Background:The decreased slowly activating delayed rectifier K+current(IKs)is the molecular basis of arrhythmia caused by myocardial hypertrophy.The aim of this study was to investigate the mechanism of IKs down-regul...Background:The decreased slowly activating delayed rectifier K+current(IKs)is the molecular basis of arrhythmia caused by myocardial hypertrophy.The aim of this study was to investigate the mechanism of IKs down-regulation related to the channel number,as well as the regulation of channel number under pathological conditions.Methods:The HEK 293 cell co-transfected with KCNQ1/KCNE1 genes was cultured conventionally.After the cells incubated with angiotensin II(AngII)(24 h),AngII(72 h),bisindolylmaleimide I(Bis),brefeldin A and dynasore,the effect and relevant mechanism of long term incubation of AngII on the IKs tail current and KCNQ1 channel total protein were investigated by electrophysiology method and western blotting.In the experiment,the Bis,brefeldin A and dynasore could inhibit the protein kinase C(PKC)activity,the forward transport of KCNQ1 channel and the endocytosis of KCNQ1 channel,respectively.At last,the Rab GTPases 11(Rab11)dominant-negative mutant dsRed-Rab11/S25N was infected into the cells to investigate the effect and relevant mechanism of long term AngII incubation on the IKs tail current and KCNQ1 channel total protein.Results:Our results showed that the decreased IKs tail current and the KCNQ1 channel total protein caused by long term AngII incubation were attenuated by Bis treatment,which inhibited PKC activity.In addition,the inhibited IKs tail current and KCNQ1 channel total protein were also alleviated by brefeldin A and dynasore treatment.At last,the expression of Rab11 dominant-negative mutant dsRed-Rab11/S25N could weak the inhibition of IKs tail current and the KCNQ1 channel total protein caused by long term AngII incubation.Conclusion:The long term incubation of AngII inhibited the IKs tail current and KCNQ1 channel total protein was achieved by PKC activation and the disorder of the channel trafficking by Rab11.展开更多
BACKGROUND Highly upregulated in liver cancer (HULC) is a long non-coding RNA (lncRNA) which has recently been identified as a key regulator in hepatocellular carcinoma (HCC) progression. However, its role in the secr...BACKGROUND Highly upregulated in liver cancer (HULC) is a long non-coding RNA (lncRNA) which has recently been identified as a key regulator in hepatocellular carcinoma (HCC) progression. However, its role in the secretion of exosomes from HCC cells remains unknown. AIM To explore the mechanism by which HULC promotes the secretion of exosomes from HCC cells. METHODS Serum and liver tissue samples were collected from 30 patients with HCC who had not received chemotherapy, radiotherapy, or immunotherapy before surgery. HULC expression in serum exosomes and liver cancer tissues of patients was measured, and compared with the data obtained from healthy controls and tumor adjacent tissues. The effect of HULC upregulation in HCC cell lines and the relationship between HULC and other RNAs were studied using qPCR and dualluciferase reporter assays. Nanoparticle tracking analysis was performed to detect the quantity of exosomes.RESULTS HULC expression in serum exosomes of patients with HCC was higher than that in serum exosomes of healthy controls, and HULC levels were higher in liver cancer tissues than in tumor adjacent tissues. The expression of HULC in serum exosomes and liver cancer tissues correlated with the tumor-node-metastasis (TNM) classification, and HULC expression in tissues correlated with that in serum exosomes. Upregulation of HULC promoted HCC cell growth and invasion and repressed apoptosis. Notably, it also facilitated the secretion of exosomes from HCC cells. Moreover, qPCR assays showed that HULC repressed microRNA-372-3p (miR-372-3p) expression. We also identified Rab11a as a downstream target of miR-372-3p. Dual-luciferase reporter assays suggested that miR-372-3p could directly bind both HULC and Rab11a. CONCLUSION Our findings illustrate the importance of the HULC/miR-372-3p/Rab11a axis in HCC and provide new insights into the molecular mechanism regulating the secretion of exosomes from HCC cells.展开更多
目的：通过调控Rab11在HeLa细胞中的表达，观察乏氧条件下Rab11对宫颈癌HeLa细胞侵袭、迁移能力的影响。方法将 HeLa 细胞分为4组：常氧对照组、常氧 Rab11siRNA 转染组、乏氧对照组、乏氧Rab11siRNA转染组。 Western blot检测各组HeLa...目的：通过调控Rab11在HeLa细胞中的表达，观察乏氧条件下Rab11对宫颈癌HeLa细胞侵袭、迁移能力的影响。方法将 HeLa 细胞分为4组：常氧对照组、常氧 Rab11siRNA 转染组、乏氧对照组、乏氧Rab11siRNA转染组。 Western blot检测各组HeLa细胞Rab11、基质金属蛋白酶（ MMP）－2、MMP－9蛋白表达， Transwell小室试验检测各组HeLa细胞侵袭、迁移能力的变化。结果乏氧条件下HeLa细胞Rab11表达量高于常氧组（P＜0．01），细胞侵袭、迁移能力增高（P＜0．01）；常氧和乏氧条件下，Rab11siRNA转染组与对照组相比，细胞侵袭能力、迁移能力下降（P＜0．01），乏氧条件下的Rab11 siRNA转染组下降更明显。结论乏氧促进HeLa细胞的侵袭、迁移，常氧及乏氧条件下下调Rab11表达均能够抑制HeLa细胞的侵袭、迁移， HeLa细胞的侵袭能力、迁移能力的变化依赖于Rab11的表达。展开更多
基金supported by the National Natural Science Foundation of China(NSFC)Youth Project(No.82204397 and No.22203063).
文摘Background:The decreased slowly activating delayed rectifier K+current(IKs)is the molecular basis of arrhythmia caused by myocardial hypertrophy.The aim of this study was to investigate the mechanism of IKs down-regulation related to the channel number,as well as the regulation of channel number under pathological conditions.Methods:The HEK 293 cell co-transfected with KCNQ1/KCNE1 genes was cultured conventionally.After the cells incubated with angiotensin II(AngII)(24 h),AngII(72 h),bisindolylmaleimide I(Bis),brefeldin A and dynasore,the effect and relevant mechanism of long term incubation of AngII on the IKs tail current and KCNQ1 channel total protein were investigated by electrophysiology method and western blotting.In the experiment,the Bis,brefeldin A and dynasore could inhibit the protein kinase C(PKC)activity,the forward transport of KCNQ1 channel and the endocytosis of KCNQ1 channel,respectively.At last,the Rab GTPases 11(Rab11)dominant-negative mutant dsRed-Rab11/S25N was infected into the cells to investigate the effect and relevant mechanism of long term AngII incubation on the IKs tail current and KCNQ1 channel total protein.Results:Our results showed that the decreased IKs tail current and the KCNQ1 channel total protein caused by long term AngII incubation were attenuated by Bis treatment,which inhibited PKC activity.In addition,the inhibited IKs tail current and KCNQ1 channel total protein were also alleviated by brefeldin A and dynasore treatment.At last,the expression of Rab11 dominant-negative mutant dsRed-Rab11/S25N could weak the inhibition of IKs tail current and the KCNQ1 channel total protein caused by long term AngII incubation.Conclusion:The long term incubation of AngII inhibited the IKs tail current and KCNQ1 channel total protein was achieved by PKC activation and the disorder of the channel trafficking by Rab11.
基金Supported by Tianjin Clinical Research Center for Organ Transplantation Project,No.15ZXLCSY00070The Nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences,No.2018PT32021
文摘BACKGROUND Highly upregulated in liver cancer (HULC) is a long non-coding RNA (lncRNA) which has recently been identified as a key regulator in hepatocellular carcinoma (HCC) progression. However, its role in the secretion of exosomes from HCC cells remains unknown. AIM To explore the mechanism by which HULC promotes the secretion of exosomes from HCC cells. METHODS Serum and liver tissue samples were collected from 30 patients with HCC who had not received chemotherapy, radiotherapy, or immunotherapy before surgery. HULC expression in serum exosomes and liver cancer tissues of patients was measured, and compared with the data obtained from healthy controls and tumor adjacent tissues. The effect of HULC upregulation in HCC cell lines and the relationship between HULC and other RNAs were studied using qPCR and dualluciferase reporter assays. Nanoparticle tracking analysis was performed to detect the quantity of exosomes.RESULTS HULC expression in serum exosomes of patients with HCC was higher than that in serum exosomes of healthy controls, and HULC levels were higher in liver cancer tissues than in tumor adjacent tissues. The expression of HULC in serum exosomes and liver cancer tissues correlated with the tumor-node-metastasis (TNM) classification, and HULC expression in tissues correlated with that in serum exosomes. Upregulation of HULC promoted HCC cell growth and invasion and repressed apoptosis. Notably, it also facilitated the secretion of exosomes from HCC cells. Moreover, qPCR assays showed that HULC repressed microRNA-372-3p (miR-372-3p) expression. We also identified Rab11a as a downstream target of miR-372-3p. Dual-luciferase reporter assays suggested that miR-372-3p could directly bind both HULC and Rab11a. CONCLUSION Our findings illustrate the importance of the HULC/miR-372-3p/Rab11a axis in HCC and provide new insights into the molecular mechanism regulating the secretion of exosomes from HCC cells.
