Cyclic Mechanical Stress Promotes NPMSCs Proliferation And Differentiation Through MiR-5100/PRTG/integrin α1 Signaling Pathway

Part Ⅰ: Expression of extracellular matrix and alteration of microRNA-5100 gene expression in rat degenerative intervertebral disc tissues under cyclic mechanical stressObjective: At present,there is still a lack of safe,effective and minimally invasive treatment for intervertebral disc degeneration(IDD).With the development of stem cell engineering,tissue engineering and molecular biology,a method to inhibit nucleus pulposus degeneration and restore the quantity and quality of nucleus pulposus cells has gradually aroused the interest of researchers in the field of intervertebral disc regeneration.At the same time,tissue engineering of lumbar intervertebral disc nucleus pulposus has become one of the most promising methods for the treatment and repair of spinal degenerative diseases.SD rats of four sizes were taken,and the models of coccygeal disc degeneration were made by coccygeal disc puncture.Nucleus pulposus cells were extracted from the coccygeal disc tissue of rats as the experimental group and the control group,and the changes of microRNA-5100 and chondroblast gene expression levels in nucleus pulposus cells and nucleus pulposus mesenchymal stem cells in the degenerative disc tissue were studied.Methods: The cyclic mechanical stress field was established,and then cultured in the self-developed cyclic mechanical stress field culture system.Finally,nucleus pulposus cells were extracted and treated with nucleus pulposus mesenchymal stem cells.The expression levels of miR-5100 and other genes in the nucleus pulposus cells of the experimental group and the control group were determined by qRT-PCR.To investigate the differentiation ability of cyclic mechanical stress under the action of chondrogenic differentiation kit.Results: The rat model was established successfully.Under the action of periodic mechanical stress,mesenchymal stem cells in nucleus pulposus transformed into nucleus pulposus cells with high expression of miR-5100,Sox,AGG and CollegenⅡ(P < 0.05).Conclusion: Under cyclic mechanical stress,not only can nucleus pulposus mesenchymal stem cells differentiate into nucleus pulposus cells,but also can promote the proliferation and differentiation of degraded nucleus pulposus cells and nucleus pulposus mesenchymal stem cells in rats and promote the high expression of miR-5100.Part Ⅱ: Periodic Mechanical Stress Stimulation of miR-5100 expression Targeting PRTG promotes proliferation and differentiation of mesenchymal stem cells in the nucleus pulposus through the integrin α1 signaling pathwayObjective: Although intervertebral disc(IVD),an indispensable part of the spine,is only a kind of fibrocartilage,its unique physiological position and structure determine that the spine can absorb and transmit mechanical load and allow spinal movement,playing an important role in intervertebral joint and movement.Intervertebral disc degeneration is the pathophysiological basis of a range of intervertebral disc diseases.Because intervertebral disc is subjected to different mechanical stresses all the time,our previous studies have found that the unique periodicity of cyclic mechanical stress and appropriate pressure cause a series of signal pathway and signal molecule changes in nucleus pulposus mesenchymal stem cells(NPMSCs),and these changes lead to an increase in their number.As we go further,we find that there are few reports and even fewer studies of these mechanisms.Therefore,based on the previous studies of our research group,this study clarified the molecular mechanism of miR-5100 and proposed that under the action of periodic mechanical stress,the expression of miR-5100 in nucleus pulposus mesenchymal stem cells increased and targeted PRTG affected the expression of chondroblast genes and transmembrane protein integrinα1 in the cells.To determine its upstream role in the cell,to determine the relationship between the upstream and downstream.To explore a new mechanism of how cyclic mechanical stress induced proliferation and differentiation of nucleus pulposus cells and activated extracellular matrix during physiological processes.Method: Under the action of periodic mechanical stress,quantitative reverse transcription polymerase chain reaction(RTq PCR)analysis was carried out to determine the expression level of miRNA-5100 gene in the experimental group and the control group.Dual luciferase assay was used to determine the relationship between miRNA-5100 and PRTG,HE staining experiment,DNA AGAR gel electrophoresis,The miRNA-5100,PRTG and Integ-Rin-α1 pathways were analyzed by PCR amplification.Conclusions: Under periodic mechanical stress,the fluorescence intensity of PRTG in the experimental group was significantly decreased(P<0.0001)after transfection of miR-5100 compared with the control group,indicating the presence of targeted binding between PRTG and miR-5100.Under the action of periodic mechanical stress,the expressions of PRTG,RUNX2,AGG and Collagen Ⅱ(extracellular matrix type Ⅱ collagen)were different between the experimental group and the control group(P < 0.05).Discussion: This study demonstrated that cyclic mechanical stress stimulated the high expression of miR-5100,which is likely to be attributed to the Integrin-α1 activation and aggregation of miR-5100 through targeted regulation of PRTG,promoting the survival and proliferation of nucleus pulposus cells in disc degenerative diseases.