Down-Regulated MIR-382 Contributes to Human Intervertebral Disc De-Generation by Targeting MMP-16

Abstract

INTRODUCTION: We have found that a number of microRNAs are differentially ex‐ pressed in IDD, amongst which miR‐382 is down‐regulated and its biologic functions have not been elucidated until now. METHODS: We collected human nucleus pulposus (NP) samples from cadavers as control and patients with IDD as degenera‐ tive NP samples. Intervertebral disc speci‐ mens were classified as no/minimal degen‐ eration and severe degeneration according to histological manifestation. A combination of methodology addressing miRNAs was used, including bioinformatics, quantitative real‐time RCR, Western blotting, immuno‐ histochemistry, in situ hybridization with locked nucleic acid, reporter gene analysis and lentiviral vector transfection. RESULTS: Bioinformatics indicated that MMP16 might be the target of miR‐382. The down‐regulation of miR‐382 and in‐ crease of MMP16 mRNA were verified using real‐time PCR. Furthermore, miR‐382 inhib‐ ited MMP16 expression by directly target‐ ing its 3’ UTR, which was abolished by mu‐ tation of the miR‐382 binding site. In vitro up‐regulation of miR‐382 in both HEK‐293 cells and human NP cells by transfection with lentiviral pre‐miR‐382 resulted in re‐ pression of MMP16; whereas knockdown of miR‐382 with lentiviral antigomiR‐382 led to overexpression of MMP16. As well, a com‐ bination of in situ hybridization and im‐ munohistochemistry indicated that miR‐382 localized in the cytoplasm of human NP cells with reverse link with MMP16. DISCUSSION: This study is the first address‐ ing the biologic functions of miR‐382 and distribution of MMP16 in IDD. Our results indicate that decreased miR‐382 directly targets MMP16 in IDD, implicating an etio‐ logic role of miR‐382 in IDD.