Screening And Validation Of Differentiauy Expressed MicroRNAs In Intervertebral Disc Degeneration

Objective:To detect miRNA expression profiling of nucleus pulposus cells (NPc) in human lumbar intervertebral disc, screen differentially expressed miRNAs between degenerative and normal NPc, predict the target genes and signaling pathways of differentially expressed miRNAs. To validate the differential expression of miR-494and miR-513a-5p in human degenerative lumbar intervertebral disc tissue. The study is to provide experimental basis for the further research of the role of miRNAs in pathogenesis of intervertebral disc degeneration.Methods:1. Three cases degenerative NP tissue and one case normal tissue as control were harvested from patients of lumbar disc herniation and lumbar fracture surgery respectively and divided them into the degenerative group and normal group. By the method of enzymatic digestion isolated nucleus pulposus cells from tissue, and cell culture were performed. When the first generation of cell crawling piece to70%to80%confluence, we removed cells crawled piece, stained of the nucleus pulposus cells, extracted total RNA of specimens. We performed a miRNA microarray gene expression experiments and analysis techniques to analyze miRNA expression profiles and screened out differentially expressed miRNAs. By MicroCosm v5TargetScan5.1, and MICRORNA.ORG softwares to predict the target genes of differentially expressed microRNAs. Then the target genes of deregulation miRNAs were subjected to gene ontology(GO) analysis, counted the number of differential genes or target genes in each GO term, and used Fisher exact test statistical methods to calculate enriching significance of differential genes or target genes in each GO term. We performed pathway significance analysis to count the number of differential genes or target genes in each pathway, using the hypergeometric distribution statistical test method to calculate the Pvalue which reflect the enriching significantly of pathway in differences in the gene or target gene distribution and enrichment of differential genes or target genes in each pathway, according to the Pvalue size to find out significant related function biological pathways to differential genes or target genes.2. Five cases degenerative NP tissue and two cases normal tissue as control were harvested from patients of lumbar disc herniation and lumbar fracture surgery respectively and divided them into the degenerative group and normal group. Removed the specimens stored in RNA preservation solution after surgery immediately, saved at4℃for one night, then toke out and separated NP tissue. Part of the specimens were verified degeneration by biopsy and staining, then another part of the specimens were detect the differential expression of miR-494and miR-513a-5p using Real-time qPCR technology. According to analytical method of A ΔCt to design the experiment, using the dye method (SYBR Green I) to do relative quantitative analysis.Results:After enzymatic digestion and culture, degenerative and normal nucleus pulposus showed a typical shape of the nucleus pulposus cells. miRNA microarray gene expression experiments and analysis found that in degenerative group there20kinds of up-regulating miRNAs (miR-21*、miR-663、miR-29c*、miR-638、miR-572、 miR-1225-5p、miR-513a-5p、miR-101、miR-1275、miR-10b、miR-923、miR-195, miR-575、miR-181d、miR-494、miR-765、miR-210、miR-497、miR-483-5p、 miR-345), In which, miR-513a-5p and miR-494showed significantly high expression, Ratio value was2.2222and2.9485respectively. The target gene of miR-513a-5p and miR-494was MKK4and JunD respectively, comprehend three softwares of MicroCosm v5, TargetScan5.1and MICRORNA.ORG forecast outcome. These two target genes were involved in the JNK signaling pathway, and the two target genes were significantly up-regulating in this pathway.Through Real-time qPCR to detect the difference of two miRNAs in the nucleus pulposus tissue found that in degenerative group miR-513a-5p and miR-494showed a significant higher expression than normal group,2-ΔΔCt were of47.3660±39.052513.5560±7.9860(P<0.05) respectively, there was statistical significance, both miRNAs high expression difference multiples were more manifest compared to gene microarray results. We validated the reliability and accuracy of the results of differential expressed genes screening by microarray.Conclusions:This is the first research of miRNA expression profiling analysis about human lumbar intervertebral disc nucleus pulposus cells by microarray technology, screened out20kinds of up-regulating miRNAs in degenerative intervertebral disc, in which two kinds of miRNAs:miR-513a-5p and miR-494showing extremely significant up-regulating. MKK4and JunD was the target gene of miR-513a-5p and miR-494has been predicted using scientific method, the pathway of JNK may be their common pathway to educe function. The differential high expression of these two miRNAs in intervertebral disc nucleus pulposus tissue has been validated by technology of Real-time of qPCR, the experiment outcomes correspond with microarray absolutely. The results of this study are useful to the further research of pathogenesis of intervertebral disc degeneration at the genetic level. It can provide experimental basis for possible future genetic diagnosis, genetic intervention and disease prevention.