Role And Mechanism Of LINC00311 In Microglial Activation After Spinal Cord Injury

AIM: To explore the role and molecular mechanism of long non-coding RNA-LINC00311 in microglial activation in nerve injury by simulating spinal cord injury with LPS-induced primary microglial model in vitro.In order to further elucidate the pathological mechanism of spinal cord injury,and provide new methods and theoretical basis for clinical treatment,this study hopes to reveal the possible regulators in spinal cord injury and provide new potential targets for the treatment of spinal cord injury.METHODS:(1)SD rats within 24 hours after SPF neonatal were selected as the cell source for in vitro test,and microglia were isolated and extracted,cultured and purified.(2)The purified microglia were digested with trypsin,FITC-CD11b/c antibody was added to the cells for fluorescence staining,and the purity of the extracted microglia was detected using a cell flow detector.(3)Microglia were transfected and screened,and after cells were transfected with si RNA-LINC00311 lentivirus,the expression level of LINC00311 was detected using q PCR experiments to verify the cell transfection efficiency.(4)The transfected cells and normal cells were divided into four groups: control group,LPS group,LPS + si RNA-NC group,and LPS + si RNA-LINC00311 group.The m RNA expression level of LINC00311 in the cells was detected by q RT-PCR;the protein expression level of Iba-1 in microglia was detected by Western blot;and the contents of TNF-α,IL-1β,and IL-6 in the supernatant of microglia were detected by ELISA.Results:(1)Microglia could be observed under an inverted microscope in isolated and extracted microglia,suggesting successful cell extraction.(2)The extracted microglia suggested in the flow identification results that the microglia extracted in this experiment were highly pure and could be used as a subsequent experiment.(3)Inverted fluorescence microscopy indicated that LINC00311 knockdown slowed down the virus had been successfully transfected into rat microglia;and real-time PCR results indicated that the m RNA expression level of LINC00311 was significantly reduced in the cells of the si RNA-LINC00311 group compared with the si RNA-NC group(P < 0.001),and the LINC00311 differential expression system in rat microglia had been established.(4)The results of real-time PCR experiments showed that the m RNA expression levels of LINC00311 in the cells of the LPS +si RNA-LINC00311 group were significantly decreased compared with those of the LPS group(P < 0.001);the results of Western blot experiments showed that the protein expression levels of Iba-1 in the cells of the LPS + si RNA-LINC00311 group were significantly decreased compared with those of the LPS group(P < 0.001);and the results of ELISA experiments showed that the contents of TNF-α,IL-1β,and IL-6in the supernatant of the cells of the LPS + si RNA-LINC00311 group were significantly decreased compared with those of the LPS group(P < 0.01,P < 0.001,P< 0.0001).CONCLUSION:(1)LINC00311 can promote microglial activation,lead to the massive release of inflammatory factors,and cause a related cascade of inflammatory reactions,aggravating the degree of injury in spinal cord injury.(2)Silencing of lnc RNA-LINC00311 in primary microglia reduced LPS-induced microglial activation and the levels of proinflammatory cytokines.Therefore,si RNA-LINC00311 is protective against spinal cord injury.