Modulation Of Microrna-155 In Human Intervertebral Disc Degeneration

Introduction.The etiology of intervertebral disc degeneration (IDD) is ascribed to various factors, one of which is the increase in apoptosis. The underlying molecular mechanisms of the increase in apoptosis remain largely unknown. Both the extrinsic and intrinsic apoptotic pathways play essential roles in the apoptosis of human nucleus pulpusos (NP) cells. Accumulating evidence has shown that the apoptotic machinery is regulated by the newly defined small noncoding RNAs, microRNAs (miRNAs). Despite miRNAs play an important role in a variety of physiologic and pathologic processes, little is known on their expression profiles and attendant roles in IDD. The purpose of this study was to investigate the role of miRNAs, in particular miR-155, in IDD.Methods.Human nucleus pulposus (NP) specimens were collected from patients with idiopathic scoliosis (n=12) and IDD (n=12) as control and degenerative NP samples. Following total RNA isolation from freshly isolated NP cells, RNA labeling and hybridization on miRNA microarray chips were completed on 6 samples. The relative expression of miRNAs was verified using real-time PCR. The prediction of putative targets of miR-155 was achieved using online miRNAs database search and computational analysis. The interaction of miR-155 and the 3'-UTRs of FADD and caspase-3 was performed using the dual luciferase reporter assay. In vitro over-expression and knockdown of miR-155 in human NP cells were achieved by transfection of NP cells with lentiviral Pre-miR-155 and antagomiR-155. Following treated with FasL, the expression levels of FADD and caspase-3 in NP cells with modulated miR-155 were detected using Western Blot. Flow cytometry with APC-Annexin V/7-AAD staining was used to detect the apoptosis in NP cells with modulated miR-155. Transmission electon microscopy was used to detect apoptosis in control and IDD samples. Moreover, miR-155, FADD and caspase-3 expression in human NP was validated using in situ hybridization, immunohistochemistry and a combination thereof.Results.We found that 29 miRNAs were differentially expressed and miR-155 was down-regulated in human degenerative NP. The deregulation of miR-155 (0.56±0.17-fold) was further verified using qRT-PCR. FADD and caspase-3 were predicted as putative targets of miR-155 using the bioinformatics methods. miR-155 inhibited FADD and caspase-3 expression by directly targeting their 3'UTRs, which was abolished by mutation of the predicted miR-155-binding-sites. Overexpression and knockdown of miR-155 in human NP cells resulted in significant deregulation and upregulation expression of FADD and caspase-3. As well, Fas-mediated apoptosis was increased when antagonizing miR-155 and decreased when using pre-miR-155. Furthermore, we presented direct evidence of NP cells undergoing apoptosis in IDD samples. Moreover, miR-155 expressed in the cytoplasm of human NP cells with reverse correlation with FADD and caspase-3. On the other hand, both control and degenerative human NP consisted of mainly viable cells (87.42±1.13%; 83.27±5.00%, p>0.05) with a variety of morphology, which were further categorized as typical NP cells, NP cells with long processes and phagocytic NP cells. Moreover, NP cells with long processes existed in degenerative NP. Phagocytic NP cells had the hallmarks of both stationary macrophages with lysosomes and NP cells with a large amount of the endoplasmic reticulum.Conclusion.To our knowledge, this is the first study addressing the underlying mechanism of apoptosis and the expression profiles of miRNAs in IDD. As well, caspase-3 is identified as a novel target of miR-155. Our results suggest that deregulated miR-155 promotes Fas-mediated apoptosis in human IDD by targeting FADD and caspase-3, implicating a role of miR-155 in the etiology of IDD. In addition, human degenerative NP consists of primarily viable cells. Furthermore, we present direct and in vivo evidence that human NP cells have phagocytic potential, which may have implications for the underlying mechanisms of cell clusters. Moreover, NP cells with long processes exist in both scoliosis and degenerative NP.