目的探讨抗增殖蛋白2(PHB2)脓毒症心肌损伤线粒体功能的调控机制。方法体外培养大鼠心肌细胞株(H9C2),分为对照组、脂多糖(LPS)组、LPS+PHB2 siRNA(si-PHB2)组。检测氧化应激指标细胞内丙二醛(MDA)水平、荧光探针检测细胞内活性氧(ROS)...目的探讨抗增殖蛋白2(PHB2)脓毒症心肌损伤线粒体功能的调控机制。方法体外培养大鼠心肌细胞株(H9C2),分为对照组、脂多糖(LPS)组、LPS+PHB2 siRNA(si-PHB2)组。检测氧化应激指标细胞内丙二醛(MDA)水平、荧光探针检测细胞内活性氧(ROS)水平;线粒体指标:三磷酸腺苷(ATP)水平、线粒体膜电位、线粒体电镜、线粒体半定量评分;免疫印迹法检测PHB2、PTEN诱导激酶1(PTNKI)、帕金蛋白(Parkin)、线粒体转录因子(TFAM)的表达。结果LPS刺激后MDA水平和ROS水平升高、ATP水平低,LPS+si-PHB2组MDA(6.21±0.39 vs 3.59±0.33,P<0.05)、细胞内的ROS(15131.37±88.72 vs 8628.67±71.95,P<0.05)的水平较LPS组升高,ATP(3.46±0.34 vs 4.52±0.25,P<0.05)和线粒体膜电位水平(0.33±0.04 vs 0.55±0.09,P<0.05)进一步降低;电镜观察显示与正常组相比,LPS组、LPS+si-PHB2组出现不同程度线粒体损伤,线粒体损伤半定量评分显示LPS+si-PHB2组的损伤较LPS组更为明显(1.42±0.10 vs 0.81±0.04,P<0.05);免疫印迹法结果显示LPS处理后PHB2、PINK1、Parkin表达上调,TFAM表达下调,LPS+si-PHB2组的线粒体自噬相关蛋白PINK1(1.33±0.06 vs 1.79±0.21,P<0.05)、Parkin(1.43±0.08 vs 1.86±0.09,P<0.05)和线粒体生物发生关键蛋白TFAM(0.29±0.01 vs 0.74±0.06,P<0.05)表达均较LPS组降低。结论LPS可促进大鼠心肌细胞PHB2表达,si-PHB2干扰后线粒体自噬蛋白和生物发生蛋白表达抑制,心肌细胞氧化应激损害和线粒体功能障碍加重,提示PHB2表达上调可能恢复线粒体稳态改善脓毒症心肌损伤的线粒体功能。展开更多
Vitamin K2 (VK2, menaquinone) is a drug for osteoporosis. VK2 acts as a cofactor for γ-glutamyl carboxylase, which catalyzes the carboxylation of specific glutamic acid residues (γ-carboxylation) of substrate protei...Vitamin K2 (VK2, menaquinone) is a drug for osteoporosis. VK2 acts as a cofactor for γ-glutamyl carboxylase, which catalyzes the carboxylation of specific glutamic acid residues (γ-carboxylation) of substrate proteins. Here we demonstrate that VK2 also regulate osteoblastgenic marker gene expression. Using VK2-immobilzed nanobeads new target proteins were purified and identified from osteoblastic cell line. They are prohibitin 1 and 2 (PHB1 & 2), respectively. To confirm the PHBs function on VK2-dependent transcription, PHB1 & 2 were knock-down and osteocalcin gene 2 transcriptions were analyzed, indicating that PHBs regulate VK2-dependent transcription. Taken together PHBs are VK2 target proteins for osteoblastgenic transcription.展开更多
We sought to investigate the underlying mechanism of action of the long noncoding RNA （IncRNA） LOC283070 in the development of androgen independence in prostate cancer. The interactions between LOC283070 and target ...We sought to investigate the underlying mechanism of action of the long noncoding RNA （IncRNA） LOC283070 in the development of androgen independence in prostate cancer. The interactions between LOC283070 and target proteins were investigated by RNA pull-down and RNA-binding protein immunoprecipitation （RIP） assays. Subceilular fractionation and quantitative reverse transcription-polymerase chain reaction （qRT-PCR） were used to detect the subcellular localization of LOC283070. Western blotting was performed to detect the expression of prohibitin 2 （PHB2）. Luciferase activity assays were performed to evaluate the effects of LOC283070 and PHB2 on the androgen receptor （AR） signaling pathway. A methyl thiazolyl tetrazolium （MTT） assay and a growth curve assay were used to test cell viability. Flow cytometry was performed to analyze cell cycles. A transwell assay was employed to test cell migration. We identified PHB2 as an interaction partner of LOC283070 in the pull-down and RIP experiments. Furthermore, we confirmed that the enrichment of L0C283070 with PHB2 in androgen-independent LNCaP （LNCaP-AI） cells was much greater than that in LNCaP cells. Moreover, the expression of PHB2 was not significantly different between the two cell lines, and the expression of LOC283070 in the nuclei of the LNCaP-AI cells was significantly greater than that in the LNCaP cells. In vitro data revealed that PHB2 overexpression significantly inhibited AR activity and cell proliferation and migration and induced accumulation of prostate cancer cells in GO/G1 phase. Moreover, the overexpression of LOC283070 fully abrogated the effects of PHB2 overexpression. In conclusion, we found that LOC283070 can bind to PHB2 located in the nucleus and inhibit its effect, and this is one of the mechanisms by which LOC283070 is involved in the transition of LNCaP cells into androgen-independent cells.展开更多
文摘目的探讨抗增殖蛋白2(PHB2)脓毒症心肌损伤线粒体功能的调控机制。方法体外培养大鼠心肌细胞株(H9C2),分为对照组、脂多糖(LPS)组、LPS+PHB2 siRNA(si-PHB2)组。检测氧化应激指标细胞内丙二醛(MDA)水平、荧光探针检测细胞内活性氧(ROS)水平;线粒体指标:三磷酸腺苷(ATP)水平、线粒体膜电位、线粒体电镜、线粒体半定量评分;免疫印迹法检测PHB2、PTEN诱导激酶1(PTNKI)、帕金蛋白(Parkin)、线粒体转录因子(TFAM)的表达。结果LPS刺激后MDA水平和ROS水平升高、ATP水平低,LPS+si-PHB2组MDA(6.21±0.39 vs 3.59±0.33,P<0.05)、细胞内的ROS(15131.37±88.72 vs 8628.67±71.95,P<0.05)的水平较LPS组升高,ATP(3.46±0.34 vs 4.52±0.25,P<0.05)和线粒体膜电位水平(0.33±0.04 vs 0.55±0.09,P<0.05)进一步降低;电镜观察显示与正常组相比,LPS组、LPS+si-PHB2组出现不同程度线粒体损伤,线粒体损伤半定量评分显示LPS+si-PHB2组的损伤较LPS组更为明显(1.42±0.10 vs 0.81±0.04,P<0.05);免疫印迹法结果显示LPS处理后PHB2、PINK1、Parkin表达上调,TFAM表达下调,LPS+si-PHB2组的线粒体自噬相关蛋白PINK1(1.33±0.06 vs 1.79±0.21,P<0.05)、Parkin(1.43±0.08 vs 1.86±0.09,P<0.05)和线粒体生物发生关键蛋白TFAM(0.29±0.01 vs 0.74±0.06,P<0.05)表达均较LPS组降低。结论LPS可促进大鼠心肌细胞PHB2表达,si-PHB2干扰后线粒体自噬蛋白和生物发生蛋白表达抑制,心肌细胞氧化应激损害和线粒体功能障碍加重,提示PHB2表达上调可能恢复线粒体稳态改善脓毒症心肌损伤的线粒体功能。
文摘Vitamin K2 (VK2, menaquinone) is a drug for osteoporosis. VK2 acts as a cofactor for γ-glutamyl carboxylase, which catalyzes the carboxylation of specific glutamic acid residues (γ-carboxylation) of substrate proteins. Here we demonstrate that VK2 also regulate osteoblastgenic marker gene expression. Using VK2-immobilzed nanobeads new target proteins were purified and identified from osteoblastic cell line. They are prohibitin 1 and 2 (PHB1 & 2), respectively. To confirm the PHBs function on VK2-dependent transcription, PHB1 & 2 were knock-down and osteocalcin gene 2 transcriptions were analyzed, indicating that PHBs regulate VK2-dependent transcription. Taken together PHBs are VK2 target proteins for osteoblastgenic transcription.
基金This work was supported by grants from the National Natural Science Foundation of China （No. 81702538, 81372764, and 81528015） and the Natural Science Foundation of Shandong Province （No. ZR201702160271, ZR2018MH030, and ZR2018MH025）.
文摘We sought to investigate the underlying mechanism of action of the long noncoding RNA （IncRNA） LOC283070 in the development of androgen independence in prostate cancer. The interactions between LOC283070 and target proteins were investigated by RNA pull-down and RNA-binding protein immunoprecipitation （RIP） assays. Subceilular fractionation and quantitative reverse transcription-polymerase chain reaction （qRT-PCR） were used to detect the subcellular localization of LOC283070. Western blotting was performed to detect the expression of prohibitin 2 （PHB2）. Luciferase activity assays were performed to evaluate the effects of LOC283070 and PHB2 on the androgen receptor （AR） signaling pathway. A methyl thiazolyl tetrazolium （MTT） assay and a growth curve assay were used to test cell viability. Flow cytometry was performed to analyze cell cycles. A transwell assay was employed to test cell migration. We identified PHB2 as an interaction partner of LOC283070 in the pull-down and RIP experiments. Furthermore, we confirmed that the enrichment of L0C283070 with PHB2 in androgen-independent LNCaP （LNCaP-AI） cells was much greater than that in LNCaP cells. Moreover, the expression of PHB2 was not significantly different between the two cell lines, and the expression of LOC283070 in the nuclei of the LNCaP-AI cells was significantly greater than that in the LNCaP cells. In vitro data revealed that PHB2 overexpression significantly inhibited AR activity and cell proliferation and migration and induced accumulation of prostate cancer cells in GO/G1 phase. Moreover, the overexpression of LOC283070 fully abrogated the effects of PHB2 overexpression. In conclusion, we found that LOC283070 can bind to PHB2 located in the nucleus and inhibit its effect, and this is one of the mechanisms by which LOC283070 is involved in the transition of LNCaP cells into androgen-independent cells.
