MiR-210 Regulates Autophagy And Apoptosis By Directly Targeting ATG7 And Bcl-2 In Human Degenerative NP Cells

Part I: Analyses of mi RNA chip and mi R-210 expression in human degenerative NP tissuesObjective: Intervertebral disc degeneration(IDD) is the most common cause that leads to low back pain. However, its pathogenesis is still not fully understood. In the current study, mi RNA chip technology was used to measure the differentially expressed mi RNAs in human degenerative nucleus pulposus(NP) tissues, thereby preliminary screening IDD-related mi RNAs. Moreover, certain differentially expressed mi RNAs were identified using quantitative real-time polymerase chain reaction(q RT-PCR), which would contribute to further exploring the role of mi RNA in the occurrence and development of IDD and also provide novel targets for the prevention and treatent of degenerative disc diseases.Methods: Five normal NP tissue specimens(Grade II) and 45 degenerative NP tissue specimens including 15 Grade III, 15 Grade IV and 15 Grade V were collected from operation. Among these degenerative NP tissue specimens, five ones were selected, which included 1 Grade III, 2 Grade IV and 2 Grade V. Sure Print Human mi RNA Microarrays(8′60K) V16 was used to detect mi RNA expression in these five degenerative NP tissue specimens and five normal NP tissue specimens. According to the results of mi RNA chip, certain differentially expressed mi RNAs were identified using q RT-PCR in all 50 specimens. In addition, the correlation between the differentially expressed mi RNA levels and Pfirrmann grade was analyzed.Results: Twenty-nine IDD-related mi RNAs were obtained by chip image scanning and data analysis. Among these mi RNAs, 20 ones were significantly upregulated(Fold change≥2), including Hsa-mi R-22-3p, Hsa-mi R-21, Hsa-mi R-338-3p,Hsa-mi R-320 a, Hsa-mi R-372, Hsa-mi R-744, Hsa-mi R-122 a, Hsa-mi R-146 a,Hsa-mi R-181 c, Hsa-mi R-508-5p, Hsa-mi R-93-5p, Hsa-mi R-133 b, Hsa-mi R-210,Hsa-let-7g-5p, Hsa-mi R-187, Hsa-mi R-451 a, Hsa-mi R-659, Hsa-mi R-30-5p,Hsa-mi R-7-5p and Hsa-mi R-155. Notably, a striking upregulation of Hsa-mi R-210 were observed(Fold change≥2). At the same time, 20 ones were significantly downregulated(Fold change ≤ 0.5), including Hsa-mi R-212-5p, Hsa-mi R-10a-5p,Hsa-mi R-31, Hsa-mi R-375, Hsa-mi R-138, Hsa-mi R-129-3p, Hsa-mi R-147 b, Hsa-mi R-33 a and Hsa-let-7c. By using q RT-PCR, we found that mi R-210 was highly expressed in human degenerative NP tissues compared with normal tissues(P<0.05), which was similar to the chip data. Moreover, mi R-210 expression levels showed a positive correlation with Pfirrmann grade(r=0.67, P<0.05).Conclusions:(1) Multiple differentially expressed mi RNAs exist in human degenerative NP tissues, among which mi R-210 is notably upregulated. These mi RNAs may be associated with the occurrence and development of IDD.(2)q RT-PCR also confirms that mi R-210 is highly expressed in human degenerative NP tissues, and its levels show a positive correlation with Pfirrmann grade.Overexpression of mi R-210 may play an important role in the pathogenesis of IDD.Part II: Isolation and culture of human normal and degenerative NP cellsObjective: To proficiently master the cultural method of human normal and degenerative NP cells, thereby establishing basis for the successful performance of the latter experiments.Methods: NP cells derived from NP tissue specimens of older lumbar disc herniation(LDH) and young lumbar vertebra fracture(LVF) patients were isolated and cultured using combined digestion of 0.25% trypsogen and 0.2% type II collagen(Col II). These cells were then passaged. Normal and degenerative NP cells in thesecond generation were selected as experimental object. Immunocytochemistry staining and toluidine blue staining was used to detect Col Ⅱ and aggrecan. The m RNA expression of Col Ⅱ and aggrecan m RNA was measured by q RT-PCR.Proliferative ability on days 1, 2, 3 and 4 after culture was analyzed using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide(MTT) method. By performing these experiments, we aimed to indentiy whether these cells were NP cells.Results: Under the inverted phase contrast microscope, cells in the primary, first and second generations were vigor and had relatively strong proliferative ability;however, catabiosis occurred in these cells after the third generation.Immunocytochemistry staining, toluidine blue staining and q RT-PCR showed that in the second generation, normal NP cells contained abundant Col II、aggrecan, but their contains in degenerative NP cells were significantly decreased(P<0.05). In addition,MTT method demonstrated that proliferative ability on days 2, 3 and 4 after culture was lower in degenerative NP cells than normal NP cells(P<0.05).Conclusions:(1)Combined digestion of 0.25% trypsogen and 0.2% Col II can successfully culture human normal and degenerative NP cells.(2) NP cells in the first and second generations are suited to perform experiments in vitro.Part III: mi R-210 target autophagy related gene ATG7 to promote ECM degradation in human degenerative NP cellObjective: Col II and aggrecan are the main components of intervertebral disk ECM, which are degraded primarily by matrix metalloproteinase(MMP)-3 and MMP-9. Decreased contents of col II and aggrecan is the classic pathologic feature of IDD. Autophagy is an important pathway to regulate the metabolism of intervertebral disk ECM. ATG7 is closely associated with the formation of phagophore, and it isalso the target gene of mi R-210. However, it is not clear whether mi R-210 is able to regulate the autophagy and ECM degradation via the ATG7 in NP cell. The aim of this study was to observe the effects of mi R-210 on autophagy and ECM degradation in human degenerative NP cell, and to explore its molecular mechanisms.Methods: Bioinformatics were used to validate the binding ability of mi R-210 and ATG7. Wide type psi CHECKTM-2-ATG7-WT 3′UTR and mutant psi CHECK?-2-ATG7-Mut 3′UTR recombinant plasmids were constructed and then cotransfected with mi R-210 mimic into HEK 293 T cells, respectively. The activities of luciferase reporter gene were detected. After culturing of human normal and degenerative NP cells, the expression of mi R-210 and ATG7 in these NP cells was measured using q RT-PCR and/or Western blot. Subsequently, human degenerative NP cells were treated with various concentrations(0, 10, 20, 40 and 80 n M) of mi R-210 for 24 h or with 40 n M mi R-210 for various time(0, 6, 12, 24 and 48 h). The expression of ATG7 m RNA and protein was examined using q RT-PCR and Western blot, respectively.After transfection of human degenerative NP cells with 40 n M mi R-210 inhibitor or mimic for 24 h, monodansylcadaverine(MDC) staning was used to observe phagophore, and the ratio of microtubule associated protein 1 light chain 3(LC3)-II to LC3-I and beclin-1 expression was determined by Western blot. Additionally, the expression of MMP-3, MMP-13, Col II and aggrecan was measured by q RT-PCR and Western blot. Human degenerative NP cells were first pretreated with ATG7 small interfering RNA(si RNA) or the autophagy inhibitor 3-methyladenine(3-MA) and then cotransfected with 40 n M mi R-210 inhibitor. Subsequently, q RT-PCR and Western blot were used to detected the expression of MMP-3, MMP-13, Col II and aggrecan, thereby futher evaluating the role of mi R-210 in regulating ECM degradation.Results: Bioinformatics analysis showed that multiple mi R-210 binding sites existed within human ATG7 m RNA 3′UTR, and the free energy(-17.5kal/mol) was lower. Cotransfection of Wide type psi CHECKTM-2-ATG7-WT 3′UTR with mi R-210 mimic significantly reduced the activities of luciferase(P<0.05). A significant upregulation of mi R-210 expression and a significant downregulation of ATG7expression were observed in human degenerative NP cells when compared with normal NP cells(P<0.05). Transfection of human degenerative NP cells with mi R-210 mimic reduced the levels of ATG7 m RNA and protein in a concentration- and time-dependent manner(P<0.05). In comparison with control group, treatment of human degenerative NP cells with mi R-210 mimic markedly reduced the formation of phagophore, autophagic cell percentage, ratio of LC3-II to LC3-I and the expression levels of beclin-1, Col II and aggrecan(P<0.05), but increased the expression levels of MMP-3 and MMP-13(P<0.05). However, an opposite effect was observed in response to mi R-210 inhibitor(P<0.05). When human degenerative NP cells were pretreated with ATG7 si RNA or 3-MA and then were transfected with 40 n M mi R-210 inhibitor, downregulation of MMP-3 and MMP-13 expression and upregulation of Col II and aggrecan expression induced by mi R-210 inhibitor were partially reversed(P<0.05).Conclusions:(1) Mi R-210 and ATG7 are highly and lowly expressed in human degenerative NP cells, respectively.(2) Mi R-210 can inhibit targetedly the expression of ATG7 m RNA and protein in human degenerative NP cells.(3)Mi R-210 decreases the autophagic levels of human degenerative NP cells by suppressing ATG7, leading to upregulation of MMP-3 and MMP-13 expression and subsequent degradation of Col II and aggrecan.Part IV: mi R-210-induced target silence of Bcl-2 promotes human degenerative NP cell apoptosisObjective: Decreased NP cell number due to apoptosis plays an important role in the occurrence and development of IDD. Bcl-2 is the important factor leading to apoptotic inhibition. However, it is still unclear whether mi R-210 can affect NP cell apoptosis. The aim of this study was to investigate the effects of mi R-210 on Bcl-2expression and apoptosis in human degenerative NP cells.Methods: Bioinformatics were used to analyze the binding ability of mi R-210 and Bcl-2. Wide type psi CHECKTM-2-Bcl-2-WT 3′UTR and mutant psi CHECK?-2-Bcl-2-Mut 3′UTR recombinant plasmids were constructed and then cotransfected with mi R-210 mimic into HEK 293 T cells, respectively. The activities of luciferase reporter gene were detected. After culturing of human normal and degenerative NP cells, the expression of mi R-210 and Bcl-2 in these NP cells was measured using q RT-PCR and/or Western blot. Subsequently, human degenerative NP cells were treated with various concentrations(0, 10, 20, 40 and 80 n M) of mi R-210 for 24 h or with 40 m M mi R-210 for various time(0, 6, 12, 24 and 48 h). The expression of Bcl-2 m RNA and protein was examined using q RT-PCR and Western blot,respectively. According to the results of concentration- and time-dependent experiments, 40 n M mi R-210 was selected and then transfected human degenerative NP cells for 24 h. Apoptotic rates were analyzed by the flow cytometry, and Western blot was used to determine the protein levels of Bax and cleaved caspase-3.Results: Bioinformatics analysis showed that multiple mi R-210 binding sites existed within human Bcl-2 m RNA 3′UTR, and the free energy(-20.5kal/mol) was lower. Cotransfection of Wide type psi CHECKTM-2-Bcl-2-WT 3′UTR with mi R-210 mimic significantly decreased the activities of luciferase(P<0.05). Upregulation of mi R-210 and downregulation of Bcl-2 were observed in human degenerative NP cells when compared with normal NP cells(P<0.05). Transfection of human degenerative NP cells with mi R-210 mimic reduced the levels of Bcl-2 m RNA and protein in a concentration- and time-dependent manner(P<0.05). In addition, treatment of human degenerative NP cells with mi R-210 mimic had no effects on Bax contents(P>0.05)but increased caspase-3 activities(P<0.05).Conclusions:(1) Mi R-210 shows the ability to directly inhibit the expression of Bcl-2 m RNA and protein in human degenerative NP cells.(2) Overexpression mi R-210 can induce human degenerative NP cell apoptosis, which is involved in decreased ratio of Bcl-2 to Bax and activation of caspase-3.