MicroRNA-10a,-210 And -563 As Specific Diagnostic Markers In Peripheral Blood For Ossification Of The Posterior Longitudinal Ligament

Background and objectiveOssification of posterior longitudinal ligament is known for us all,refers to the longitudinal ligament heterotopic Ossification behind cervical vertebra and compression of the spinal cord,resulting in a corresponding neural dysfunction,such as the limbs and trunk sensory,motor and sphincter of oddi dysfunction,appear even quadriparesis and incontinence.OPLL has a population specificity,and the literatures report that the disease has a high incidence in east Asian countries.The disease was first reported by Japanese.Therefore,it is also known as "Japanese disease".The literatures report that the disease is related to endocrine hormones,mechanical stress stimulation and genetic factors.However,the single factor cannot explain the pathogenesis of ossification of the cervical spine posterior longitudinal ligament.Scholars believe that gene regulation is closely related to the ossification of posterior longitudinal ligament,so it is particularly important to explore OPLL specific gene changes and explore its pathogenesis.In addition,OPLL is concealed and there is no early specific symptom,no effective early screening and diagnosis method.Diagnostic gold standard of OPLL is cervical CT.However,the disadvantages of longer appointment time,large radiation dose and high price restrict the early diagnosis of OPLL.CT examination frequency grow more than 8 times around the world in the past 2 decades.The radiation accepted by patients also increases accordingly.Now multislice CT application becomes increasingly prevalent in our country,for its fast scanning speed and high reconstruction image quality,which has improved the diagnosis accuracy and efficiency.However,CT examination brings greater radiation,increases the risk of cancer,proved to be an urgent problem to be solved.Now more literatures report that OPLL is related to gene expression and their upstream regulatory factors seem to play an important role in the regulation mechanism of OPLL.MicroRNA(miRNA)plays a vital role in many regulatory mechanisms.MiRNA is a class of highly conservative non-coding RNAs with a length of about 22 nt,which has the function of regulating gene expression after transcription.At present,more than 1000 human miRNAs have been found to regulate gene expression.At least one third of them participate in various physiological and pathological processes,such as growth and development,immune response,and tumor occurrence.In the field of tumor occurrence,there is a large number of experimental analyses indicate that up-regulation or down-regulation of certain specific miRNA expression is closely related to tumor occurrence.Based on the above phenomenon,we designed relevant experiments to screen the specific miRNAs of OPLL,so as to provide a new method and new way for the diagnosis of non-invasive and efficient diagnosis of OPLL.Part ?: OPLL-specific miRNAs screening.The part of this paper consists of experimental results in the early stage,we used high-throughput sequencing technology to screen the differentially expressed in OPLLCs miRNAs,and,identified the five significant increase of miRNAs(miR-10a-3p,miR-10a-5p,563,885-5p and 210-3p)and five significantly lower miRNAs(miR-129-3p,199b-5p,212-3p,218-3 and 196b-5p).Subsequently,real-time PCR was used to verify the results,and a strong correlation was found between various miRNAs and OPLL,suggesting that miRNAs with differential expression may be related to the occurrence and development of OPLL.It provides a new thinking and working basis for the study of the mechanism of ossification of the posterior longitudinal ligament and the targeted therapy.Part ?: Verification and stability test of peripheral blood miRNA.Methods :(1)Five healthy volunteers were selected according to the imaging findings and clinical symptoms,and the peripheral blood was collected separately.(2)Five up-regulated miRNAs were verified by qPCR;(3)The stability of miRNAs in serum and plasma was verified by freeze-thaw experiment.Results :(1)Five healthy volunteers were extracted from the peripheral vein with 2 tubes(one tube with EDTA inside prepared for separation of plasma;the other one prepared for separation of serum)in the morning with an empty stomach.(2)In serum and plasma,the five most significant miRNAs(miR-10a-3,miR-10a-5p,miR-563,miR-210-3p,and miR-885-5p)could be detected,and the miRNAs expression levels showed no significant difference.(3)miR-563,miR-10a-3p and internal reference U6 showed no significant decrease after repeated freeze-thaw,indicating that it was stable in the external environment.Conclusion: We used qPCR to prove that there are many differentially expressed miRNAs in the peripheral blood of normal population.And they are widely available in serum and plasma,which can be used for follow-up experiments.In addition,the freeze-thaw experiment indicated that some miRNAs were stable in the external environment,which provided the possibility for miRNAs to be the diagnostic OPLL specific biomarker.Part ?: Detection of peripheral blood OPLL diagnostic miRNA.Methods:(1)According to the imaging findings and clinical symptoms,we divided objects into 3 groups: OPLL group,Intervertebral disc degeneration(IDD)group and normal control group;(2)Collect peripheral blood for all objects of study;(3)Real-time PCR analysis was implied for miRNAs expression.(4)Statistical analysis of differences in miRNAs among the three groups;(5)Determine the specific miRNAs for OPLL diagnosis,and draw the AUC curve to determine the diagnostic specificity of miRNAs.Results :(1)According to the image findings and clinical symptoms,there were 68 patients in OPLL and 45 patients with IDD.There were no significant differences in the general parameters of age and gender in the three groups.(2)Peripheral blood was extracted from three groups of patients in the morning with an empty stomach(using EDTA to collect blood vessels);(3)The expression levels of miR-10a-3p,miR-196b-5p and miR-129-2-3p were significantly different in OPLL group and non-OPLL group,and there was no significant difference between the IDD group and OPLL group.The expression of miR-218-1-3p was significantly different in OPLL group and IDD group,and there was no significant difference between OPLL group and normal control group.The expression levels of miR-10a-5p,miR-563 and miR-210-3p were significantly different in the three groups.(4)AUC proved to be 0.950 when mir-10a-5p,mir-563 and mir-210-3p combined together.Conclusion: MiR-10a-5p,miR-563 and miR-210-3p have significant differences in the expression levels in the three groups,and the AUC of the three miRNAs is 0.950,indicating that the combination of the three can be used as a specific biomarker for the diagnosis of OPLL.SummaryOPLL is a common spinal ligament ossification disorder in clinical work,refering the spinal cord compressed,resulting in a heavier nerve dysfunction.No symptoms or mild symptoms presented in the early stages of the disease.In the late stage of severe compression of the spinal cord,there are symptoms of walking disorder or incontinence.The gold standard for diagnosing the disease is high resolution thin-layer CT,but CT examination is radiative.In recent decades,with the development and application of circular marker detection technology,miRNA has been greatly expanded and identified.It has been confirmed that miRNA abnormalities are closely related to tumor occurrence,viral infection and other diseases,but the relationship between them is not clear.Aiming at the above problems,we innovatively start with microRNAs and find the connection between them.By using the advantage of high-throughput technology,the miRNAs of OPLL differential expression were selected from the posterior longitudinal ligament cells,and the miRNAs expressed by the difference expression through real-time PCR were basically consistent with the high-throughput sequencing results.Subsequently,peripheral blood in patients with OPLL further validated the expression level in serum and plasma of the up-regulated five kinds of micrornas(miR-10a-3p,miR-10a-5p,miR-563,miR-210-3p,miR-885-5p),showed the miRNA widely exists in peripheral blood,stable structure,and can be detected.Finally,we compared the selected 10 miRNAs in different spinal diseases to define OPLL specific biomarkers: miR-10a-5p,miR-563 and miR-210-3p.Three kinds of microRNAs in combination with ROC curve analysis showed AUC 0.950,providing another way for the diagnosis of OPLL.Our research has not only provided a preliminary theoretical basis for the diagnosis of OPLL but also provides the explanation ligament ossification development direction.