scholarly journals Effects of microRNA-27a Targeting Smad1 on Intervertebral Disc Degeneration and Biological Characteristics of Nucleus Pulposus Cells

2020 ◽  
Author(s):  
Heng-Tao Tang ◽  
Song Zhang ◽  
Xin-Chang Lu ◽  
Tong-Yu Geng
Keyword(s):  
Cell Proliferation ◽  
Inflammatory Response ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Biological Characteristics ◽  
Luciferase Reporter ◽  
Ecm Proteins ◽  
Reporter Assays

Abstract Objective: The present study aimed to analyze the expression of microRNA-27a (miR-27a) in intervertebral disc degeneration (IDD) and its effect on the biological characteristics of nucleus pulposus (NP) cells. Methods: An IDD rat model was established, and the expression of miR-27a and Smad1 in the intervertebral disc tissue was detected. An oxygen and glucose deprivation (OGD) NP cell model was established to simulate the IDD microenvironment, and the effects of downregulated miR-27a on the proliferation, apoptosis, inflammatory response, and extracellular matrix (ECM) proteins of OGD-NP cells were analyzed. The target relationship of miR-27a and Smad1 was verified by luciferase reporter assays, and siRNA-Smad1 was transfected to reverse the experiment. Results: The level of miR-27a in the IDD model group was significantly increased, whereas that of Smad1 was decreased compared with the sham group (P<0.05). Inhibition of miR-27a improved cell proliferation, and inhibited apoptosis, degradation of the ECM, and inflammatory response of OGD-NP cells compared with the OGD group (P<0.05). The results of the double luciferase reporter assays indicated that Smad1 was the target gene of miR-27a. Smad1 silencing reversed the increase in ECM proteins induced by inhibition of miR-27a; However, it did not affect cell proliferation and apoptosis. Conclusion: The expression levels of miR-27a were upregulated in IDD and it may be involved in the progression of IDD by promoting the apoptosis of NP cells and ECM degradation by targeting Smad1.

scholarly journals Expression of miR-195 and its target gene Bcl-2 in human intervertebral disc degeneration and their effects on nucleus pulposus cell apoptosis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Xue-Lin Lin ◽  
Zhao-Yun Zheng ◽  
Qing-Shan Zhang ◽  
Zhen Zhang ◽  
You-Zhi An
Keyword(s):  
Cell Proliferation ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Rat Model ◽  
Cell Apoptosis ◽  
Intervertebral Disc Degeneration ◽  
Target Gene ◽  
Blank Group ◽  
Tnf Α

Abstract Objective To investigate the expression of miR-195 and its target gene Bcl-2 in intervertebral disc degeneration (IVDD) and its effect on nucleus pulposus (NP) cell apoptosis. Methods The expressions of miR-195 and Bcl-2 in NP tissues of IVDD patients were quantified by qRT-PCR and western blotting, respectively. NP cells were divided into blank group, TNF-α group, TNF-α + miR-NC group, TNF-α + siBcl-2 group, and TNF-α + miR-195 inhibitors + siBcl-2 group. Cell proliferation was detected by MTT assay, cell apoptosis evaluated by flow cytometry, and mitochondrial membrane potential (MMP) tested by JC-1 staining. Moreover, the function of miR-195 on IVDD in vivo was investigated using a puncture-induced IVDD rat model. Results IVDD patients had significantly increased miR-195 expression and decreased Bcl-2 protein expression in NP tissues. The expression of miR-195 was negatively correlated with the expression of Bcl-2 in IVDD patients. Dual-luciferase reporter gene assay indicated that Bcl-2 was a target gene of miR-195. In comparison with blank group, TNF-α group showed decreased cell proliferation and MMP, increased cell apoptosis, upregulated expression of miR-195, Bax, and cleaved caspase 3, and downregulated Bcl-2 protein, while these changes were attenuated by miR-195 inhibitors. Additionally, siBcl-2 can reverse the protective effect of miR-195 inhibitors on TNF-α-induced NP cells. Besides, inhibition of miR-195 alleviated IVDD degeneration and NP cell apoptosis in the rat model. Conclusion MiR-195 was significantly upregulated in NP tissues of IVDD patients, and inhibition of miR-195 could protect human NP cells from TNF-α-induced apoptosis via upregulation of Bcl-2.

scholarly journals MiR-125a Rs12976445 Polymorphism is Associated with the Apoptosis Status of Nucleus Pulposus Cells and the Risk of Intervertebral Disc Degeneration

2016 ◽  
Vol 38 (1) ◽  
pp. 295-305 ◽  
Author(s):  
Jin Feng Ma ◽  
Li Na Zang ◽  
Yong Ming Xi ◽  
Wen Jiu Yang ◽  
Debo Zou
Keyword(s):  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Minor Allele ◽  
Expression Level ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Degenerative Diseases ◽  
Nucleus Pulposus Cells

Background: Spinal degenerative diseases are a major health problem and social burden worldwide. Intervertebral disc degeneration (IDD) is the pathological basis of spinal degenerative diseases and is characterized by loss of nucleus pulposus cells due to excessive apoptosis caused by various factors. MicroRNAs (miRNAs) have been reported to be functionally involved in the control of apoptosis. Methods: computational analysis and luciferase assay were used to identify the target of miR-125a, and cell culture, transfection were used to confirm such relationship. Sequencing was used to determine the genotype of each participant. Results: We confirmed the previous report that the presence of the minor allele (T) of rs12976445 polymorphism significantly downregulated the expression level of miR-125a in nucleus pulposus cells, leading to less efficient inhibition of its target gene. We also validated TP53INP1 as a target of miR-125a in nucleus pulposus cells using a dual luciferase reporter system, and the transfection of miR-125a significantly reduced the expression of TP53INP1. The expression level of TP53INP1 was significantly lower in nucleus pulposus cells genotyped as CT or TT than in those genotyped as CC, and the apoptosis rate was consistently lower in the CC group than in the nucleus pulposus cells collected from individuals carrying at least one minor allele of rs12976445 polymorphism. To study the association between rs12976445 polymorphism and the risk of IDD, we enrolled 242 patients diagnosed with IDD and 278 normal controls, and significant differences were noted regarding the genotype distribution of rs12976445 between the IDD and the control groups (OR = 2.69, 95% C.I. = 1.88-3.83, p < 0.0001). In summary, rs12976445 polymorphism is significantly associated with the risk of IDD in the Chinese population. Conclusion: The present study indicated that miR-125a is a promising potential target for patients with IDD in clinical practice.

scholarly journals Expression of miR-195 and its target gene Bcl-2 in human intervertebral disc degeneration and their effects on nucleus pulposus cell apoptosis

2021 ◽  
Author(s):  
Xue-Lin Lin ◽  
Zhao-Yun Zheng ◽  
Qing-Shan Zhang ◽  
Zhen Zhang ◽  
You-Zhi An
Keyword(s):  
Cell Proliferation ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Rat Model ◽  
Cell Apoptosis ◽  
Intervertebral Disc Degeneration ◽  
Target Gene ◽  
Blank Group ◽  
Tnf Α

Abstract Objective: To investigate the expression of miR-195 and its target gene Bcl-2 in intervertebral disc degeneration (IVDD) and its effect on nucleus pulposus (NP) cell apoptosis.Methods: The expressions of miR-195 and Bcl-2 in NP tissues of IVDD patients were quantified by qRT-PCR and Western blotting, respectively. NP cells were divided into Blank group, TNF-α group, TNF-α + miR-NC group, TNF-α + siBcl-2 group, and TNF-α + miR-195 inhibitors + siBcl-2 group. Cell proliferation was detected by MTT assay, cell apoptosis evaluated by flow cytometry, and mitochondrial membrane potential (MMP) tested by JC-1 staining. Moreover, the function of miR-132 on IVDD in vivo was investigated using a puncture-induced IVDD rat model.Results: IVDD patients had significantly increased miR-195 expression and decreased Bcl-2 protein expression in NP tissues. The expression of miR-195 was negatively correlated with the expression of Bcl-2 in IVDD patients. Dual-luciferase reporter gene assay indicated that Bcl-2 was a target gene of miR-195. In comparison with Blank group, TNF-α group showed decreased cell proliferation and MMP, increased cell apoptosis, up-regulated expression of miR-195, Bax and cleaved caspase 3, and down-regulated Bcl-2 protein, while these changes were attenuated by miR-195 inhibitors. Additionally, siBcl-2 can reverse the protective effect of miR-195 inhibitors on TNF-α-induced NP cells. Besides, inhibition of miR-195 alleviated IVDD degeneration and NP cell apoptosis in the rat model.Conclusion: MiR-195 was significantly up-regulated in NP tissues of IVDD patients, and inhibition of miR-195 could protect human NP cells from TNF-α-induced apoptosis via upregulation of Bcl-2.

scholarly journals Mechanism of Long Noncoding RNA HOTAIR in Nucleus Pulposus Cell Autophagy and Apoptosis in Intervertebral Disc Degeneration

2022 ◽  
Vol 2022 ◽  
pp. 1-18
Author(s):  
Shujun Zhang ◽  
Sheng Song ◽  
Wei Cui ◽  
Xueguang Liu ◽  
Zhenzhong Sun
Keyword(s):  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Noncoding Rna ◽  
Intervertebral Disc Degeneration ◽  
Intervertebral Discs ◽  
Luciferase Reporter ◽  
Blue Staining ◽  
Tunel Staining ◽  
Lncrna Hotair

Objective. Intervertebral disc degeneration (IDD) contributes to cervical and lumbar diseases. Long noncoding RNAs (lncRNAs) are implicated in IDD. This study explored the mechanism of lncRNA HOTAIR in IDD. Methods. Normal and degenerative nucleus pulposus (NP) cells were isolated from NP tissues obtained in intervertebral disc surgery. Cell morphology was observed by immunocytochemistry staining and toluidine blue staining. NP cell markers were detected by RT-qPCR. Proliferation was detected by MTT assay. Autophagy-related proteins were detected by Western blot. Autophagosome was observed by monodansylcadaverine fluorescence staining. Apoptosis was detected by TUNEL staining and flow cytometry. si-HOTAIR and/or miR-148a inhibitor was introduced into degenerative NP cells. Binding relationships among HOTAIR, miR-148a, and PTEN were predicted and verified by dual-luciferase reporter assay and RNA pull-down. Finally, IDD rat models were established. Rat caudal intervertebral discs were assessed by HE staining. Expressions of HOTAIR, miR-148a, and PTEN were determined by RT-qPCR. Results. HOTAIR was highly expressed in degenerative NP cells p < 0.05 . si-HOTAIR inhibited degenerative NP cell apoptosis and autophagy p < 0.05 . HOTAIR upregulated PTEN as a sponge of miR-148a. miR-148a was poorly expressed in degenerative NP cells. miR-148a deficiency partially reversed the inhibition of si-HOTAIR on degenerative NP cell autophagy and apoptosis (all p < 0.05 ). In vivo assay confirmed that si-HOTAIR impeded autophagy and apoptosis in intervertebral disc tissues, thus improving pathological injury in IDD rats (all p < 0.05 ). Conclusion. LncRNA HOTAIR promoted NP cell autophagy and apoptosis via promoting PTEN expression as a ceRNA of miR-148a in IDD.

scholarly journals Expression of miR-195 and its target gene Bcl-2 in human intervertebral disc degeneration and their effects on nucleus pulposus cell apoptosis

2020 ◽  
Author(s):  
Xue-Lin Lin ◽  
Zhao-Yun Zheng ◽  
Qing-Shan Zhang ◽  
Zhen Zhang ◽  
You-Zhi An
Keyword(s):  
Cell Proliferation ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Intervertebral Disc Degeneration ◽  
Target Gene ◽  
Blank Group ◽  
Tnf Α

Abstract Objective: To investigate the expression of miR-195 and its target gene Bcl-2 in intervertebral disc degeneration (IVDD) and its effect on nucleus pulposus (NP) cell apoptosis.Methods: The expressions of miR-195 and Bcl-2 in NP tissues of IVDD patients were quantified by qRT-PCR and Western blotting respectively. NP cells were divided into Blank group, TNF-α group, TNF-α + miR-NC group, TNF-α + siBcl-2 group, and TNF-α + miR-195 inhibitors + siBcl-2 group. Cell proliferation was detected by MTT assay, cell apoptosis evaluated by flow cytometry, mitochondrial membrane potential (MMP) tested by JC-1 staining, and the expression of apoptosis-related proteins quantified by Western blotting. Results: Compared with controls, IVDD patients had significantly increased miR-195 expression and decreased Bcl-2 protein in NP tissues. The expression of miR-195 was negatively correlated with the expression of Bcl-2 in NP tissues of IVDD patients (r = - 0.89, P < 0.001). Dual-luciferase reporter gene assay indicated that Bcl-2 was a target gene of miR-195. In comparison with Blank group, TNF-α group showed decreased cell proliferation and MMP, increased cell apoptosis, up-regulated expression of miR-195, Bax and cleaved caspase 3, and down-regulated Bcl-2 protein, these changes were attenuated by miR-195 inhibitors. Additionally, siBcl-2 can reverse the protective effect of miR-195 inhibitors on TNF-α-induced NP cells. Conclusion: IVDD patients had increased miR-195 expression in NP tissues, and inhibiting miR-195 can specifically up-regulate Bcl-2 expression to curb apoptosis of TNF-α-induced NP cells.

scholarly journals MiR-25 Protects Against Apoptosis of Nucleus Pulposus Cells by Targeting SUMO2 in Intervertebral Disc Degeneration

2020 ◽  
Author(s):  
Lei Changbin ◽  
Li Jiang ◽  
Tang Guang ◽  
Wang Jiong ◽  
Hongsheng Lin
Keyword(s):  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Luciferase Reporter ◽  
Nucleus Pulposus Cells ◽  
P53 Signaling ◽  
Pulldown Assay ◽  
Signaling Axis ◽  
Incorporation Assay

Abstract Background MiR-25 was reported to be down-regulated in patients with intervertebral disc degeneration (IDD). However, the potential role of miR-25 in IDD remained unclear. Therefore, the present study aimed to investigate the effects of miR-25 on human intervertebral disc nucleus pulposus cells (NPCs).Methods We evaluated the expression of miR-25 and small ubiquitin-related modifier 2 (SUMO2) in human nucleus pulposus (NP) tissues by real-time PCR and western blotting. Then, the target relationship between miR-25 and SUMO2 was validated by luciferase reporter assay and biotin-coupled miRNA pulldown assay. The potential roles of miR-25 in NPC proliferation and apoptosis were confirmed using CCK-8 assay, EdU incorporation assay, and flow cytometry.Results MiR-25 was lowly expressed in the patients with IDD. In addition, miR-25 facilitated the growth of NPCs by increasing cell proliferation and inhibiting apoptosis. Furthermore, we elucidated that SUMO2 was a target gene of miR-25, and was regulated by miR-25 through p53 signaling pathway. Restore of SUMO2 expression abrogated the effects of miR-25 on NPCs.Conclusion MiR-25 promoted the proliferation, inhibited the apoptosis of NPCs, and suppressed the development of IDD via SUMO2-mediated p53 signaling axis.

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