Regulation Of MiR-81a-5p In OPLL And The Underlaying Mechanism

BackgroundOssification of the posterior longitudinal ligament(OPLL)is a common spinal ligament ectopic ossification(HO)disease.Due to the limited space in the spinal canal,with the growth of ossification,the corresponding level of spinal cord or nerve root is likely to be damaged by compression,which will lead to physical sensory and motor dysfunction,and ultimately seriously affect human health and quality of life.An epidemiological investigation found that OPLL occurs mainly in the cervical and thoracic spine.Once the spinal cord function of the cervical and thoracic spine is impaired,the neurological dysfunction caused is often widespread,and it seriously affects the quality of life and even the survival time of patients.The current effective treatment is to relieve the direct compression of the spinal cord by surgical removal of the ossification directly or by indirect decompression by expanding the spinal canal volume.However,the risk of cervical and thoracic surgery is high.The ossification and dura mater may have adhesion,so the surgeon needs to choose not to remove or partially resect the ossification according to his own experience,which also places high requirements on the operator's experience and technology.After the operation,there is a certain probability that the ossification continues to grow or even accelerates,making the patient face the risk of secondary surgery.OPLL is a multifactorial disease and may result from age,obesity,diet,diabetes,mechanical stimulation caused by inappropriate posture or abnormal spine activity.Whatever the cause of the disease,it will eventually manifest as the formation of local ectopic ossification of the posterior longitudinal ligament and a gradually increasing pathological process.Therefore,finding the mechanism of ossification and growth in OPLL has been one of the research hotspots in this field.Previous studies have shown that many ossification-related pathways are activated during the ossification of the posterior longitudinal ligament,including the TGF-?/BMP cell pathway,the Wnt/?-catenin pathway,the JAK-STAT pathway,and the PI3K/AKT pathway.These also play a role in fracture healing and bone development.Therefore,we speculate that the activation of these pathways may have more upstream and specific regulatory factors.MiRNAs play a wide regulatory role in human development,diseases,and tumors.The normal osteogenesis process in vivo is also regulated by a variety of miRNAs.After comparing PLL and OPLL cells,my research group found that there was a significant difference in miRNA expression profiles between the two and initially found that miR-10a played an important role in the process of ossification of the posterior longitudinal ligament.The regulation of miRNAs on cell functions is a complex network.Therefore,a deeper study of the regulatory role of miRNAs in OPLL and its mechanism will help to further understand the pathogenesis of OPLL.At the same time,miRNA as a small molecule also has potential for clinical translation.Based on the above understanding,my group found that the expression profile of miRNAs in the posterior longitudinal ligament of OPLL and non-OPLL patients was significantly different through sequencing,and found that miR-10a-3p played an important role in the posterior longitudinal tropical osteogenesis.Therefore,we hypothesized that the miRNA regulatory network was abnormal during the ossification of the posterior longitudinal ligament.Through further analysis of the previous sequencing data,it was found that the expression of miR-181a-5p in the posterior longitudinal ligament tissue of OPLL patients was significantly increased,and it was also one of the top ten miRNAs.Showing a close relationship with ossification-related pathways,our miR-181a-5p may play an important role in the ossification of the posterior longitudinal ligament.In this study,we explored the biological characteristics of miR-181a-5p in primary posterior longitudinal ligament cells,and applied molecular biology methods to analyze and verify the target genes of miR-181a-5p.The regulation mechanism in the process of chemical transformation provides a possible molecular target for the treatment of posterior longitudinal ligament ossification.Part 1:Expression and function of miR-181a in ossification of posterior longitudinal ligamentMethod:(1)Ossification-related miRNA/mRNA interaction network was constructed after bioinformatics analysis of PLL and OPLL miRNA and mRNA sequencing data(GSE69787).After collection of longitudinal ligament tissue,the expression of miR-181a in the difference between the non-OPLL OPLL(hereinafter referred to as PLL)patients verified by RT-qPCR.(2)Collect the patient's posterior longitudinal ligament tissue specimen of posterior longitudinal ligament for primary cell culture.The expression of osteogenic related genes and miR-181a in primary cells was detected after osteogenic induction culture.(3)MiRNA mimics or inhibitors were used to manipulate the expression of miR-181a-5p/3p in PLL cells and OPLL cells.After 21 days of osteogenic induction culture,the osteogenesis ability was evaluated by alizarin red and ALP staining,and the expression changes of mRNA and protein levels of ossification-related genes were detected by RT-qPCR and western-blot respectively.Results:(1)Among the miRNA/mRNA ossification-related networks,miR-181a-5p is the most abundantly expressed in OPLL cells,and is located at the 7th position among all differentially expressed miRNAs,and is the 4th position among all up-regulated miRNAs.RT-qPCR analysis of 10 cases of OPLL-derived and PLL-derived ligament tissue revealed that the expression levels of miR-181a-5p and-3p in OPLL ligament were significantly higher than those in PLL-derived ligament tissue.(2)Under osteogenic induction culture of posterior longitudinal ligament cells,RT-qPCR detection confirmed that the expression of ossification-related genes(RUNX2,OSX,OPN,ALP)gradually increased,and RT-qPCR detection revealed the expression of miR-181a-5p and-3p were increased.(3)After transfection of miR-181a-5p mimics with PLL cells,calcium nodule formation and alkaline phosphatase activity were increased.The mRNA and protein expression levels of ossification-related genes were significantly higher than those of the control group and the negative control group.Over-expression of miR-181a-3p in PLL cells showed no significant changes.In contrast,after OPLL was transfected with miR-181a-5p inhibitors,the osteogenesis of OPLL cells decreased,while the osteogenesis of OPLL cells that inhibited miR-181a-3p did not change significantly.Conclusions:(1)Bioinformatics analysis shows that miR-181a-5p is closely related to ossification-related pathways in OPLL.miR-181a-5p is highly expressed in OPLL cells.The expression of miR-181a-5p and-3p in the posterior longitudinal ligament tissue of cervical spine was higher in patients with OPLL than in non-OPLL.(2)This expression pattern was also found in primary cells isolated from longitudinal tissue.(3)Overexpression/inhibition of miR-181a-5p significantly affects the expression of ossification-related genes in cervical ligament cells after osteogenic induction culture.These results indicate that miR-181a-5p and-3p may be involved in the regulation of ossification in OPLL.Part 2 Prediction and identification of miR-181a target genesMethods:(1)According to the results of previous bioinformatics analysis,the expression of predicted target genes of miR-181a was detected by RT-qPCR in PLL and OPLL cells,respectively.(2)MiR-181a-5p and-3p are overexpressed in PLL cells,and target gene expression changes are predicted by RT-qPCR;(3)PBX1 and ACAN wild type(WT)and mutation(MUT)plasmid were construed by using pMir-Reporter vector.Then the relationship between miR-181a-5p and-3p and target genes was verified by the dual luciferase reporter gene system.(4)RT-qPCR and Western-blot were used to detect changes in the expression of target genes in posterior longitudinal ligament cells after overexpression and inhibition of miRNA,and to analyze their correlation with miR-181a-5p and-3p expression.Results:(1)RT-qPCR detected a total of 8 possible target genes(PBX1,ACAN,S1PR1,GLI2,HAND2,TLL1,HOXA11,RPS6KA3).Four of them(PBX1,ACAN,S1PR1,HOXA11,RPS6KA3)had decreased expression in OPLL cells.(2)RT-qPCR results showed that overexpression of miR-181a-5p in PLL cells could reduce the mRNA expression levels of PBX1 and ACAN in these 8 candidate target genes.Overexpression of miR-181a-3p had no significant effect on the expression levels of these eight candidate target genes.Overexpression of miR-181a-5p can reduce the PBX1 WT and ACAN WT Firefly luciferase/Renialla luciferase ratio,but miR-181a-3p did not significantly change the Firefly luciferase/Renialla luciferase ratio.(4)RT-qPCR and Western-blot results showed that PBX1 and ACAN expression levels in PLL cells significantly decreased at mRNA levels and protein levels after over-expressed miR-181a-5p,but over-expressed miR-181a-3p did not show these effects.Conclusions:PBX1 and ACAN are the target genes of miR-181a-5p,and miR-181a-3p has no targeted regulatory effect on PBX1 and ACNA.Part 3:MiR-181a-5p Regulating Osteogenesis of Longitudinal Ligament Cells by targeting PBX1Methods:(1)The clinical tissues of posterior longitudinal ligament of cervical spine of PLL and OPLL patients were collected,and the expression of PBX1 and AC AN mRNA in tissues was detected by in situ hybridization histochemistry(ISHH).(2)Down-regulate the expression of PBX1 and ACAN in OPLL cells and observe the changes in the expression of ossification-related genes.(3)The rescue assay further verified the regulation of miR-181a-5p targeting PBX1 on the osteogenesis of posterior longitudinal ligament cells.(4)The effect of PBX1 on the nucleosome methylation level in the promoter region of ossification-related genes was studied by co-immunoprecipitation method.Results:(1)ISHH results showed that the expression levels of PBX1 and ACAN in the posterior longitudinal ligament tissue of patients with PLL were significantly higher than those in the control group.(2)The results of RT-qPCR and western-blot showed that siPBX1 significantly upregulated the expression of ossification-related genes in PLL and OPLL cells.siACAN has no effect on the expression of ossification-related genes in PLL and OPLL cells,except that ALP and OCN have an up-regulating effect.(3)When inhibiting the expression of PBX1 or ACAN,alizarin red and ALP staining showed that mineral deposition mineral deposition and ALP activity increased after osteogenesis induction in OPLL cells.MiR-181a-5p knockdown OPLL cells down-regulated mineral deposition and ALP activity after osteogenesis induction.Next,we performed PBX1 or ACAN knockdown in miR-181a-5p-inhibited osteo-induced OPLL cells and evaluated the ossification phenotype.We found that although miR-181a-5p inhibition attenuated the expression of ossification-related genes,lowered the ALP activities and reduced mineral deposition,PBX1,but not ACAN,knockdown significantly rescued these changes.(4)ChIP test was performed after miR-181a-5p mimic were transfected into PLL cells.The results showed that in the OCN and OSX promoter regions,the binding of PBX1 was reduced,the binding of RUNX2 was increased,and the binding of HOXA10 was not significantly changed,the levels of histone H3K9 acetylation was increased,the levels of histone 2-methylation decreased,and transcriptional activity increased.ChIP test performed by using OPLL cells transfected with miR-181a-5p inhibitor showed the opposite result.Conclusions:MiR-181a-5p can target PBX1 and ACAN in posterior longitudinal ligament cells,but mainly regulates osteogenesis of posterior longitudinal ligament cells by targeting PBX1.MiR-181a-5p/PBX1 regulates nucleosome methylation in the promoter region of ossification-related genes(OCN and OSX)to affects downstream gene expression.Part 4:MicroRNA-181a-5p promotes ossification by targeting PBX1 signaling in vivoMethods:After 4 days of infection with lentivirus,PLL cells stably expressing miR-181a-5p mimic,miR-181a-5p inhibitor,siPBX1,and siNC were constructed and then co-cultured with Bio-Oss Collagen scaffold for 2 days.The framework of the heterotopic ossification model is to implant a scaffold with cells subcutaneously on the back of nude mice.After 6 weeks,micro-CT was used to observe the osteogenesis effect in vivo,and the expression of ossification-related genes and miR-181a-5p target gene PBX1 in transplanted materials were detected by immunohistochemistry.The miR-181a-5p agomir and antagomir were constructed and injected into the tail vein of OPLL tool mice(ttw mice),respectively.After 18 weeks,the ossification of the posterior longitudinal ligament of each group of ttw mice was detected by micro-CT to evaluate the miR-181 In-vivo effects of the course of OPLL.To further verify the mechanism of miR-181a-5p regulating OPLL,we detected the expression of ossification-related genes and miR-181a-5p target gene PBX1 in the posterior longitudinal ligament tissue by immunohistochemical methods.Result:Micro-CT data of subcutaneous ectopic ossification specimens in nude mice showed that overexpression of miR-181 a-5p or interference with PBX1 can cause significant increases in BV/TV and BMD values in the transplanted material.When miR-181a-5p expression is inhibited,these parameters are significantly reduced.In addition,immunohistochemical results showed that over-expression of miR-181a-5p significantly up-regulated the expression of ossification-related genes OCN in the specimens,while down-regulating PBX1 expression and inhibiting miR-181a-5p had opposite results.These results indicate that miR-181 a-5p promotes osteogenic differentiation of posterior longitudinal ligament cells in a heterotopic ossification model in nude mice.Conclusion:MiR-181a-5p can down-regulate the expression of the target gene PBX1 in vivo,thereby promoting the expression of ossification-related genes,and ultimately leading to the occurrence of posterior longitudinal ligament ossification.