AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS...AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.展开更多
目的:探讨膀胱癌细胞中细胞色素c氧化酶亚基7A2(COX7A2)基因的表达,及其与顺铂联用对膀胱癌J82细胞增殖、凋亡及线粒体功能影响。方法:采用生物信息学方法分析COX7A2在膀胱癌患者中的表达,并在J82细胞中进行验证。功能实验分为对照组(...目的:探讨膀胱癌细胞中细胞色素c氧化酶亚基7A2(COX7A2)基因的表达,及其与顺铂联用对膀胱癌J82细胞增殖、凋亡及线粒体功能影响。方法:采用生物信息学方法分析COX7A2在膀胱癌患者中的表达,并在J82细胞中进行验证。功能实验分为对照组(仅转染阴性对照siNC)、siRNA组(仅转染COX7A2的siRNA)、对照组+顺铂组(先转染阴性对照后用顺铂处理)和siRNA+顺铂组(先敲低COX7A2后用顺铂处理)。CCK-8、Transwell迁移能力测试和克隆增殖实验检测对照组和siRNA组中J82细胞的增殖、迁移能力。采用相应试剂盒检测各组细胞的ATP水平、活性氧(ROS)水平及线粒体膜电位(ΔΨm),以评估线粒体功能。流式细胞术检测各组细胞凋亡,以反映细胞的线粒体状态与对顺铂治疗的响应性关系。进一步通过癌症治疗响应基因标识数据库(CTR-DB),分析COX7A2与接受顺铂联合治疗的膀胱癌患者预后的关系。结果:生物信息学分析与生存曲线显示,COX7A2在膀胱癌患者中高表达并且与患者预后不良有关联。COX7A2在J82细胞中呈高表达(P<0.05)。在未经顺铂处理时,与对照组相比,siRNA组J82细胞增殖、迁移和克隆形成能力均显著下降(均P<0.001),而线粒体的ATP表达减少(P<0.01)、ROS表达量增多(P<0.0001)、MMP发生去极化(P<0.0001),凋亡水平增加(P<0.05);顺铂处理后,与对照组+顺铂相比,siRNA+顺铂组ATP表达减少(P<0.01)、ROS表达量增多(P<0.0001)、MMP发生去极化(P<0.0001),线粒体功能受损,凋亡水平增加(P<0.001)。CTR-DB数据库生信分析显示,5例接受顺铂+多柔比星+甲氨蝶呤+长春碱联合治疗的膀胱癌患者中,有应答者比无应答者COX7A2中位RNA表达量低(中位表达量:4501 vs 5009),12例铂类药物联合治疗的膀胱癌患者中有应答者比无应答者COX7A2中位RNA表达量低(中位表达量:2947 vs 3035),由于样本量有限,虽观察到趋势但无统计学意义。结论:敲低COX7A2可通过损伤线粒体功能,抑制膀胱癌细胞增殖与迁移,并可能由此增强细胞对顺铂诱导凋亡的敏感性。展开更多
基金Supported by National Natural Science Foundation of China(No.81600754)。
文摘AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.
文摘目的:探讨膀胱癌细胞中细胞色素c氧化酶亚基7A2(COX7A2)基因的表达,及其与顺铂联用对膀胱癌J82细胞增殖、凋亡及线粒体功能影响。方法:采用生物信息学方法分析COX7A2在膀胱癌患者中的表达,并在J82细胞中进行验证。功能实验分为对照组(仅转染阴性对照siNC)、siRNA组(仅转染COX7A2的siRNA)、对照组+顺铂组(先转染阴性对照后用顺铂处理)和siRNA+顺铂组(先敲低COX7A2后用顺铂处理)。CCK-8、Transwell迁移能力测试和克隆增殖实验检测对照组和siRNA组中J82细胞的增殖、迁移能力。采用相应试剂盒检测各组细胞的ATP水平、活性氧(ROS)水平及线粒体膜电位(ΔΨm),以评估线粒体功能。流式细胞术检测各组细胞凋亡,以反映细胞的线粒体状态与对顺铂治疗的响应性关系。进一步通过癌症治疗响应基因标识数据库(CTR-DB),分析COX7A2与接受顺铂联合治疗的膀胱癌患者预后的关系。结果:生物信息学分析与生存曲线显示,COX7A2在膀胱癌患者中高表达并且与患者预后不良有关联。COX7A2在J82细胞中呈高表达(P<0.05)。在未经顺铂处理时,与对照组相比,siRNA组J82细胞增殖、迁移和克隆形成能力均显著下降(均P<0.001),而线粒体的ATP表达减少(P<0.01)、ROS表达量增多(P<0.0001)、MMP发生去极化(P<0.0001),凋亡水平增加(P<0.05);顺铂处理后,与对照组+顺铂相比,siRNA+顺铂组ATP表达减少(P<0.01)、ROS表达量增多(P<0.0001)、MMP发生去极化(P<0.0001),线粒体功能受损,凋亡水平增加(P<0.001)。CTR-DB数据库生信分析显示,5例接受顺铂+多柔比星+甲氨蝶呤+长春碱联合治疗的膀胱癌患者中,有应答者比无应答者COX7A2中位RNA表达量低(中位表达量:4501 vs 5009),12例铂类药物联合治疗的膀胱癌患者中有应答者比无应答者COX7A2中位RNA表达量低(中位表达量:2947 vs 3035),由于样本量有限,虽观察到趋势但无统计学意义。结论:敲低COX7A2可通过损伤线粒体功能,抑制膀胱癌细胞增殖与迁移,并可能由此增强细胞对顺铂诱导凋亡的敏感性。
