Dysregulated miR-127-5p contributes to type II collagen degradation by targeting matrix metalloproteinase-13 in human intervertebral disc degeneration.

BACKGROUND: Intervertebral disc degeneration (IDD) is a chronic disease associated with the degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMP)-13 is a major enzyme that mediates the degradation of ECM components. MMP-13 has been predicted to be a potential target of miR-127-5p. However, the exact function of miR-127-5p in IDD is still unclear. OBJECTIVE: We designed this study to evaluate the correlation between miR-127-5p level and the degeneration of human intervertebral discs and explore the potential mechanisms. METHODS: miR-127-5p levels and MMP-13 mRNA levels were detected by quantitative real-time polymerase chain reaction (qPCR). To determine whether MMP-13 is a target of miR-127-5p, dual luciferase reporter assays were performed. miR-127-5p mimic and miR-127-5p inhibitor were used to overexpress or downregulate miR-127-5p expression in human NP cells, respectively. Small interfering RNA (siRNA) was used to knock down MMP-13 expression in human NP cells. Type II collagen expression in human NP cells was detected by qPCR, western blotting, and immunofluorescence staining. RESULTS: We confirmed that miR-127-5p was significantly downregulated in nucleus pulposus (NP) tissue of degenerative discs and its expression was inversely correlated with MMP-13 mRNA levels. We reveal that MMP-13 may act as a target of miR-127-5p. Expression of miR-127-5p was inversely correlated with type II collagen expression in human NP cells. Moreover, suppression of MMP-13 expression by siRNA blocked downstream signaling and increased type II collagen expression. CONCLUSION: Dysregulated miR-127-5p contributed to the degradation of type II collagen by targeting MMP-13 in human IDD. Our findings highlight that miR-127-5p may serve as a new therapeutic target in IDD.

Inhibitory effect of silencing STAT3 gene with short hairpin RNA mediated by polyamidoamine dendrimers on growth of prostate cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the possibility of using generation 5 polyamidoamine dendrimers (G5-PAMAM-D) as gene vector for eukaryotic expression plasmid of siRNA in prostate carcinoma in vitro and vivo.</p><p><b>METHODS</b>Firstly, eukaryotic expression vector of siRNA pSilencing 4.1-EGFP-shRNA, specific for enhanced green fluorescent protein (EGFP), pSilencing 4.1-STAT3-shRNA for signal transducers and activators of transcription 3 (STAT3) was constructed. pEGFP-C1 and pSilencing 4.1-EGFP-shRNA were cotransfected into prostate cancer cells PC-3 and 22Rv1 with G5-PAPAM-D as vector, and to observe silencing of EGFP. Next, pSilencing 4.1-STAT3-shRNA was transfected into PC-3 and 22Rv1 cells by G5-PAPAM-D, Western blotting and apoptosis staining was used to detect silencing of STAT3 and growth inhibition. Thirdly, BALB/C mice subcutaneous tumor model was made with PC-3 cells. Polyplex of G5-PAMAM-D and pSilencing 4.1-STAT3-shRNA was injected intratumorally. The tumor volume was measured and recorded.</p><p><b>RESULTS</b>Fluorescence detection and Western blotting analysis demonstrated that G5-PAMAM-D was able to deliver Silencing 4.1-EGFP-shRNA and pSilencing 4.1-STAT3-shRNA into the two prostate cancer cell lines, and shRNA was expressed to induce silence of EGFP and STAT3. MTT results showed that proliferation of prostate cancer cells was suppressed by G5-PAMAM-D/pSilencing 4.1-STAT3-shRNA and induced apoptosis of PC-3 cells in vitro. Human prostate cancer in mice was successfully formed by inoculation of PC-3 cells into male BABL/C mice. In G5-PAMAM-D/pSilencing 4.1-STAT3-shRNA treated group, the tumor volume was shrank remarkably at 9 days after treatment and tumor growth was retarded compared with control groups.</p><p><b>CONCLUSION</b>GS-PAMAM-D nanoparticles can be used to deliver plasmid vector expressing shRNA into prostate cancer cells effectively in vitro and vivo. It appears to be a promising gene vector for RNA interference therapy in prostate cancer.</p>