| Intervertebral disc degeneration(IVDD)is considered to be the pathologic basis of spinal degenerative disease;the degenerative disease of intervertebral disc caused by disc degeneration is a series of clinical syndromes characterized by neck,shoulder,lower back and leg pain,which is one of most common disease in clinic.IVDD not only brings great pain to patients,but also seriously affects their quality of life and health,it also causes huge economic burden on society and families.At present,the clinical treatments of IVDD are mainly to relieve the clinical symptoms by conservative and surgical treatment,which can not prevent,delay or reverse the pathological process of IVDD,and there are no ideal preventive and therapeutic measures.Therefore,it is of great significance to explore the mechanism and slow down the development of IVDD.IVDD is a very complex multi-factor process,and its specific molecular biological mechanism has not yet been fully elucidated.IVDD is characterized by the balance between survival and damage of nucleus pulposus cells.The excessive apoptosis of nucleus pulposus cells is considered to be an important cause of IVDD and plays an important role in the development of intervertebral disc degeneration.Moreover,the apoptosis and decrease in the number of nucleus pulposus cells will lead to the decrease of extracellular matrix synthesis,thus accelerating the process of IVDD.Recent studies have shown that there is a close relationship between inflammatory factors and IVDD.TNF-?,as one of the most important inflammatory factors,can induce apoptosis of nucleus pulposus cells.MicroRNAs(miRNAs/miR),a kind of endogenous noncoding single-stranded RNAs composed of about 20-24 nucleotides,can regulate intracellular gene posttranscriptional expression by interacting with the 3'-untranslated region(3'-UTR)of the target mRNA resulting in translational repression or degradation,which is an important part of gene regulatory network.MiRNAs play essential roles in the regulation of various biological processes such as growth,differentiation,proliferation and apoptosis of normal cells in vivo.Studies have shown that miRNA-mediated post-transcriptional regulation mechanism can affect the process of IVDD.However,the role of miRNAs in regulating apoptosis of nucleus pulposus cells under inflammatory conditions is still unclear.Literature research and summary found that miR-199 may be involved in cell apoptosis by interaction with downstream target genes(apoptosis-related genes,such as MAP3K5,etc).Further bioinformatics analysis software was used to conclude that there were complementary binding sequences between miR-199 and MAP3K5.However,the mechanism of apoptosis of nucleus pulposus cells caused by tumor necrosis factor-?(TNF-?)has not been reported.In this study,we detected and screened TNF-? and miRNAs in clinical specimens and cell models to clarify the differential expression of miRNAs in normal and degenerative intervertebral discs under the action of TNF-?,and discover the relationship between miR-199 and TNF-? in the development of IVDD.Through establishing a human nucleus pulposus cell injury model induced by TNF-?,we verified the role of miR-199 in TNF-?-induced apoptosis of human nucleus pulposus cells,predicted its target gene and clarified its biological role and molecular biological mechanism in TNF-?-induced apoptosis of human nucleus pulposus cells.This will provide a sensitive and specific new molecular diagnostic marker for the early diagnosis of IVDD and scientific evidence of early therapy for IVDD disease.Part ? The expression of TNF-? and miRNAs in nucleus pulposus of human intervertebral discObjective: To investigate the relationship between TNF-? and miRNAs in development of IVDD through analyz the differential expression of TNF-? and miRNAs in normal and IVDD nucleus pulposus tissues.Methods: Each five nucleus pulposus specimens from IVDD patients with degenerated intervertebral disc and young spinal burst fracture patients with no obvious degenerated intervertebral disc in the third hospital of Hebei medical university were collected.The differentially expressed miRNAs were screened by high throughput analysis and the changes of TNF-? in the two groups were detected by ELISA.Results: The result of high-throughput annlysis shows that 30 miRNAs in nucleus pulposus cells of two groups are deregulated,of which 18 miRNAs are down-regulated in IVDD group(including miR-199? miR-502 and miR-203 etc.)and 12 miRNAs are up-regulated(including miR-33b?miR-451 and miR-22 etc.).Among them,the expression of miR-199 was significantly different(Fold change>2).The expression of TNF-? in human degenerative disc nucleus pulposus tissue was significantly higher than that in normal disc nucleus pulposus tissue,and the difference was statistically significant(P<0.05).Conclusion: High throughput screening and qRT-PCR confirmed that the expression of miR-199 in degenerated nucleus pulposus tissue was significantly down-regulated compared with that in normal nucleus pulposus tissue.Compared with nucleus pulposus tissue of normal intervertebral disc,the expression level of TNF-? in degenerative nucleus pulposus tissue was significantly increased.The expression changes of TNF-? and miR-199 indicate that they are closely related to IVDD and may be involved in the occurrence and development of IVDD.Part ? Study of the effect of TNF-? on the expression of miRNAs and apoptosis in human nucleus pulposus cells in vitroObjective: The model of human nucleus pulposus cell injury(apoptosis)induced by TNF-? was established in vitro.High-throughput analysis was performed to screen the differential expression of miRNAs in normal and injured nucleus pulposus cells.The result was verified by the molecular biology.And to explore the effect of miR-199 on human nucleus pulposus cell injury.Methods: Human nucleus pulposus cells were cultured in TNF-?(50ng/ml)condition in vitro.The cell viability was detected by MTT assay,LDH activity by ELISA and mir-199 expression by real-time quantitative PCR at 0h,6h,12 h,24h,48 h differrnt time points under TNF-? treatment.The differentially expressed miRNAs of two groups of Human nucleus pulposus normal control and treated by TNF-? for 24 h.Human nucleus pulposus cells were infected with AAV(recombinant adenovirus-associated virus)-miR Control(miR Ctrl),AAV-anti-miR Control(anti-miR Ctrl),AAV-miR-199(miR-199),AAV-anti-miR-199(anti-miR-199).The expression of miR-199 of infected nucleus pulposus cells was detected by real-time quantitative PCR and was compared to the control group.The cell viability,LDH viability and the expression of apoptotic relative proteins of the Bcl-2 and casepase family were detected after human nucleus pulposus cells were infected with AVV-miR-199 and AVV-anti-miR-199 under TNF-? treatment and were compared to the control group.The potential role of miR-199 in TNF-?-induced human nucleus pulposus cells was further elucidated.Results: The result of high-throughput annlysis shows that 30 miRNAs in nucleus pulposus cells of two groups are deregulated,of which 18 miRNAs are down-regulated in IVDD group(including miR-199? miR-224 and miR-223,etc.)and 12 miRNAs are up-regulated(including miR-33b?miR-498 and miR-1983,etc).Results show that the cell viability and the expression of miR-199 was decreased and the LDH viability was highly increased in Human nucleus with the time of TNF-? treatment(P<0.05).There were no obvious differences in cell viability,LDH viability and miR-199 expression between TNF-? treatment for 24 h and 48 h.Human nucleus pulposus cells transfected with miR-199 obviously increased miR-199 expression,and the expression of miR-199 in Human nucleus pulposus cells with miR-199 inhibitor(anti-miR-199)was significantly decreased(P<0.05).Compared with the control group,TNF-? treated group showed cell activity was significantly decrease(P<0.05),LDH activity was significantly increased(P<0.05),apoptotic rate was significantly increased(P<0.05),the expression of apoptotic relative proteins(Bax,Bcl-2,and Cleaved-caspase-3)and the ratio of Bax/Bcl-2 were significantly increased(P<0.05).Compared with the TNF-? treated group,anti-miR-199 group showed cell activity was significantly decrease(P<0.05),LDH activity was significantly increased(P<0.05),apoptotic rate was significantly increased(P<0.05),the expression of apoptotic relative proteins(Bax,Bcl-2,and Cleaved-caspase-3)and the ratio of Bax/Bcl-2 were significantly increased(P<0.05).Overexpression of miR-199 could revers the above effects.Conclusion: In this part of the study,it is confirmed that TNF-? could induce apoptosis of human nucleus pulposus cells in a time-dependent manner and there is a significant difference in the expression of miR-199 in TNF-? treated human nucleus pulposus cells.Thus it suggests that miR-199 maybe involved in the IVDD.It is clear that the downregulation of miR-199 promotes human nucleus pulposus cells apoptosis under TNF-? treatment.It is concluded that miR-199 may be the key target of TNF-?-induced apoptosis in human nucleus pulposus cells.Part ? The predict and verify of miR-199 target genes and the study of regulation mechanisObjective: The interaction and mechanism between miR-199 and its target gene MAP3K5 were predicted and verified,so as to further clarify the role of miR-199 in TNF-?-induced apoptosis of human nucleus pulposus cells.Methods: The DIANA bioinformatic resources were performed to predict the target genes of miR-199 and the validation of binding site between miR-199 and target genes was determined by dual-luciferase reporter gene assay.After the target gene was determined,the interaction between miR-199 and target gene was further verified.The targeted regulatory relationship and the molecular biological mechanism between miR-199 and target gene MAP3K5 were investigated through experiments.After human nucleus pulposus cells were transfected with AAV-miR Control(miR Ctrl),AAV-anti-miR Control(anti-miR Ctrl),AAV-miR-199(miR-199),AAV-anti-miR-199(anti-miR-199),the expression of mRNA of MAP3K5 was detected by real-time quantitative PCR and protein of MAP3K5 by Western blot detection.The expression of mRNA and protein of MAP3K5 in the TNF-?-induced group and control group was detected.After human nucleus pulposus cells were co-transfected with AAV-miR Control(miR Ctrl),AAV-miR-199(miR-199)and MAP3K5 expression plasmid or empty plasmid under TNF-? treatment,the expression of mRNA and protein of MAP3K5,cell viability,LDH viability,apoptotic rate and the expression of apoptotic relative proteins(Bax?Bcl-2?caspase-3 and Cleaved-caspase-3)were detected.Results: MAP3K5 is a direct target gene of miR-199 by the predictive analysis of DIANA bioinformatics resources,which plays an important role in TNF-?-induced Human nucleus pulposus cell injury.Results of dual-luciferase reporter gene assay: miR-199 binds to the predicated sites on the 3'-UTR of MAP3K5 to inhibit its expression.Compared with the miR Ctrl group,the mRNA and protein of MAP3K5 expression level in the miR-199 group was significantly decreased(P<0.05),but the anti-miR-199 group was significantly increased(P<0.05).Compared with the control group,the mRNA and protein of MAP3K5 expression level in the TNF-?-induced group was significantly increased(P<0.05).Compared with the miR Ctrl group,the mRNA and protein of MAP3K5 expression level in the miR-199+NC group was significantly increased under TNF-? treatment(P<0.05),but which in miR-199+MAP3K5 group was significantly increased compared with miR-199+NC group under TNF-? treatment(P<0.05).Compared with the control group,the cell viability,LDH viability,apoptotic rate and the expression of apoptotic relative proteins(Bax?Bcl-2?caspase-3 ? Cleavedcaspase-3)were significantly decrease under TNF-? treatment(P<0.05),but compared with the miR-199+MAP3K5 group,those results metioned above showed the oppsite trend in miR-199+NC group.Conclusion: MAP3K5 is a direct target gene of miR-199 by the predictive analysis of bioinformatics resources,dual-luciferase reporter gene assay and Western blot detection,and miR-199 binds to the predicated sites on the 3'-UTR of MAP3K5 to inhibit its expression.It is confirmed that miR-199 regulates TNF-?-induced human nucleus pulposus cell apoptosis by through targeting MAP3K5. |
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