| Objective: To explore the correlation between mi R-340-5p expressed in nucleus pulposus(NP)and abnormal expression of fibromodulin(FMOD)in annulus fibrosus(AF)during intervertebral disc degeneration(IDD),and to clarify that mi R-340-5p expressed in degenerated NP cells can cause AF degeneration by down-regulating the expression of FMOD,thus accelerating the occurrence and development of IDD.Methods: 1.Collect tissue samples of clinical IDD(5 samples from each of the control group,the simple herniation group,and the degenerative herniation group),and use techniques such as hematoxylin-eosin(HE)staining,Western blot(WB),and real-time fluorescent quantitative PCR(q RT-PCR)to observe the degeneration of intervertebral discs and detect the expression of extracellular matrix(ECM)in NP and AF cells,Preliminary correlation analysis was conducted between the expression of mi R-340-5p and FMOD and the occurrence of IDD;2.Using the rat caudal vertebra to construct an IDD degeneration model,NP and AF cells were obtained and isolated from the rat.The morphology and ECM distribution of the isolated NP and AF cells were observed using an inverted microscope and immunofluorescence staining.Set up a control experiment:(1)blank control group;(2)Overexpression negative control group(OE mi RNA-NC group);(3)Mi R-340-5p overexpression group(OE-mi R-340-5p group);(4)Knockdown negative control group(KO-mi RNA-NC group);(5)In the mi R-340-5p knockdown group(KO-mi R-340-5p group),after coculturing with normal rat AF cells,Western Blot and q RT-PCR were used to detect the expression of ECM and m RNA in NP and AF cells,and to analyze the effect of mi R-340-5p elevation/decrease on the expression of FMOD in AF cells.3.Construct AF cells overexpressing FMOD(OE-FMOD)and set up a control experiment:(1)blank control group;(2)OE-mi R-340-5p group;(3)Negative control group with OE-mi R-340-5p+FMOD overexpression(OE-mi R-340-5p+OE-NC group);(4)In the OE-mi R-340-5p+FMOD overexpression group(OE-mi R-340-5p+OE-FMOD group),the expression of EMC and its m RNA in intervertebral discs was detected using Western Blot and q RT-PCR,respectively,to further verify the regulatory relationship between mi R-340-5p and FMOD in degenerative discs.Results: 1.HE staining observed that in clinical samples,compared with the control group,the intervertebral disc tissue structure of the simple herniation group and the degenerative herniation group was loose and disordered,with the degenerative herniation group being the most significant;Western blot showed that compared with the control group,the protein expression levels of FMOD,type II collagen fibers(Col II),and proteoglycan in the intervertebral disc tissue of the simple herniation group and the degenerative herniation group decreased,while the type I collagen fibers(Col I)significantly increased,with the most significant in the degenerative herniation group;QRT-PCR showed that the expression level of mi R-340-5p increased with the aggravation of degeneration,while the expression level of FMOD gradually decreased.2.After the lentivirus was transfected to construct rat NP cells with OE-mi R-340-5p/KO-mi R-340-5p,fluorescence microscopy observation showed that the virus had successfully transfected into the cells.The results of q RT-PCR confirmed that the differential expression of mi R-340-5p in NP cells had been established.After co culture of cells in each group,Western blot results showed that the expression level of ECM protein in AF and NP cells in OE-mi R-340-5p group was significantly lower than that in KO-mi R-340-5p group;The results of q RT-PCR showed that the m RNA expression level of mi R-340-5p in AF and NP cells in the OE-mi R-340-5p group was significantly higher than that in the KO-mi R-340-5p group,while the m RNA expression level of ECM was significantly lower.3.After constructing OE-FMOD AF cells,fluorescence microscopy observation showed that the virus had successfully transfected into the cells,and q RT-PCR results showed that OE-FMOD AF cells had been established.After co culture,Western blot results showed that in NP cells,compared with the Control group,the protein expression levels of Col I in the OE-mi R-340-5p group,OE-mi R-340-5p+OE-NC group,and OE-mi R-340-5p+OE-FMOD group were significantly increased,while the protein expression levels of other ECMs were significantly decreased;In AF cells,the protein expression levels of FMOD,Col I,Col II,and Aggrescan were decreased.In the OE-mi R-340-5p+OE-FMOD group,the protein expression levels of FMOD,Col I,and Col II were also significantly decreased,while there was no significant difference in the protein expression level of Aggrescan.The results of q RT-PCR showed that in NP cells,except for the blank Control group,the m RNA expression levels of mi R-340-5p and Col I were significantly increased in the other three groups,while the m RNA expression levels of FMOD,Col II,and Aggrescan were significantly decreased;In AF cells,the m RNA expression level of mi R-340-5p was significantly increased in the OE-mi R-340-5p group and the OE-mi R-340-5p+OE-NC group,while the ECM expression level was significantly decreased.In the OE-mi R-340-5p+OE-FMOD group,the m RNA expression level of mi R-340-5p was significantly increased in AF cells,while the m RNA expression level of FMOD and Col I was significantly decreased.Conclusion: 1.In the intervertebral disc tissue,the abnormal expression of mi R-340-5p and FMOD is related to the degeneration of intervertebral disc tissue;2.The mi R-340-5p expressed in degenerated NP tissue inhibits the synthesis of FMOD in the inner AF and accelerates the degeneration of intervertebral disc. |
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