Aim： To investigate the regulatory function of Cox7a2 on steroidogenesis and the mechanism involved in TM3 mouse Leydig cells. Methods： The cDNA of Cox7a2 was cloned from TM3 mouse Leydig cells. It was subcloned to ...Aim： To investigate the regulatory function of Cox7a2 on steroidogenesis and the mechanism involved in TM3 mouse Leydig cells. Methods： The cDNA of Cox7a2 was cloned from TM3 mouse Leydig cells. It was subcloned to pDsRed- Express-N 1 and transfected back into TM3 mouse Leydig cells for Cox7a2 overexpression by transient gene transfection. Steroidogenesis affected by overexpressed Cox7a2 was studied by ELISA. To elicit the mechanism of this effect, expression of steroidogenic acute regulatory （STAR） protein and reactive oxygen species （ROS） were examined by Western blot and fluorometer, respectively. Results： The cDNA of Cox7a2 （249 bp） was cloned from Leydig cells and confirmed by DNA sequencing. After constructed pDsRed-Express-N1-Cox7a2 was transfected back into TM3 mouse Leydig cells, Cox7a2 inhibited not only luteinizing hormone （LH）-induced secretion of testosterone but also the expression of StAR protein. At the same time, Cox7a2 increased the activity of ROS in TM3 mouse Leydig cells. Conclusion： Cox7a2 inhibited LH-induced StAR protein expression, and consequent testosterone production, at least in part, by increasing ROS activity in TM3 mouse Leydig cells.展开更多
Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell l...Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was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
文摘Aim： To investigate the regulatory function of Cox7a2 on steroidogenesis and the mechanism involved in TM3 mouse Leydig cells. Methods： The cDNA of Cox7a2 was cloned from TM3 mouse Leydig cells. It was subcloned to pDsRed- Express-N 1 and transfected back into TM3 mouse Leydig cells for Cox7a2 overexpression by transient gene transfection. Steroidogenesis affected by overexpressed Cox7a2 was studied by ELISA. To elicit the mechanism of this effect, expression of steroidogenic acute regulatory （STAR） protein and reactive oxygen species （ROS） were examined by Western blot and fluorometer, respectively. Results： The cDNA of Cox7a2 （249 bp） was cloned from Leydig cells and confirmed by DNA sequencing. After constructed pDsRed-Express-N1-Cox7a2 was transfected back into TM3 mouse Leydig cells, Cox7a2 inhibited not only luteinizing hormone （LH）-induced secretion of testosterone but also the expression of StAR protein. At the same time, Cox7a2 increased the activity of ROS in TM3 mouse Leydig cells. Conclusion： Cox7a2 inhibited LH-induced StAR protein expression, and consequent testosterone production, at least in part, by increasing ROS activity in TM3 mouse Leydig cells.
文摘Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was
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。
