Pro-apoptotic effect of a tumor suppressor gene WWOX on ovarian cancer stem cells and its mechanism

doi:10.3969/j.issn.2095-4344.2017.29.012

Abstract

Abstract:

BACKGROUND: WWOX, a tumor suppressor gene, can affect the growth of ovarian cancer stem cells; however, there is no report on whether its mechanism of action is related to Hedgehog signaling pathway.
OBJECTIVE: To investigate the effect and mechanism of overexpression of WWOX on the apoptosis of ovarian cancer stem cells.
METHODS: pcDNA3.1-WWOX (pcDNA3.1-WWOX group) and pcDNA4.0-WWOX (pcDNA4.0-WWOX group) were transferred into ovarian cancer stem cells, respectively; and meanwhile, pcDNA3.1 (pcDNA3.1 group) and pcDNA4.0 (pcDNA4.0 group) were transferred into the cells. A non-transfection group (only with Lipofectamine2000) was set up. After cultured 48 hours, the levels of WWOX in the pcDNA3.1-WWOX group and pcDNA4.0-WWOX group were detected using western blot assay, and the cell proliferation and apoptosis were detected using MTT assay and flow cytometry, respectively. Western blot assay was also used to detect the levels of Hedgehog signaling pathway associated proteins, SHH, PTCH1, Gli-1, SMO and apoptosis-related protein Cleaved Caspase-3 in the cells. Cyclopamine, Hedgehog signaling pathway inhibitor, was used in ovarian cancer stem cells without transfection (cyclopamine group) and after the transfection of WWOX overexpression vector (WWOX+cyclopamine group) followed by 48 hours of culture, and then MTT, flow cytometry and western blot detections were performed.
RESULTS AND CONCLUSION: (1) The expression level of WWOX in the pcDNA4.0-WWOX group was significantly higher than that in the pcDNA3.1-WWOX group (t=27.84, P=0.00). The ovarian cancer stem cells which were transfected with pcDNA4.0-WWOX were used to overexpress WWOX in the late experiment. (2) Overexpression of WWOX could inhibit the proliferation of ovarian cancer stem cells and promote the apoptosis of ovarian cancer stem cells. (3) Overexpression of WWOX could inhibit the expression of Gli-1, PTCH1, SMO and SHH in ovarian cancer stem cells, and promote the expression of Cleaved Caspase-3. (4) Cyclopamine could inhibit the expression of SHH, PTCH1, Gli-1, SMO, and promote the expression of Cleaved Caspase-3. Cyclopamine had obvious inhibitory effect on Hedgehog signaling pathway. (5) Cyclopamine could enhance the apoptosis induced by overexpression of WWOX in ovarian cancer stem cells , and enhance the inhibition of proliferation of ovarian cancer stem cells induced by overexpression of WWOX. To conclude, WWOX effects on proliferation and apoptosis of ovarian cancer stem cells may be related to the inhibition of Hedgehog signaling pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neoplastic Stem Cells, Ovarian Neoplasms, Hedgehog Proteins, Apoptosis, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Wang Yi-yi, Zhang Xiang-ning, Dong Hao, Gao Lei, Cui Xiao-rong, Wang Chun-yan, Ma Rui-lin. Pro-apoptotic effect of a tumor suppressor gene WWOX on ovarian cancer stem cells and its mechanism[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(29): 4660-4665.

Figures/Tables 2

2.1 转染后细胞中WWOX表达水平 pcDNA3.1-WWOX组、pcDNA4.0-WWOX组细胞中WWOX表达水平均明显高于未转染组。pcDNA3.1组、pcDNA4.0组细胞中WWOX表达水平与未转染组相比差异无显著性意义。pcDNA4.0-WWOX组细胞中WWOX表达水平明显高于pcDNA3.1-WWOX组，差异有显著性意义，见图1。后期选用转染pcDNA4.0-WWOX的卵巢癌干细胞继续研究。 2.2 WWOX对细胞增殖凋亡的影响转染pcDNA4.0-WWOX的卵巢癌干细胞存活率与未转染组相比明显下降，而凋亡率明显升高，差异均有显著性意义(t=18.74，P=0.00；t=21.56，P=0.00)。过表达WWOX能够促进卵巢癌干细胞凋亡，抑制卵巢癌干细胞增殖，见图2。 2.3 WWOX对卵巢癌干细胞中SHH、PTCH1、Gli-1、SMO、Cleaved Caspase-3水平的影响过表达WWOX的卵巢癌干细胞中SHH、PTCH1、Gli-1、SMO水平与未转染组相比均明显下降，而Cleaved Caspase-3水平明显升高。WWOX可能通过抑制Hedgehog信号通路促进卵巢癌干细胞凋亡，见图3。 2.4 Hedgehog信号通路抑制剂对细胞中SHH、PTCH1、Gli-1、SMO、Cleaved Caspase-3水平的影响与对照组相比，WWOX+环巴胺组细胞中SHH、PTCH1、Gli-1、SMO水平下降，Cleaved Caspase-3水平升高。与对照组相比，环巴胺组细胞中SHH、PTCH1、Gli-1、SMO表达水平明显下降，Cleaved Caspase-3水平明显升高。WWOX+环巴胺组细胞中SHH、PTCH1、Gli-1、SMO表达水平明显低于环巴胺组，Cleaved Caspase-3水平明显高于环巴胺组，见图4。环巴胺能够有效抑制卵巢癌干细胞中Hedgehog信号通路的激活，过表达WWOX和环巴胺对卵巢癌干细胞中Hedgehog信号通路的激活均具有抑制作用。 2.5 Hedgehog信号通路抑制剂对WWOX诱导的细胞增殖凋亡的影响环巴胺组和WWOX+环巴胺组细胞存活率下降，凋亡率增高。WWOX+环巴胺组细胞存活率明显低于环巴胺组，而凋亡率明显高于环巴胺组，见图5。环巴胺能够增强WWOX诱导的卵巢癌干细胞凋亡作用，增强WWOX诱导的卵巢癌干细胞增殖抑制作用。"

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