Increased MicroRNA-210 Expression Inhibits Autophagy In Human Degenerated Nucleus Pulposus Cell

Part ?.The expression of microRNA-210 and autophagy in human degenerated nucleus pulposus cellsObjective: Human degenerated nucleus pulposus cells were obtained from patients with intervertebral disc degeneration(IDD)and from lumbar vertebral fracture patients that were used as normal control(NC).In order to investigate the expression of microRNA-210 and autophagy in human degenerated nucleus pulposus cells.Method: Human degenerated nucleus pulposus cells were obtained from 12 patients with intervertebral disc degeneration(IDD)(n=24,16 male 8 female,average age 51.4 years,range 39-60 years)and from lumbar vertebral fracture patients(n=6,5 male 1 female,average age 22.2 years,range18-25 years)that were used as normal control(NC).Western blot was used to detect the expression of LC3?/? and Beclin-1 both in 24 human degenerated NP cell samples and 6 normal NP cell samples.Using qRT PCR to detect the expression of microRNA-210 both in 24 human degenerated NP cell samples and 6 normal NP cell samples.Results: In human degenerated NP cell samples,the expression of LC3?/? and Beclin-1 was significantly decreased compared with healthy controls,and the expression of microRNA-210 was significantly increased(1.01±0.11 to 0.64±0.11,t=8.239,P<0.01).Conclusions: Compared with healthy controls,the expression of LC3?/? and Beclin-1 was significantly decreased and the expression of microRNA-210 was significantly increased in human degenerated NP cell samples.Part ?.The effect of micro RNA-210 on extracellular matrix metabolism via regulating autophagy of degenerated nucleus pulposus cellsObjective: To investigate the effect of micro RNA-210 on extracellular matrix metabolism and autophagy of degenerated nucleus pulposus cells,and verify that autophagy is involved in extracellular matrix metabolism of degenerated nucleus pulposus cells.Method:(1)Human degenerated nucleus pulposus cells were transfected with micro RNA-210 mimic,micro RNA-210 inhibitor,mimic control and inhibitor control,q RT PCR and western blot were used to detect the expression of LC3?/?,Beclin-1,Collagen?,aggrecan,matrix metalloproteinase 3(MMP-3)and matrix metalloproteinase 13(MMP-13).(2)Treating human degenerated nucleus pulposus cells with autophagy inhibitor 3-Methyladenine(3-MA),q RT PCR was used to detect the expression of Collagen?,aggrecan,matrix metalloproteinase 3(MMP-3)and matrix metalloproteinase 13(MMP-13).Results:(1)Overexpression of micro RNA-210 significantly downregulated the expression of LC3?/?,Beclin-1,Collagen? and aggrecan,whereas significantly upregulated the expression of matrix metalloproteinase 3(MMP-3)and matrix metalloproteinase 13(MMP-13).Knockdown of micro RNA-210 significantly upregulated the expression of LC3?/?,Beclin-1,Collagen? and aggrecan,whereas significantly downregulated the expression of matrix metalloproteinase 3(MMP-3)and matrix metalloproteinase 13(MMP-13).(2)Treated with autophagy inhibitor 3-Methyladenine(3-MA)significantly downregulated the expression of Collagen? and aggrecan,whereas significantly upregulated the expression of matrix metalloproteinase 3(MMP-3)and matrix metalloproteinase 13(MMP-13).Conclusions:micro RNA-210 inhibits autophagy in human degenerated nucleus pulposus cells,whereas facilitates degradation of extracellular matrix metabolism.Autophagy is involved in extracellular matrix metabolism,which abrogates extracellular matrix loss.Part ?.ATG7 is a target gene of micro RNA-210 and involved in extracellular matrix metabolismObjective: To demonstrate micro RNA-210 directly binding with the 3'UTR of ATG7 which is involved in extracellular matrix metabolism.Method:(1)Took advantage of Target Scan to predict its target gene and binding site.Dual luciferase assay with a p MIR-REPORT vector containing either the putative micro RNA-210 binding site(wild type,WT)or the mutant binding site(MUT)was performed to confirmed the prediction.Human nucleus pulposus cells were transfected with micro RNA-210 mimic,micro RNA-210 inhibitor,mimic control and inhibitor control,then use dual luciferase reporter system to detect luciferase activity.(2)Human nucleus pulposus cells were transfected with ATG7 si RNA and micro RNA-210 inhibitor,then the expression of Collagen?,aggrecan,matrix metalloproteinase 3(MMP-3)and matrix metalloproteinase 13(MMP-13)were detected by q RT PCR and western blot.Results:(1)As bioinformatics software predicted,a binding site existed between micro RNA-210 and the 3'UTR of ATG7.Upregulating micro RNA-210 significantly attenuated luciferase activity of the reporter gene in wild type,but not mutant.Downregulating micro RNA-210 significantly increased luciferase activity of the reporter gene in wild type,but not mutant.(2)Knockdown of ATG7 abrogated the effects of micro RNA-210 inhibitor on Collagen?,aggrecan,matrix metalloproteinase 3(MMP-3)and matrix metalloproteinase 13(MMP-13).Conclusions: micro RNA-210 inhibits autophagy to promote extracellular matrix degradation via directly binding with the 3'UTR of ATG7 in human degenerated nucleus pulposus cells.