RESUMEN
BACKGROUND:MicroRNAs (miRNAs) play an important role in a variety of diseases. Investigation of miRNA expression profile in degenerative lumbar scoliosis is beneficial for understanding its pathogenesis, providing a novel therapeutic target. Therefore, we tested the hypothesis that miRNAs promote intervertebral disc degeneration through the interleukin-10/STAT3 signaling pathway, a potential regulator of intervertebral disc degeneration. OBJECTIVE:To compare the differentialy expressed miRNAs in the intervertebral disc tissues from patients with degenerative lumbar scoliosis and normal controls and to identify specific miRNAs in degenerative lumbar scoliosis folowed by functional validation. METHODS: An initial screening of miRNA expression in nucleus pulposus tissues by miRNA Solexa Sequencing was performed in samples from 10 patients with degenerative lumbar scoliosis and 10 controls, respectively. Subsequently, differentialy expressed miRNAs were validated using qRT-PCR. The level of differentialy expressed miRNAs in degenerative nucleus pulposus tissues was investigated. Then, functional analysis of the miRNAs in regulating type II colagen expression was carried out. Western blot and luciferase reporter assay were used to further confirm the target gene. RESULTS AND CONCLUSION: We identified 30 miRNAs that were differentialy expressed (16 upregulated and 14 downregulated) in patients with degenerative lumbar scoliosis compared with controls. Folowing qRT-PCR confirmation, Has-let-7f was significantly down-regulated in degenerative nucleus pulposus tissues as compared with controls. Moreover, its level was correlated with the severity of disc degeneration. Overexpression of Has-let-7f promoted type II colagen expression in nucleus pulposus cels. Knockout of interleukin-10 induced effects on nucleus pulposus cels similar to Has-let-7f. Bioinformatics target prediction identified interleukin-10 as a putative target of Has-let-7f. Furthermore, luciferase reporter assays demonstrated that Has-let-7f altered the expression of STAT3 and matrix metaloproteinase-2. These findings indicate that the downregulation of Has-let-7f induces type II colagen loss by directly targeting inleukin-10, thereby resulting in intervertebral disc degeneration and degenerative lumbar scoliosis. Has-let-7f is likely to be a novel therapeutic target for degenerative lumbar scoliosis.
RESUMEN
BACKGROUND:MicroRNAs are widely involved in the regulation of protein expression, and play a critical role in many physiological and pathological processes in the body. But microRNA expression profile in degenerative lumbar scoliosis is rarely reported and understood. OBJECTIVE:To compare the microRNA expression profile in the normal intervertebral disc and degenerative lumbar scoliosis and to identify degenerative lumbar scoliosis-specific microRNAs, folowed by functional validation. METHODS: Total RNA samples were extracted from the nucleus pulposus tissues of 57 patients with degenerative lumbar scoliosis as experimental groups and the normal nucleus pulposus tissues of 42 patients with lumbar fractures as control group. An initial screening of differentialy expressed microRNAs in the nucleus pulposus tissues by microRNA microarray was performed in 10 samples from each group. Subsequently, differentialy expressed microRNAs were validated using real-time quantitative RCR. The level of differentialy expressed microRNAs in the degenerative nucleus pulposus tissues was investigated. Then, the functional analysis of microRNAs in regulating colagen II expression was carried out. Western blot and luciferase reporter assay were also used to detect target genes. RESULTS AND CONCLUSION:We identified 22 microRNAs that were differentialy expressed (17 upregulated and 5 downregulated) in degenerative lumbar scoliosis patients compared with the controls. Folowing real-time quantitative RCR confirmation, miR-491-5p was significantly down-regulated in degenerative nucleus pulposus tissues in comparison with the controls. Moreover, its level was closely correlated with the pathological grading of disc degeneration. Overexpression of miR-491-5p promoted type II colagen expression in nucleus pulposus cels. Bioinformatics target prediction identified matrix metaloproteinase-9 as a putative target of miR-491-5p. Furthermore, luciferase reporter assays demonstrated that miR-491-5p directly targeted matrix metaloproteinase-9 and affected its protein expression in nucleus pulposus cels. These results show that the downregulation of miR-491-5p induces type II colagen loss by directly targeting matrix metaloproteinase-9, thereby resulting in degeneration of the intervertebral disc and degenerative lumbar scoliosis. This study also underscores the potential of miR-491-5p as a novel therapeutic target in degenerative lumbar scoliosis.