| Objective：To investigate expression changes of microRNA (miRNA) and proteinase in humannormal and degenerated nucleus pulposus. Applying the bioinformatics prediction methodsto find the corresponding relationship between them and explore their effect on discdegeneration. Finally, we provide an initial theoretical support for the in-depth explorationof microRNA regulating the corresponding proteinase in human degenerated intervertebraldiscs.Methods：①Control group (Normal): Normal nucleus pulposus consisted of five cases (male3,female2), which were obtained from patients who suffered from traumatic spinal fracturesand underwent spine surgery in the Second Affiliated Hospital of Soochow University inthe period of April2013to November2013.The mean age was40.60(30~50).②Experimental group (IVDD): Degenerated nucleus pulposus consisting of twentycases (male10, female10) were obtained from patients with lumbar disc herniation in theSecond Affiliated Hospital of Soochow University during the same period, the mean agewas45.20(16~65).All of the specimens were carried out paraffin sections routinely and HE staining wasapplied to evaluate morphologic changes and diagnosis the pathology of the specimens; thetarget genes of microRNA—mRNA of proteinase were predicted by the bioinformaticsprediction methods, then real-time quantitative PCR was used to detect the expressionchanges of microRNA and corresponding proteinase gene in the two groups of the nucleus pulposus.Results：1. HE staining of nucleus from patients with spinal fractures revealed many smallcells, namely chondrocyte-like cells, which were round and sitting in the cartilage lacunaand interspersed in the extracellular matrix. A small number of cell clusters were alsofound. The collagen fibers surrounded the chondrocyte-like cells regularly forming anetwork structure. However, nucleus of patients with lumbar disc herniation mainlyshowed decreased number of cells, with cytoplasmic vacuolar, cell lysis and the cell imagewas not clear, apoptotic cells and cell aggregation phenomena also increased, disorganizedcollagen fibers and cracks appeared. HE staining showed that specimens from controlgroup were normal, with no obvious degenerated signs; IVDD group revealed obviousdegenerated changes.2. RT-PCR results were expressed as mean±standard deviation, statistical analysiswas carried out by two-tailed Student’s t-test (two-group comparisons), statisticalsignificance was determined by a P-value less than0.05. Bioinformatics predicted thattarget gene of human miR-663b, miR-625-5p was MMP-2and ADAMTS-4respectively,and target genes of miR-623was both the MMP-3and MMP-13. RT-PCR results showedthat miR-663b expression level reduced significantly in the degenerated nucleus, butmiR-625-5p and miR-623changes in the two samples were not statistically significant;mRNA expression levels of MMP-2, ADAMTS-4, MMP-3and MMP-13increasedsignificantly.Conclusion：In the degenerated disc, gene expression levels of proteinase MMP-2, ADAMTS-4,MMP-3and MMP-13increased significantly, suggesting their involvement in theextracellular matrix degradation, ultimately resulted in disc degeneration; miR-663bexpression decreased in degenerated disc, and had a corresponding relationship with theproteinase MMP-2mRNA, this result suggested that the downregulation of miR-663b maycontribute to the upregulation of MMP-2mRNA. The detection of expression levels of several microRNA and proteinase in degenerated nucleus pulposus can provide atheoretical basis for the future to further explore the regulatory mechanisms between them. |
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