The Role And Regulation Mechanism Study Of MicroRNA-10a-3p In Ossification Of Posterior Longitudinal Ligament

Background and objective Ossification of posterior longitudinal ligament(OPLL)is a common spinal disease,refers to the posterior longitudinal ligament ossification and compression of the spinal cord,easy to cause patients with limbs and trunk sensation,exercise and sphincter dysfunction,and even lead to limb paralysis and incontinence,is a serious harm to human health diseases.OPLL has a population-specific disease,the disease reported in the East Asian countries a higher incidence,scholars believe that genetic factors in the occurrence and development of OPLL played an important role,so to explore OPLL-specific gene changes and regulatory network is particularly important.At present,many osteogenic genes are reported to be closely related to the development of OPLL,and their upstream regulatory factors seem to play an important role in the regulation mechanism of OPLL.Micro RNA(miRNA)is a factor worth exploring.It is reported that miRNA can not only regulate the expression of many genes,but also can change the state of cells.As an important post-transcriptional regulation,miRNA can participate in the process of gene expression in nearly 1/3 of human growth.And so has a wide range of roles.Mi RNAs have a very complex regulatory network,the study found a variety of diseases in the presence of miRNA expression.In order to decipher the upstream regulation network of OPLL,we intend to use the advantages of high-throughput sequencing and bioinformatics to analyze the differentially expressed miRNAs in the posterior longitudinal ligament ossification ligament cells and normal posterior longitudinal ligament cells,the role of differentially expressed miRNAs in osteogenic differentiation of posterior longitudinal ligament cells was studied.There have been many reports that miRNAs can regulate the processes of osteogenesis of many cells,and these osteogenesis processes are precisely regulated by miRNAs,which regulates miRNAs as upstream critical factors in bone formation processes.Thus,the differentially expressed miRNAs in OPLL patients also affect the activation or inhibition of certain ossification-related factors downstream,as well as in the process of ligament ossification.The attendant question is whether the above-described differential expression of miRNAs leads to the promotion and inhibition of osteogenesis of the posterior longitudinal ligament cells through which intermediate signaling pathways With these questions,we designed the following cell experiments and animal experiments in order to explore the upstream and in vivo environment OPLL development of the upstream regulatory factors and its regulatory network for the diagnosis and treatment of OPLL provide a theoretical basis.Part ?: High-throughput sequencing of miRNAs with differentially expressed osteolytic ligaments in the posterior longitudinal ligamentMethods:(1)Intraperitoneal culture of posterior longitudinal ligament cells in vitro.(2)Extraction and isolation of total RNA.(3)Construction of small RNA library and high-throughput sequencing.(4)Mi RNA differential expression analysis.(5)GO and KEGG significant enrichment analysis.(6)Differential expression miRNA validation.Results:(1)There were no obvious abnormalities in the morphology of OPLL ligament cells and normal control ligament cells(P<0.05).(2)There were no obvious abnormalities in the morphology of OPLL ligament cells and normal control ligament cells.2)After the original sequence was processed,the number of small RNA sequences available for analysis in the two groups was 2066542 and 1833901,respectively.The length of the sequence is mainly concentrated in 18~22nt,indicating that the sequence quality is higher.(3)There are 218 miRNAs with differences of more than 2 times,including 144 up-regulated miRNAs and 74 down-regulated miRNAs,with miRNAs with more than 4 times(P <0.01).The GO terminology was associated with the up-regulated gene,and it was found that OPLL cells expressed more ossification than PLL cells,and there were 52 miRNAs with differences of more than 8 times.(4)We usedGO analysis,a total of 65 significant(P <0.01)(5)Real-time PCR showed that the differentially expressed miRNAs were consistent with the high-throughput sequencing results.Conclusion: We used next-generation sequencing techniques(NGS)to assess miRNA and m RNA expression changes.A large number of differentially expressed miRNAs,represented by miR-10a-3p,were found.The results of real-time PCR also demonstrated miRNA expression changes consistent with high-throughput sequencing results.These differentially expressed miRNAs may be involved in the development and progression of OPLL,and provides a new target for OPLL diagnosis or treatment.Part ?: Mi R-10a-3p promotes osteogenic differentiation of posterior longitudinal ligament cellsMethods:(1)Transient transfection of posterior longitudinal ligament cells using transfection reagent,and osteogenesis differentiation of posterior longitudinal ligament cells.(2)Alkaline phosphatase staining and alizarin red staining were used to detect osteogenic ability of posterior longitudinal ligament cells.(3)The expression level of the target protein in the sample was quantitatively analyzed by western blot.Results:(1)Overexpression of miR-10a-3p promoted PLLCs osteogenesis differentiation;(2)inhibition of miR-10a-3p inhibition of OPLLCs osteogenic differentiation.Conclusion: Overexpression of miR-10a-3p can increase the ossification induction level of PLLCs,enhance the activity of alkaline phosphatase and calcium deposition in the posterior longitudinal ligament cells.The expression of miR-10a-3p plays an important role in osteogenesis differentiation of posterior longitudinal ligament cells,and its overexpression in OPLL may play a role in promoting ossification of ligamentous ligamentous cells.Part ?: The mechanisms of miR-10a-3p promoting osteogenic differentiation of ossification ligament cells.Methods:(1)Targetscan 6.0 was used and the potential target genes of miR-10a-3p were significantly enriched by GO and KEGG function.(2)The target gene was verified by luciferase reporter assay.(3)Ch IP analysis of miR-10a-3p target gene downstream pathway.Results:(1)The target genes of miR-10a-3p were predicted to be ID3 and HAND2.(2)Overexpression of miR-10a-3p in PLLCs could reduce the expression of ID3 and inhibit miR-10a-3p in OPLLCs could increase it.(3)Ch IP analysis found that overexpression miR-10a-3p,RUNX2 and OCN/ALP binding in PLLCs significantly increased,and in OPLLCs inhibition of miR-10a-3p,RUNX2 the combination with OCN/ALP was significantly reduced.Conclusion: By predicting the target gene of miR-10a-3p and verifying it,we finally found that the target gene was ID3.The results of Ch IP analysis showed that miR-10a-3p could regulate the ossification level of ligament cells by targeting ID3,promote the combination of RUNX2 and ossification related genes,and promote the development of OPLL.We finally found the function and mechanism of miR-10a-3p in PLL and OPLL ligament cells in vitro.Part ?: The study of miR-10a-3p promotes posterior longitudinal ligament ossification in vivo.Methods:(1)The stable cell lines overexpressing miR-10a-3p were co-incubated with collagen scaffolds and osteogenic differentiation was performed.(2)Micro-CT analysis of samples was performed(BMD,mg / cc)and the ratio of new bone volume to existing tissue volume(BV/TV).(3)HE staining and immunohistochemistry were used to validate the in vitro experimental results.Results:(1)Mi R-10a-3p had the function of promoting the osteogenesis of posterior longitudinal ligament cells in vivo.(2)HE staining showed that OPLLCs had strongosteogenic ability in vivo group formed a larger bone cell defect range and more tissue extracellular matrix stratified bone than other cell type groups.The overexpression of miR-10a-3p in the PLL group resulted in greater lamellar bone formation,whereas inhibition of miR-10a-3p in the OPLL group resulted in reduced bone formation and OCN-treated immunohistochemistry showed that overexpression of miR-10a-3p.Leading to more positive staining of cells,whereas inhibition of miR-10a-3p resulted in a decrease in positive staining cells.However,ID3 showed more positive staining cells after inhibition of miR-10a-3p,whereas overexpression of miR-10a-3p showed less.Conclusion: Mi R-10a-3p plays an important role not only in simple in vitro environment for osteogenic differentiation of posterior longitudinal ligament cells.Under the condition of complex biological in vivo,miR-10a-3p has negative effect on ID3 recognized.SummaryWith the development and application of high-throughput technology,the miRNAs have been greatly expanded and identified in recent years.In this study,the differential expression of ossification in the posterior longitudinal ligament was found by high-throughput sequencing and bioinformatics analysis miRNAs and possible target genes.In our study,we combined with in vitro osteogenic differentiation of ligamentous cells and in vivo bone formation assays found that miR-10a-3p plays an important role in osteogenic differentiation of posterior ligament cells and shows that the target gene ID3 of miR-10a-3p between PLL and OPLL was significantly different,which could play an active role in the development and progression of OPLL.We overexpress miR-10a-3p in PLLCs and inhibit its expression in OPLLCs.The results showed that miR-10a-3p could actively regulate the ossification level of ligament cells by targeting ID3,and promote the expression of RUNX2 and ossification-related genes Combination.In vivo trials also yielded the same conclusion that we identified the upstream regulatory factor for thedevelopment of OPLL,that miR-10a-3p exhibited its function through the ID3 / RUNX2 axis,and for the first time revealed that the miR-10a-3p / ID3 / RUNX2 axis in OPLL.