| Part I Screening of differentially expressed mi RNAs andvalidationof overexpressed mi R-21 in human nucleuspulposus tissuesObjective: To explore the roles of mi RNAs(mi RNAs) in the pathogenesis of intervertebral disc(IVD) degeneration(IDD), we detected mi RNAs expression profiles, screened and validated differentially expressed mi RNAs in human nucleus pulposus(NP) tissues.Methods: Specimens from 5 patients with IDD and 5 with idiopathic scoliosis were selected for Sure Print human mi RNA microarrays(V16.0) analysis, then differentially expressed mi RNAs derived from the microarray data were screened out. In addition, the overexpressed mi R-21 was then validated by quantitative reverse transcription polymerase chain reaction(q RT-PCR) in48 specimens from NP tissues of differential Pfirrmann grade.Results: The microarray data demonstrated that 20 mi RNAs were found to be upregulated(fold change>2.0) and 9 mi RNAs were found to be downregulated(fold change<0.5) in the IDD group compared with the idiopathic scoliosis. Among these differentially expressed mi RNAs, has-mi R-21, has-mi R-122 a, has-mi R-210 and has-mi R-30-5p showed significantly high expression, while expression of has-mi R-212-5p and has-mi R-33 a were markedly decreased(P<0.01). The q RT-PCR results validated the overexpression of mi R-21, moreover, the expression levels of mi R-21 were increased with increasing gradeof IVD degeneration.Summary: mi R-21 expression displays higher levels in human degenerative NP tissues and were increased with increasing gradeof IVD degeneration, implying that mi R-21 may involve in the pathophysiological processes of IDD. Part II Mi R-21 suppressescellular autophagy by targetingPTEN in human nucleus pulposus cellsObjective: The aim of current section is to investigate the roles of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(m TOR) signaling pathway in mi R-21 induced cellular autophagy via targeting phosphatase and tensin homologue(PTEN) in nucleus pulposus cells.Methods: The online bioinformatics software were performed to validatethe binding sequence between mi R-21 and PTEN. Human degenerative NP cells from tissueswere isolated and cultured, and then were subjected to various treatment during monolayer cells. The effects of mi R-21 on target gene PTEN m RNA and protein levels were detected by q RT-PCR and Western blot technologies in human degenerative NP cells. Monodansylcadaverine(MDC) staining and Western blottechnology were performed for detecting the effects of mi R-21 in autophagosome and autophagy related proteins in NP cells. Treatment of NP cells with LY249002(PI3K/Akt inhibitor) or rapamycin(m TOR inhibitor)after transfecting mi R-21 mimic and inhibitor into NP cells by lipofectamine 2000, the protein and(or) m RNA expression levels of related downstream factors were detected by Western blot and(or) q RT-PCR technologies.Results: The prediction results of multiple online bioinformatics software validated that mi R-21 had a theoretical binding sites with PTEN 3'-UTR and lower free energy with it. Furthermore, the expression of PTEN protein was dramatically reduced in mi R-21 mimic group and was markedly increased in mi R-21 inhibitor group compared with blank control(P<0.05), however, there were no significant difference for the PTEN m RNA level in all groups(P>0.05).MDC staining shown that numbers of autophagosome were obviously increased in mi R-21 inhibitor group compared with blank control, while it almost be not observed in mi R-21 mimic group(P<0.05). Western blot results illustrated that PTEN protein level was not affected by LY249002 and rapamycin(P>0.05). Whereas, when expressionof PTEN protein was downregulated by mi R-21 mimic, expression levels of PI3 K, Akt, m TOR and P62 protein were noteworthily upregulated compared with the blank control group(P<0.05), on the contrary, ratio LC3-II/LC3-I was reduced(P<0.05). Of note, the change trend of P62 and ratio LC3-II/LC3-I were reversed by LY249002 and rapamycin(P<0.05). The m RNAlevels of P62 and LC3 detected by q RT-PCR were consistent with the change trend of their protein.Summary:PTEN was target gene of mi R-21 in human degenerative tissues. Mi R-21 can silence PTEN, lead to PI3K/Akt/m TOR signaling pathway activate, and then inhibiting NP cellular autophagy and exacerbating the pathological process of IDD. Conclusion:1. The expression level of mi R-21 is remarkly increased in humandegenerative NP tissues.2. Overexpressed of mi R-21 suppresses the expression levelsofcellular autophagy through directly targeting PTEN andactivating PI3K/ Akt/m TOR signaling pathway in humandegenerative NP cells. |
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