Current research indicates that beta cell loss in type 2 diabetes may be attributed to beta cell dedifferentiation rather than apoptosis;however,the mechanisms by which this occurs remain poorly understood.Our previou...Current research indicates that beta cell loss in type 2 diabetes may be attributed to beta cell dedifferentiation rather than apoptosis;however,the mechanisms by which this occurs remain poorly understood.Our previous study demonstrated that elevation of microRNA-24(miR-24)in a diabetic setting caused beta cell dysfunction and replicative deficiency.In this study,we focused on the role of miR-24 in beta cell apoptosis and dedifferentiation under endoplasmic reticulum(ER)stress conditions.We found that miR-24 overabundance protected beta cells from thapsigargin-induced apoptosis at the cost of accelerating the impairment of glucosestimulated insulin secretion(GSIS)and enhancing the presence of dedifferentiation markers.Ingenuity?Pathway Analysis(IPA)revealed that elevation of miR-24 had an inhibitory effect on XBP1 and ATF4,which are downstream effectors of two key branches of ER stress,by inhibiting its direct target,Irela.Notably,elevated miR-24 initiated another pathway that targeted Mafa and decreased GSIS function in surviving beta cells,thus guiding their dedifferentiation under ER stress conditions.Our results demonstrated that the elevated miR-24,to the utmost extent,preserves beta cell mass by inhibiting apoptosis and inducing dedifFerentiation.This study not only provides a novel mechanism by which miR-24 dominates beta cell turnover under persistent metabolic stress but also offers a therapeutic consideration for treating diabetes by inducing dedifferentiated beta cells to re-differentiation.展开更多
Background： MicroRNA-24 （miR-24） plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but littl...Background： MicroRNA-24 （miR-24） plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy. Methods： Twelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randonlly divided into two groups： a transverse aortic constriction （TAC） group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay, lmmunofluorescence labeling was used to measure the cell surface area, and 3H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes （NRCMs） with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P 〈 0.05 was considered as the threshold for significance. Results： The expression of miR-24 was abnormally increased in TAC rat cardiac tissue （ t =-2.938, P 〈 0.05）. TargetScans algorithm-based prediction demonstrated that CDKN 1B （p27, Kip 1 ）, a cell cycle regulator, was a putative target of miR-24, and was confirmed by luci ferase assay. The expression of p27 was decreased in TAC rat cardiac tissue （t = 2.896, P 〈 0.05）. The overexpression of miR-24 in NRCMs led to the decreased expression of p27 （t = 4.400, P 〈 0.01 ）, and decreased G0/G 1 arrest in cell cycle and cardiomyocylc hypertrophy. Conclusion： MiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression.展开更多
目的探讨微小RNA\|24(miR-24)对过氧化氢(H_(2)O_(2))诱导的HLE-B3细胞凋亡的影响,分析其与线粒体中第二个线粒体衍生的胱天蛋白酶激活因子/低等电位点的凋亡抑制蛋白的直接结合蛋白(the second mitochondria-derived activator of casp...目的探讨微小RNA\|24(miR-24)对过氧化氢(H_(2)O_(2))诱导的HLE-B3细胞凋亡的影响,分析其与线粒体中第二个线粒体衍生的胱天蛋白酶激活因子/低等电位点的凋亡抑制蛋白的直接结合蛋白(the second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI,Smac/Diablo)-XIAP-caspase-9/3凋亡途径的关系。方法将HLE-B3细胞分为对照组、H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组。先按照miR-24抑制剂、miR-24 NC试剂盒说明书转染H_(2)O_(2)+miR-24抑制剂组和H_(2)O_(2)+miR-24 NC组细胞24 h,然后向H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组HLE-B3细胞中加入200μmol·L^(-1)H_(2)O_(2)干预24 h,收集细胞用于后续实验;将正常培养的HLE-B3细胞设置为对照组。采用实时荧光定量PCR检测各组HLE-B3细胞miR-24表达情况,CCK-8法检测各组HLE-B3细胞生长情况,用流式细胞仪检测各组细胞凋亡情况,计算各组细胞存活率和细胞凋亡率。采用荧光探针JC-1法检测线粒体膜电位变化情况(利用红绿荧光强度比反映线粒体膜电位);采用蛋白免疫印迹法检测各组HLE-B3细胞线粒体和细胞质分级中Smac/Diablo及细胞质分级中XIAP、caspase-9和caspase-3蛋白表达情况。对所得数据进行统计学分析。结果当H_(2)O_(2)浓度大于200μmol·L^(-1)时,细胞存活率均小于50%,本实验以200μmol·L^(-1)作为最适H_(2)O_(2)浓度。对照组、H_(2)O_(2)组、H_(2)O_(2)+miR-24 NC组和H_(2)O_(2)+miR-24抑制剂组细胞miR-24相对表达量分别为1.04±0.02、2.73±0.09、2.69±0.07和1.15±0.06;与对照组相比,H_(2)O_(2)组细胞中miR-24表达升高(P<0.05);与H_(2)O_(2)组和H_(2)O_(2)+miR-24 NC组相比,H_(2)O_(2)+miR-24抑制剂组中miR-24表达降低(均为P<0.05),但H_(2)O_(2)组与H_(2)O_(2)+miR-24 NC组中miR-24表达差异无统计学意义(P>0.05)。与H_(2)O_(2)组和H_(2)O_(2)+miR-24 NC组相比,H_(2)O_(2)+miR-24抑制剂组细胞存活率显著�展开更多
基金the National Key Research and Development Program of China(2016YFC1304804)the National Natural Science Foundation of China(81420108007)to X.H.+1 种基金the National Natural Science Found ation of China(81670703)China Postdoctoral Science Foundation(2016M590479)to Y.Z.
文摘Current research indicates that beta cell loss in type 2 diabetes may be attributed to beta cell dedifferentiation rather than apoptosis;however,the mechanisms by which this occurs remain poorly understood.Our previous study demonstrated that elevation of microRNA-24(miR-24)in a diabetic setting caused beta cell dysfunction and replicative deficiency.In this study,we focused on the role of miR-24 in beta cell apoptosis and dedifferentiation under endoplasmic reticulum(ER)stress conditions.We found that miR-24 overabundance protected beta cells from thapsigargin-induced apoptosis at the cost of accelerating the impairment of glucosestimulated insulin secretion(GSIS)and enhancing the presence of dedifferentiation markers.Ingenuity?Pathway Analysis(IPA)revealed that elevation of miR-24 had an inhibitory effect on XBP1 and ATF4,which are downstream effectors of two key branches of ER stress,by inhibiting its direct target,Irela.Notably,elevated miR-24 initiated another pathway that targeted Mafa and decreased GSIS function in surviving beta cells,thus guiding their dedifferentiation under ER stress conditions.Our results demonstrated that the elevated miR-24,to the utmost extent,preserves beta cell mass by inhibiting apoptosis and inducing dedifFerentiation.This study not only provides a novel mechanism by which miR-24 dominates beta cell turnover under persistent metabolic stress but also offers a therapeutic consideration for treating diabetes by inducing dedifferentiated beta cells to re-differentiation.
基金This work was supported by grant from National Natural Science Foundation of China （No. 91339105, and No. 81625001）.
文摘Background： MicroRNA-24 （miR-24） plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy. Methods： Twelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randonlly divided into two groups： a transverse aortic constriction （TAC） group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay, lmmunofluorescence labeling was used to measure the cell surface area, and 3H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes （NRCMs） with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P 〈 0.05 was considered as the threshold for significance. Results： The expression of miR-24 was abnormally increased in TAC rat cardiac tissue （ t =-2.938, P 〈 0.05）. TargetScans algorithm-based prediction demonstrated that CDKN 1B （p27, Kip 1 ）, a cell cycle regulator, was a putative target of miR-24, and was confirmed by luci ferase assay. The expression of p27 was decreased in TAC rat cardiac tissue （t = 2.896, P 〈 0.05）. The overexpression of miR-24 in NRCMs led to the decreased expression of p27 （t = 4.400, P 〈 0.01 ）, and decreased G0/G 1 arrest in cell cycle and cardiomyocylc hypertrophy. Conclusion： MiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression.
