卵巢癌干细胞的生长,其作用机制是否与Hedgehog信号通路有关则未见相关报道。目的:研究过表达WWOX对卵巢癌干细胞凋亡的作用及机制。方法:转染WWOX过表达载体pcDNA3.1-WWOX(pcDNA3.1-WWOX组)和pcDNA4.0-WWOX(pcDNA4.0-WWOX组)至卵巢癌干细胞中,同时转染空载体pcDNA3.1(pcDNA3.1组)和pcDNA4.0(pcDNA4.0组),设置未转染组(只加入转染试剂Lipofectamine2000)。培养48 h后,Western blot检测细胞中WWOX水平,MTT检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Western blot检测细胞中Hedgehog信号通路相关蛋白SHH、PTCH1、Gli-1、SMO和凋亡相关蛋白Cleaved Caspase-3的表达水平。用Hedgehog信号通路抑制剂环巴胺作用于未转染的卵巢癌干细胞(环巴胺组)和转染WWOX过表达载体的卵巢癌干细胞(WWOX+环巴胺组),培养48 h后,再进行MTT、流式细胞术、Western blot检测。结果与结论:①pcDNA4.0-WWOX组细胞中WWOX表达水平明显高于pcDNA3.1-WWOX组(t=27.84,P=0.00)。后期选用转染pcDNA4.0-WWOX的卵巢癌干细胞过表达WWOX;②过表达WWOX能够抑制卵巢癌干细胞增殖,促进卵巢癌干细胞凋亡;③过表达WWOX能够抑制卵巢癌干细胞中SHH、PTCH1、Gli-1、SMO表达,促进Cleaved Caspase-3表达;④环巴胺能够抑制SHH、PTCH1、Gli-1、SMO表达,促进Cleaved Caspase-3表达。环巴胺对Hedgehog信号通路具有明显的抑制作用;⑤环巴胺能够增强过表达WWOX诱导的卵巢癌干细胞凋亡,增强过表达WWOX对卵巢癌干细胞增殖的抑制作用;⑥结果表明,WWOX对卵巢癌干细胞的增殖抑制作用和促凋亡作用可能与抑制Hedgehog信号通路有关。
[Abstract]:Background: studies have shown that WWOX can affect the growth of ovarian cancer stem cells and whether its mechanism is related to the Hedgehog signaling pathway has not been reported. Objective: to study the effect and mechanism of overexpression of WWOX on apoptosis of ovarian cancer stem cells. Methods: WWOX overexpression vectors pcDNA3.1-WWOX (pcDNA3.1-WWOX group) and pcDNA4.0-WWOX (pcDNA4.0-WWOX group) were transfected into ovarian cancer stem cells, and the empty vectors pcDNA3.1 (pcDNA3.1 group) and pcDNA4.0 (pcDNA4.0 group) were transfected. After 48 h culture, the level of WWOX in cells was detected by Western blot, and the cell proliferation was detected by flow cytometry. The expression of Hedgehog signal transduction related protein SHH,PTCH1,Gli-1,SMO and apoptosis-related protein Cleaved Caspase-3 was detected by flow cytometry and Western blot was used to detect the expression of Hedgehog signal pathway related protein SHH,PTCH1,Gli-1,SMO and apoptosis-related protein Cleaved Caspase-3. Cyclobaramine, a signal pathway inhibitor of Hedgehog, was used to treat the untransfected ovarian cancer stem cells (cyclophosphamide group) and ovarian cancer stem cells transfected with WWOX overexpression vector (WWOX cyclophosphamide group). After 48 hours of culture, MTT, flow cytometry was used to detect the results of MTT, blot. Results and conclusion the expression of WWOX in the cells of the pcDNA4.0-WWOX group was significantly higher than that in the pcDNA3.1-WWOX group. The over-expression of WWOX;2 and WWOX in ovarian cancer stem cells transfected with pcDNA4.0-WWOX in later stage could inhibit the proliferation of ovarian cancer stem cells, and promote the apoptosis of ovarian cancer stem cells. The overexpression of WWOX could inhibit the expression of SHH,PTCH1,Gli-1,SMO and promote the expression of Cleaved Caspase-3 in ovarian cancer stem cells. 4 Cyclobaramine could inhibit the expression of SHH,PTCH1,Gli-1,SMO and promote the expression of Cleaved Caspase-3. Cyclobaramine has a significant inhibitory effect on Hedgehog signaling pathway. Cyclobaramine can enhance apoptosis of ovarian cancer stem cells induced by overexpression of WWOX and inhibit proliferation of ovarian cancer stem cells induced by overexpression of WWOX. The results suggested that the inhibitory effect of WWOX on the proliferation and apoptosis of ovarian cancer stem cells may be related to the inhibition of Hedgehog signaling pathway.
【作者单位】：山东大学临床医学院;山东大学齐鲁医院妇产科;滨州医学院消化内科;
【分类号】：R737.31

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