Inhibition of TNFR1 Attenuates LPS Induced Apoptosis and Inflammation in Human Nucleus Pulposus Cells by Regulating the NF-KB and MAPK Signalling Pathway.

Intervertebral disc degeneration (IDD) is accompanied by nucleus pulposus (NP) cell apoptosis, inflammation, and extracellular matrix degradation. Tumour necrosis factor receptor 1 (TNFR1) is a receptor of TNF-α, and is deeply involved in the processes of IDD. However, the effect of TNFR1 inhibition on IDD is not clear. Herein, we report that TNFR1 was increased in LPS-treated HNPCs. The aim of this study was to investigate the potential therapeutic effect of TNFR1 siRNA and selective antagonists of TNFR1 (GSK1995057) on HNPC damage. The results showed that the blockade of TNFR1 by TNFR1 siRNA and GSK1995057 effectively suppressed the cell viability loss, apoptosis, and inflammation induced by LPS in HNPCs. Furthermore, we found that TNFR1 siRNA and GSK1995057 inhibited activation of the NF-KB and MAPK signalling pathways in LPS-stimulated HNPCs. In summary, the blockade of TNFR1 effectively suppressed LPS-induced apoptosis and inflammation in HNPCs through the NF-KB and MAPK signalling pathways. This revealed that the blockade of TNFR1 may provide a potential therapeutic treatment for IDD.

miR­519d­3p promotes TGFß/Smad mediated postoperative epidural scar formation via suppression of BAMBI.

To investigate the role of microRNA (miR)­519d­3p in postoperative epidural scar formation and its regulation of the bone morphogenetic protein and activin membrane­bound inhibitor (BAMBI), miR­519d­3p and BAMBI expression levels in the lumbar disc of patients who had undergone laminectomy were detected with reverse transcription­quantitative polymerase chain reaction and western blotting. The results demonstrated that miR­519d­3p expression was significantly increased, whereas BAMBI expression was sharply reduced in the lumbar discs of patients suffering from epidural scars. Subsequently, the miR­519d­3p mimic was transfected into primary fibroblasts isolated from epidural scar tissues. Flow cytometric and Cell Countin Kit­8 analyses indicated that overexpression of miR­519d­3p promoted the proliferation of fibroblasts, the production of tumor necrosis factor­α and interleukin (IL)­1α, and the expression of type I collagen (col I), α­smooth muscle actin (α­SMA) and fibronectin (FN). Downregulation of miR­519d­3p by the miR­519d­3p antagomir transfection had the opposite effect. Bioinformatics and luciferase reporter gene analyses demonstrated that BAMBI is a target gene of miR­519d­3p: miR­519d­3p directly binds to the 3'­untranslated region of BAMBI mRNA and suppressed BAMBI protein expression. Finally, the pcDNA­BAMBI vector and BAMBI small interfering RNA were respectively transfected into primary fibroblasts to overexpress and knockdown the BAMBI gene. It was demonstrated that BAMBI overexpression suppressed fibroblast proliferation, TNF­α and IL­1α production, and the expression of col I, α­SMA and FN proteins, whereas, BAMBI knockdown had the opposite effect. In conclusion, it was noted that BAMBI is a target of miR­519d­3p and miR­519d­3p promotes transforming growth factor ß/mothers against decapentaplegic homolog 9­mediated postoperative epidural scar formation via suppression of BAMBI.

RASSF1-AS1, an antisense lncRNA of RASSF1A, inhibits the translation of RASSF1A to exacerbate cardiac fibrosis in mice.

Cardiac fibrosis is associated with various cardiovascular diseases and can eventually lead to heart failure. Dysregulation of long non-coding RNAs (lncRNAs) are recognized as one of the key mechanisms of cardiac diseases. However, the roles and underlying mechanisms of lncRNAs in cardiac fibrosis have not been explicitly defined. Here, we investigated the role of an antisense (AS) lncRNA from the Ras association domain-containing protein 1 isoform A (RASSF1A) gene locus, named RASSF1-AS1, in the development of cardiac fibrosis. Cardiac fibrosis mouse model was established by isoproterenol injection. We found that RASSF1A protein was downregulated, whereas RASSF1-AS1 was markedly upregulated during cardiac fibrosis. Overexpression and knockdown of mouse primary cardiac fibroblasts showed that RASSF1-AS1 negatively regulated RASSF1A expression at the post-transcriptional level. According to the landscape analysis and sense-AS binding evaluation, RASSF1-AS1 partially overlaps with RASSF1A messenger RNA (mRNA) at the exon2 region. RNA pull-down and luciferase activity assays confirmed that RASSF1-AS1 directly bound to RASSF1A mRNA and suppressed its translation. Furthermore, wild-type RASSF1-AS1 had a promoting effect on nuclear factor-κB activation and cardiac fibrosis, but mutated RASSF1-AS1, in which the binding region was deleted, had no effect. In conclusion, RASSF1-AS1 inhibits the translation of RASSF1A to exacerbate cardiac fibrosis in mice, indicating a potential application of RASSF1-AS1 as a therapy target for cardiac fibrosis.

MicroRNA-146a Ameliorates Inflammation via TRAF6/NF-κB Pathway in Intervertebral Disc Cells.

BACKGROUND Intervertebral disc degeneration (IDD) has been widely recognized as a major contributor to low back pain. Accumulating evidence suggests that IDD is linked to various pro-inflammatory cytokines and metabolites. Recently, numerous studies have demonstrated that microRNAs (miRNAs) play a pivotal role in the development of most disorders, including degenerative disc diseases. Previous reports have revealed that miRNA-146a (miR-146a) could attenuate neuropathic pain in the spinal cord. The aim of this study was to investigate the role of miR-146a in the inflammatory response of IDD. MATERIAL AND METHODS Quantitative real-time (RT)-PCR was performed to investigate the levels of miR-146a in the PBMCs (peripheral blood mononuclear cells) of patients with IDD. Human nucleus pulposus (NP) cells were transiently transfected with miR-146a mimic; control NP cell transfections lacked miR-146a. Then all NP cells were treated with LPS (10 µM) to induce inflammation. The mRNA levels of miR-146a in NP cells were determined by RT-PCR. In addition, the mRNA and protein expression levels of tumor necrosis factor (TNF), receptor-associated factor 6 (TRAF6), and nuclear factor (NF)-κB in NP cells were evaluated by quantitative RT-PCR and Western blot analysis, respectively. RESULTS We found that miR-146a was significantly downregulated in the PBMCs of patients. Moreover, overexpression of miR-146a significantly decreased the levels of pro-inflammatory cytokines in LPS-stimulated NP cells. The mRNA and protein levels of TRAF6 and NF-κB were downregulated by miR-146a overexpression. CONCLUSIONS These results suggest that overexpression of miR-146a could promote IDD through the TRAF/NF-κB pathway. Our findings also highlight miR-146a as a novel possible therapeutic target for IDD.