The Role And Mechanism Of Mechanical Sensitive CircRNA₀022382 In The Degeneration Of Endplate Chondrocytes

Objective: To construct a model of endplate chondrocytes induced degeneration in vitro,and to study the mechanism of mechanically sensitive Circ RNA₀022382 on degeneration of endplate chondrocytes under a certain tension.Methods: Take the endplate chondrocytes of patients undergoing cervical spine surgery in our department were collected.P2 generation of endplate chondrocytes were randomly divided into the control group and the afterburner group.The FX-5000 cell strain loading system was used to construct endplate chondrocytes in vitro Tension model?10% elongation?0.5Hz?8h/d?,the control group was only cultured in normal condition;Detection of expression difference of circrnas in endplate chondrocytes before and after afterloading by circrna microarray?The experimental contents of the chip include RNA quality detection,fluorescent labeling probe preparation,chip hybridization,image acquisition and data analysis,etc?.Select high fold circ RNAs for q PCR repeat verification;Bioinformatics software?Miranda,RNAhybrid,Target Scan,and RBPmap?was used to predict the mi RNAs and RBPs of circ RNA₀022382 with sequence matching,combining related literature to further target targeting molecules;The target Micro RNA-765 was selected for animal in vitro experiments,and cell staining was used to observe the phenotype of chondrocytes before and after traction stimulation.Cell proliferation and apoptosis and changes in cytoskeleton of endplate cartilage were detected by CCK-8 method,flow cytometry,and and phalloidin staining;Realtime RT-PCR and western blot were used to detect changes in genes and protein levels of proteoglycan?ACAN?,type II collagen?COL2A1?,and SOX9 genes in endplate chondrocyte-associated genes mimic and inhibit Micro RNA-765.Results: The expression levels of ACAN and COL2A1,SOX9 and cartilage-related marker genes in endplate chondrocytes induced by tension loading show a gradually down-regulation change with the extension of tension time,which significantly affects the synthesis of extracellular matrix of endplate Inhibition.The cytoskeleton morphology of endplate chondrocytes changed significantly after being stretched.The original polygonal shape was stretched into a spindle shape,and the corresponding changes in the apoptosis level of the cells also occurred.There is no significant effect on the proliferation of endplate chondrocytes.Circ RNA microarray was used to screen the chondrocytes of endplate after tension loading,and found that a total of 16 Circ RNAs were up-regulated and 12 Circ RNAs were down-regulated;Bioinformatics software analyzed that Circ RNA₀022382 can specifically bind to Micro RNA-765 to regulate the expression of cartilage marker genes;Micro RNA-765 expression levels were significantly inhibited after overexpression of Circ RNA₀022382.On the contrary,the expression of microrna-765 was significantly increased by inhibiting Circ RNA₀022382,and the expression of ACAN,COL2A1 and Sox9 related genes in endplate chondrocytes was increased by inhibiting Circ RNA₀022382 and over expressing microrna-765;Alleviates degeneration of endplate chondrocytes.Conclusion: Stimulation of mechanical tension under certain conditions can accelerate the degeneration of chondrocytes in the endplate;Inhibition of Circ RNA₀022382 and overexpression of Micro RNA-765 can alleviate the degree of degeneration of endplate chondrocytes.