The Role Of MiR-129-5p In Intervertebral Disc Degeneration And Its Epigenetic Regulation

The detailed mechanisms of intervertebral disc degeneration (IDD) remain not defined.IDD is characterized by changes in extracellular matrix (ECM), amongst which theincrease of collagenⅠand decrease of collagen Ⅱ might be of critical importance. Wehave addressed the microRNAs expression profiles in human IDD, amongst whichmiR-129-5p was down-regulated. COL1A1(Gene of collagenⅠ) and ITGA1(Gene ofintegrin) were predicted as putative targets of miR-129-5p by bioinformatics methods. Themolecular mechanisms of the increase of collagen Ⅰin NP remain enigmatic. Moreover,it has been noted that integrin acts as the receptor of collagenⅠupon human NP cells. Asthe key molecule that simultaneously regulates ligand (collagen Ⅰ) and recepter (integrinα1), miR-129-5p inevitably plays important role in the pathogenesis of IDD. Thedistribution of miR-129-5p, COL1A1and ITGA1in normal and degenerative nucleuspulposus (NP) was identified, the direct modulation between miR-129-5p andCOL1A1/ITGA1was investigated in vitro, the in vivo modulation of miR-129-5p in IDDpathogenesis was investigated and the upstream regulation of miR129-5p was verified. Asthe key molecular, miR-129-5p regulation mechanism in IDD pathogenesis was revealed in this study. The upstream and downstream regulation of miR-129-5p was elucidated aswell. The whole study were devided into four parts:Part1：Expression of miR-129-5p, COL1A1and ITGA1in nucleus pulposusBackground and objective. We have addressed the microRNAs expression profiles inhuman IDD, amongst which miR-129-5p was down-regulated. COL1A1(Gene ofcollagen Ⅰ) and ITGA1(Gene of integrin) were predicted as putative targets ofmiR-129-5p by bioinformatics methods. The increase of collagenⅠis recognized as one ofthe featured mechanisms of IDD. Although as the receptor of collagenⅠupon human NPcells, the role of Integrin α1in IDD remains not reported. To investigate the roles ofmiR-129-5p, COL1A1and ITGA1in IDD, we firstly identify the expression anddistribution of miR-129-5p, COL1A1and ITGA1mRNA, as well as their encoded proteinin normal and degenetive NP.Methods. Human nucleus pulposus(NP) samples were collected from cadavers as controland patients with IDD as degenerative NP samples, qRT-PCR was employed to determinthe expression of miR-129-5p, COL1A1and ITGA1mRNA. Fibrillar collagen in tissuesections was detected by Picric Sirius red staining. Standard HE staining was performed todetermin the degeneration grade of NP. Immunofluorescence and western blot wasperformed to determin the expression and distribution of collagenⅠand integrin α1.Fluorescence in situ hybridization(FISH)-immunofluorescence double labeling wasperformed to observe the co-expression between miR-129-5p and collagenⅠ/integrin α1.Results. Picric Sirius red staining revealed the transformation from collagen II to collagenI in degenerative NP. The expression of miR-129-5p was down-regulated in degenerativeNP, while the expression of ITGA1and COL1A1mRNA was up-regulated,as well as theirencoded protein. FISH-immunofluorescence double labeling showed thatmiR-129-5p-positive NP cells were integrin α1and collagen I-negative, and vice versa.Conclusion. The expression between miR-129-5p and ITGA1or COL1A1mRNA, as wellas their encoded protein, shows a tendency of negative regulation. Part2：In vitro study of miR-129-5p modulates COL1A1and ITGA1in nucleuspulposusBackground and objective. The tendency of nective regulation between miR-129-5p andITGA1or COL1A1mRNA was observed in part1. The next question is the interreactionbetween miR-129-5p and ITGA1or COL1A1mRNA is directly or through certain signalpathways? The3’UTR of COL1A1and ITGA1mRNA were predicted as putative targetsof miR-129-5p through bioinformatics methods, which provided theoretical foundation ofthe direct modulation between miR-129-5p and ITGA1or COL1A1mRNA. The objectiveof this part is to verify the direct modulation between miR-129-5p and ITGA1/COL1A1mRNA, and to investigate the changes of cytobiology function while miR-129-5p isregulated.Methods. Luciferase reporter assay was performed to validate the direct interrationbetween miR-129-5p and ITGA1/COL1A1mRNA3’UTR. Lentivirus was employed toup/down-regulate the expression of miR-129-5p in NP cells, followed by western blottingto determin the chamge of COL1A1and ITGA1level. After confirming that miR-129-5pexists in MG-63and U-2OS by qRT-PRC, lentivirus was employed to up/down-regulatethe expression of miR-129-5p in MG-63and U-2OS, followed by wound healing test andtrans-well test to investigate the changes of cytobiology function.Results.3’UTR of ITGA1and COL1A1mRNAare direct targets of miR-129-5p.Up-regulation/down-regulation of miR-129-5p resulted in repression/over-expression ofintegrin α1and collagenⅠin primarily cultured NP cells. As revealed by wound healingassay, the spreading of MG-63and U-2OS into the incised area was faster in themiR-129-5p up-regulation group than in down-regulation group. As demonstrated intrans-well assay, the migration and invasion of MG-63and U-2OS incresed inmiR-129-5p up-regulation group while decreased in miR-129-5p down-regulation groupcompare to the control group.Conclusion. miR-129-5p inhibits integrin α1and collagen I expression by directlytargeting their3’-UTRs of mRNA, which leads to the alteration of cytobiology function. Part3：In vivo study of miR-129-5p modulates COL1A1and ITGA1in nucleuspulposusBackground and objective. As part1and part2revealed, miR-129-5p can inhibit theexpression of COL1A1and ITGA1by directly targeting the3’UTRs of their mRNAs.Different from in vivo study, the cellular composition is simple, the culture condition canbe easily controlled and the influencing factors are relative simple, in in vitro study. Theobjective of this part is to investigate the in vivo modulation function of miR-129-5p inIDD using C57mouse tail annulus puncturing model.Methods. Using qRT-PCR, we confirmed the miR-129-5p expressed in various organs ofC57mice. miR-129-5p level was up/down-regulated through adeno-associated virus (AAV)in C57mouse tail annulus puncturing model. Western blot for collagenⅠand integrin α1was performed in3,6,12weeks after puncturing. Disk height index (DHI) was calculated,standard HE staining and FISH-immunofluorescence double labeling was performed toinvestigate the degeneration grade of the disc at each time point.Results. The expression of CollagenⅠand Integrin α1incresed with time at3,6,12weeksafter puncturing. Up/down-regulation of miR-129-5p led to decrease/increase ofexpression of CollagenⅠand Integrinα1at each time point.%DHI was higher andhistology score was lower in the up-regulation of miR-129-5p group than in thedown-regulation group.Conclusion. Down-regulation of miR-129-5p was directly connected with deterioration ofIDD and up-regulation would alleviate the degree of IDD.Part4：Epigenetic regulation of miR-129-5p in nucleus pulposusBackground and objective. Part1-part3revealed the direct downstream regulation ofmiR-129-5p in IDD with in vitro and in vivo vilidation, while the upstream regulation ofmiR129-5p is not identified. The objective of this part is to investigate the upstreamregulation of miR129-5p.Methods. Human NP samples were collected from cadavers(n=5) as control and patients with IDD (n=5) as degenerative NP samples. Altogether four CpG islands was foundwithin the range of10kb away from the promoter regions of miR-129-1and miR-129-2.MeDIP-PCR was performed in the four CpG islands.5-Aza-2’-deoxycytidine(5Aza-CdR) was used to treat primarily cultured NP cells in vitro, followed by qRT-PCRto determin the expression of miR-129-5p, COL1A1and ITGA1mRNA.Results. The degree of DNA methylation was significant elevated (P<0.05) at the twoCpG islands locate on miR-129-2chr11:43596990-43597336andchr11:43602545-43603215. Treated with5Aza-CdR, the expression of miR-129-5p wasincreased while the expression of ITGA1and COL1A1mRNA was decreased in NP cells.Conclusion. The aberrant down-regulation of miR-129-5p in degenerative NP isassociated with DNA hypermethylation of the two CpG islands locate on miR-129-2chr11:43596990-43597336and chr11:43602545-43603215.