BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchym...BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.展开更多
背景:miR-34a是一种起抑癌作用的miRNA,它能调节多个靶基因的表达,有可能成为治疗肿瘤的新靶点。目的:探讨miR-34a及其靶基因SIRT1对结肠癌干细胞体外增殖、凋亡、侵袭能力的影响。方法:采用无血清悬浮培养法从人结肠癌细胞株HCT116中...背景:miR-34a是一种起抑癌作用的miRNA,它能调节多个靶基因的表达,有可能成为治疗肿瘤的新靶点。目的:探讨miR-34a及其靶基因SIRT1对结肠癌干细胞体外增殖、凋亡、侵袭能力的影响。方法:采用无血清悬浮培养法从人结肠癌细胞株HCT116中富集肿瘤干细胞,流式细胞仪检测CD44^+/CD133^+细胞所占的比例进行鉴定。利用脂质体转染法将miR-34a模拟物转染结肠癌干细胞,并设miRNA无序阴性对照物和未转染组。CCK-8增殖实验、细胞凋亡实验、细胞侵袭实验检测结肠癌干细胞增殖、凋亡、侵袭情况。采用q RT-PCR和Western blot法检测miR-34a转染后细胞中SIRT1 m RNA和蛋白的表达水平。结果与结论:(1)无血清悬浮培养法富集的肿瘤干细胞球中含有较高比例的CD44^+/CD133^+表型细胞,所占比例为(78.3±6.7)%;(2)miR-34a转染组的miR-34a表达量高于未转染组和miR-34a对照组(P<0.05);(3)与未转染组和miR-34a对照组比较,miR-34a转染组的细胞增殖能力明显降低(P<0.05);细胞凋亡率明显升高(P<0.05);侵袭细胞数明显降低(P<0.05);(4)miR-34a过表达后,miR-34a转染组的SIRT1 m RNA和蛋白的表达水平均明显低于未转染组和miR-34a对照组(P<0.05);(5)结果表明,过表达miR-34a可抑制结肠癌干细胞增殖、侵袭并诱导凋亡,可能与其下调SIRT1的表达有关。展开更多
基金Supported by the National Natural Science Foundation of China,No.82172462,No.81972136the Traditional Chinese Medicine Science and Technology Development Plan Project of Jiangsu Province,No.YB2020085Cross Cooperation Project of Northern Jiangsu People’s Hospital,No.SBJC21014.
文摘BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.
文摘背景:miR-34a是一种起抑癌作用的miRNA,它能调节多个靶基因的表达,有可能成为治疗肿瘤的新靶点。目的:探讨miR-34a及其靶基因SIRT1对结肠癌干细胞体外增殖、凋亡、侵袭能力的影响。方法:采用无血清悬浮培养法从人结肠癌细胞株HCT116中富集肿瘤干细胞,流式细胞仪检测CD44^+/CD133^+细胞所占的比例进行鉴定。利用脂质体转染法将miR-34a模拟物转染结肠癌干细胞,并设miRNA无序阴性对照物和未转染组。CCK-8增殖实验、细胞凋亡实验、细胞侵袭实验检测结肠癌干细胞增殖、凋亡、侵袭情况。采用q RT-PCR和Western blot法检测miR-34a转染后细胞中SIRT1 m RNA和蛋白的表达水平。结果与结论:(1)无血清悬浮培养法富集的肿瘤干细胞球中含有较高比例的CD44^+/CD133^+表型细胞,所占比例为(78.3±6.7)%;(2)miR-34a转染组的miR-34a表达量高于未转染组和miR-34a对照组(P<0.05);(3)与未转染组和miR-34a对照组比较,miR-34a转染组的细胞增殖能力明显降低(P<0.05);细胞凋亡率明显升高(P<0.05);侵袭细胞数明显降低(P<0.05);(4)miR-34a过表达后,miR-34a转染组的SIRT1 m RNA和蛋白的表达水平均明显低于未转染组和miR-34a对照组(P<0.05);(5)结果表明,过表达miR-34a可抑制结肠癌干细胞增殖、侵袭并诱导凋亡,可能与其下调SIRT1的表达有关。
