Correlation Study Between Nucleic Acid Markers In Peripheral Blood And The Prognosis Of Ossification Of Posterior Longitudinal Ligament

Background and objectiveOssification of posterior longitudinal ligament(OPLL)is a severe degenerative spinal disease characterized by abnormally ossified posterior longitudinal ligament of the cervical spine that compresses the spinal cord and nerve roots,leading to neurological dysfunction.Clinical symptoms mainly include sensory and motor dysfunction of limb and trunk,sphincter dysfunction,and neck stiffness and pain.Severe ossified ligament can lead to quadriplegia and incontinence,greatly reducing the quality of life in patients with OPLL.Epidemiological studies have shown that OPLL is more likely to occur in men and its average age of onset is 50 years.The incidence of OPLL has significant racial differences,and the incidence rate of East Asian population is higher.The survey data shows that the incidence rate is 0.1%-4.3% among different ethnic groups.At present,scholars consider that OPLL is a disease caused by a combination of genetic factors and environmental factors,but the specific pathogenesis is still unclear.Most patients with OPLL have no obvious clinical symptoms or only mild symptoms such as hand numbness and neck discomfort in the early stage,which failed to pay enough attention.Due to the growth of ossification or cervical trauma,patients may appear symptoms of cervical myelopathy or radiculopathy.At this moment,the condition is often serious and the prognosis is poor.Therefore,early diagnosis and treatment of OPLL have a great impact on its prognosis.The clinical diagnosis of OPLL is mainly based on the patient's symptoms,signs and imaging findings.Although the high-resolution CT is golden standard for the diagnose of OPLL,CT examination has many disadvantages such as radiation damage,expensive equipment and high inspection cost,which is not conducive to early screening test of OPLL.Therefore,the search for an effective,simple,safe,and economical method for early screening and diagnosis of OPLL is an urgent problem to be solved.In addition,scholars generally believe that surgery is the only effective treatment for OPLL patients with severe cervical spondylosis symptoms.However,when patients have no symptoms or only mild cervical spondylosis symptoms,whether surgery should be performed has been controversial.Therefore,it is of great significance to explore the prognostic factors of OPLL for guiding clinical treatment,reasonably choosing the surgical timing and evaluating the prognosis of the disease.At present,a large number of studies have found that multiple genes play a role in the etiology and pathogenesis of OPLL.Epigenetics is an important way of regulating gene expression.It can change the expression of genes without changing the DNA sequence,including miRNA and DNA methylation.MicroRNAs are a class of non-coding single-stranded RNAs with 18-22 nt in length that regulate gene expression after transcription and play an important role in the pathogenesis of tumors,autoimmune diseases,cardiovascular diseases,and osteoarthritis.DNA methylation is that a methyl group covalently binds to the 5th carbon atom of cytosine to form 5-methylcytosine.In mammals,DNA methylation occurs primarily in CpG dinucleotides,which regulates gene transcription by affecting the binding between transcription factors and chromatin.The studies have shown that DNA methylation plays a role in the pathogenesis and progression of various diseases such as cancer,aging,diabetes,autoimmune diseases,and cardiovascular diseases.These studies suggest that there may be abnormally expressed miRNAs and DNA methylation in the patients with OPLL,which may influence the occurrence and development of OPLL by regulating gene expression.Therefore,we designed the following experiments to explore the relationship between miRNA and DNA methylation and the prognosis of OPLL.Part ?: MiRNAs high-throughput sequencing in peripheral blood of OPLLMethods:(1)Collecting the posterior longitudinal ligament tissue and peripheral blood of surgical treatment of OPLL patients and non-OPLL patients;(2)MiRNA sequencing was performed on tissue and peripheral blood samples by using high-throughput sequencing technology;(3)The bioinformatics analysis was performed to screen out miRNAs with significantly different expression in OPLL;(4)The different expression of miR-218 in peripheral blood of OPLL was verified by real-time PCR.Results:(1)In the posterior longitudinal ligament tissue of OPLL,the expression of miR-10a-5p,miR-10a-3p,miR-563,miR-885-5p and miR-210-3p was significantly up-regulated.However,the expression of miR-199b-5p,miR-196b-5p,miR-129-3p,miR-218-3p and miR-212-3p was significantly down-regulated.(2)In peripheral blood of OPLL,the expression of miR-484 and miR-4731-5p was significantly up-regulated.However,the expression of miR-218-5p and miR-9-3p was significantly down-regulated.(3)The results of the two experiments were taken as intersections.Only miR-218 was significantly different expression in tissues and peripheral blood of OPLL.(4)Thereal-time PCR revealed a significant down-regulation expression of OPLL-specific miR-218.Conclusion: There is a significant difference in miR-218 expression in the posterior longitudinal ligament tissue and peripheral blood of patients with OPLL.Compared with the control group,the expression of miR-218 in peripheral blood of patients with OPLL was significantly down-regulated,and the low expression of miR-218 may be related to the pathogenesis and progression of OPLL.Part ?: DNA methylation high-throughput sequencing in peripheral blood of OPLLMethods:(1)Collecting peripheral blood from 20 patients with OPLL and 20 healthy subjects;(2)Extracting DNA from peripheral blood cells;(3)Constructing CpG methylation library;(4)Conducting CpG precise methylation sequencing;(5)Different DNA methylation regions(DMRs)were found by data analysis and performed functional annotation analysis.Results:(1)A total of 250,640 DMRs were obtained by the methylation data analysis of two groups,and 197 significant DMRs were screened out.(2)DMR annotation found out 197 genes,and GO analysis screened three genes related to bone development:BMP5,BMPR1 B and FGFR1.In the OPLL group,the methylation levels of BMP5 and BMPR1 B genes were significantly decreased,and the methylation level of FGFR1 was significantly increased.Conclusion: Hypomethylation of BMP5 and BMPR1 B and hypermethylation of FGFR1 may play a role in the pathogenesis and progression of OPLL through gene expression regulation.Part ?: Correlation study between miRNA and DNA methylation in peripheral blood and the prognosis of OPLLMethods:(1)Collecting peripheral blood from 52 surgical patients with OPLL;(2)Evaluating neurological function of patients preoperatively and at 1-year follow-up;(3)Detecting the expression level of miR-218 in peripheral blood by utilizing the high-throughput sequencing;(4)Detecting the expression levels of BMP5,BMPR1 B and FGFR1 methylation in peripheral blood by using the CpG precise methylation sequencing;(5)To analyze the correlation between the expression levels of nucleic acid markers in peripheral blood and the recovery of neurological function after surgery.Results:(1)Compared with the group with poor neurological recovery,the expression level of miR-218 in preoperative peripheral blood of patients in the group with better neurological recovery was significantly higher,while the level of FGFR1 methylation was significantly lower.(2)The multivariate logistic regression showed that the expression level of miR-218 was a protective factor for unfavourable prognosis of patients with OPLL,while the level of FGFR1 methylation was a risk factor for unfavourable prognosis.Conclusion: The expression levels of miR-218 and FGFR1 methylation in peripheral blood of patients with OPLL are related to the recovery of postoperative neurological function.They can be used as the potential biomarkers of prognostic evaluation for OPLL.SummaryCervical OPLL is a serious degenerative spinal disease with complicated pathogenesis and unclear etiology.At present,the diagnosis and prognosis assessment of OPLL are mainly based on the patient's symptoms and imaging findings,and no effective body fluid test indicators have been found for the diagnosis and treatment of OPLL.As the important mechanism of epigenetics,miRNA and DNA methylation play an important role in the pathogenesis and progression of a variety of diseases.Meanwhile,they can be used for the diagnosis,prognosis assessment and targeted therapy of diseases,but the correlation study between miRNA or DNA methylation and the prognosis of OPLL has not been reported.We used high-throughput sequencing technology to find a significantly down-regulated miR-218 in OPLL patients' tissues and peripheral blood,which may be related to the pathogenesis of OPLL.By conducting CpG precise methylation sequencing of peripheral blood in patients with OPLL,we found that the methylation level of BMP5 and BMPR1 B were significantly decreased,while the methylation level of FGFR1 was increased.The differentially expressed DNA methylations may play a role in the pathogenesis of OPLL by affecting the process of ossification.In addition,by analyzing the correlation between the expression levels of nucleic acid markers in preoperative peripheral blood of OPLL and the improvement of postoperative neurological function,we found that the expression level of miR-218 is a protective factor for unfavourable prognosis of patients with OPLL,while the FGFR1 methylation level is a risk factor for unfavourable prognosis,which suggested thatmiR-218 and FGFR1 methylation can be used as the potential biomarkers of prognostic assessment for OPLL.