| Objective:Lower back pain(LBP)is one of the most common spinal disorders in today’s society and has profoundly affected people’s work,school and life.Etiologists have shown that intervertebral disc degeneration(IDD)and its secondary pathologies are the main cause of pain.It is estimated that IDD causes a global economic loss of more than 65 billion euros per year.The intervertebral disc is a special structure without blood vessels and is in a special environment of ischemia and hypoxia in its normal physiological state,and homeostasis of the disc microenvironment is the basis for maintaining normal disc function.Currently,there is no effective non-surgical treatment for IDD.The main reason is that the specific pathogenesis of IDD is unknown,and no effective specific target has been identified.The pathological changes of IDD are mainly manifested by disc herniation,interbody instability and spinal stenosis.Currently,the progression of IDD cannot be delayed at the source by pharmacological treatment,so the clinical treatment of it is still based on symptomatic treatment and interventional therapy.This study will explore the mechanism by which Lnc RNA JPX affects the HIF-1α/Hippo-YAP signaling axis and thus regulates the proliferation and apoptosis of HNPC by targeting mi R-18a-5p to further understand the biological properties of intervertebral disc degeneration and thus provide new ideas and methods for its treatment.Methods:1.The data set GSE132182 gene data downloaded from GEO database was analyzed by bioinformatics analysis of 2.5 month healthy mouse medullary nucleus cells and 21 month senescent degenerated mouse medullary nucleus cells to simulate the degeneration of intervertebral discs in vitro,and the target Lnc RNAs were identified by bioinformatics analysis and screening of differentially expressed Lnc RNAs.2.In cellular experiments,real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the expression of Lnc RNA JPX in HNPC of normoxia(Nx)and hypoxia(Hx)groups,and the proliferation and apoptosis of HNPC of Nx and Hx groups were detected by cell proliferation and activity assay(CCK-8)and flow cytometry(FCM).A stable HNPC cell line overexpressing(ov)Lnc RNA JPX was constructed under Nx conditions to compare its effect of ov-JPX on HNPC function under Nx and Hx conditions.3.The Starbase database and Bi Bi Serv database were used to analyze the predicted mi RNAs bound to Lnc RNA JPX,and the double luciferase assay and RNA pull down assay to confirm the targeting relationship between Lnc RNA JPX and mi R-18a-5p.4.Since HIF-1α is a key gene affecting IDD,the targeting relationship between mi R-18a-5p and HIF-1α was analyzed using Target Scan database and verified by dual luciferase assay.Since HIF-1α is correlated with Hippo-YAP signaling pathway and Hippo-YAP is involved in the regulation of IDD,we used protein immunoblotting(Western Blot)to detect the difference of HIF-1α,YAP and p-YAP expression under Hx and Nx conditions;and we added mi R-18a-5p inhibitor.The differences in HIF-1α,YAP and p-YAP expression were detected in HNPC with the addition of mi R-18a-5p inhibitor(inhibitor).Meanwhile,we constructed three si RNAs targeting HIF-1α,and co-transfected si-HIF-1α with mi R-18a-5p inhibitor into HNPC to detect the protein levels of HIF-1α,YAP,p-YAP and the proliferation and apoptosis of HNPC.5.mi R-18a-5p inhibitor,ov-JPX and si-HIF-1α were co-transfected with HNPC,and the protein levels of HIF-1α,YAP,p-YAP and HNPC proliferation and apoptosis were detected.Results:1.Lnc RNA JPX was found to be significantly downregulated in myeloid cells of senescent degenerating mice at 21 months after analysis of the dataset by GEO and was identified as the target RNA.2.Lnc RNA JPX expression was found to be down-regulated in Nx by RT-q PCR,indicating that Nx conditions inhibit Lnc RNA JPX.CCK-8 assay with flow cytometry HNPC proliferation was found to be decreased and apoptosis was increased in the Nx group.However,transfection of HNPC with overexpression of Lnc RNA JPX plasmid(ov-JPX),using CCK-8 with flow cytometry assay,revealed that the cells in the ov-JPX group increased proliferation and decreased apoptosis,this result suggested that ov-JPX could antagonize the effect of Nx on HNPC,thus increasing the proliferation and decreasing the apoptosis of HNPC.3.Using Starbase database and Bi Bi Serv database,Lnc RNA JPX has a potential binding target with mi R-18a-5p,as demonstrated by dual luciferase assay and RNA pull down assay,Lnc RNA JPX can directly bind to mi R-18a-5p.4.Using Target Scan database analysis,HIF-1α can be used as a mi R-18a-5p as a downstream target.mi R-18a-5p could directly target HIF-1α as demonstrated by dual luciferase assay.HIF-1α was correlated with Hippo-YAP signaling pathway as confirmed by KEGG pathway enrichment analysis and confirmed by Western blot assay: compared with Hx group,HIF-1α and YAP in Nx condition protein levels were decreased and p-YAP expression was upregulated,i.e.,Nx had an inhibitory effect on HIF-1α and YAP proteins and an elevating effect on p-YAP;after transfection with mi R-18a-5p inhibitor using Western blot analysis,compared with the NC group in the Nx condition,transfection with mi R-18a-5p inhibitor in HNPC HIF-1α and YAP protein levels were up-regulated and p-YAP levels were decreased,this result suggested that inhibition of mi R-18a-5p could antagonize the effect of Nx on apoptosis.Using si RNA technology to interfere with HIF-1α,the expression and protein levels of HIF-1α were inhibited by RT-q PCR and Western blot,especially the inhibition effect of si-HIF-1α-2 was the most significant.Meanwhile,mi R-18a-5p inhibitor was co-transfected with si-HIF-1α-2 in HNPC,and the Western blot assay showed that HIF-1α and YAP protein levels decreased and p-YAP levels increased in the mi R-18a-5p inhibitor+si-HIF-1α group compared with the NC group.The results of CCK-8 and flow cytometry experiments showed that the proliferation rate of HNPC was decreased and the apoptosis rate was increased in the mi R-18a-5p inhibitor+si-HIF-1α group compared with the NC group.The above results confirmed that mi R-18a-5p negatively regulated the expression of HIF-1α.5.Western blot results showed that the HIF-1α and YAP protein levels increased and p-YAP levels decreased in the mi R-18a-5p inhibitor group compared with the Nx group;CCK-8 and flow cytometry experiments showed that compared with the Nx group Compared with the Nx group,the proliferation rate of HNPC increased and the apoptosis rate decreased in the mi R-18a-5p inhibitor group.After co-transfection of mi R-18a-5p inhibitor with ov-JPX,the results of Western blot assay showed that the HIF-1α and YAP protein levels increased and the p-YAP level decreased in the ov-JPX group compared with the mi R-18a-5p inhibitor group;the results of CCK-8and flow cytometry assay showed that the HNPC proliferation rate increased and the apoptosis rate decreased in the mi R-18a-5p inhibitor group compared with the Nx group.The proliferation rate of HNPC increased and apoptosis rate decreased in the ov-JPX group compared with the mi R-18a-5p inhibitor group.Meanwhile,after co-transfection of si-HIF-1α with mi R-18a-5p inhibitor+ov-JPX,the results of Western blot experiments showed that the levels of HIF-1α and YAP protein decreased and the levels of p-YAP increased in the si-HIF-1α group compared with the mi R-18a-5p inhibitor+ov-JPX group;The results of CCK-8 and flow cytometry experiments showed that the value-added rate of HNPC decreased and the apoptosis rate increased in the si-HIF-1α group compared with mi R-18a-5p inhibitor+ov-JPX.The above results indicated that mi R-18a-5p inhibitor could increase the expression of HIF-1α and YAP proteins,decrease the expression of p-YAP,increase the proliferation ability of HNPC and attenuate its apoptosis;ov-JPX could enhance the effect of mi R-18a-5p inhibitor;however,after co-transfection of si-HIF-1α,the previous effect was reversed.Conclusions: 1.Lnc RNA JPX is a key gene in IDD,overexpressed JPX increases HNPC proliferation and reduces apoptosis under Nx conditions.2.Lnc RNA JPX regulates Hippo-YAP signaling pathway through mi R-18a-5p/HIF-1α axis and regulates HNPC proliferation and apoptosis. |
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