Previous research has shown that SMAD7 is demonstrated to be a downstream target of miR-424-5p in cancer cells . Considering the critical role of SMDA7 in OA development, we then addressed the potential role of miR-424-5p in OA tissues. Initially, rat cartilage tissues in sham and OA groups were stained with HE to verify the occurrence of OA and the morphology of tissues were estimated to be altered in OA (Fig. 1A). Then mRNA and protein levels of SMAD7 were further detected to assess the OA development. The results showed that mRNA level of SMAD7 decreased in rat cartilage tissues with OA compared to sham (Fig. 1B). In agreement with expression variety in tissues, mRNA and protein levels of SMAD7 were reduced in OA in contrast to sham of rat cartilage tissues (Fig. 1C). and chondrocytes (Fig. 1D). To further corroborate association between miR-424-5p and SMAD7 in chondrocytes, levels of miR-424-5p were checked and the correlation between them was analyzed statistically. Notably, levels of miR-424-5p were negatively correlated to levels of SMAD7 (r=-0.8421, p < 0.001, Fig. 1E), hinting the putative regulation of miR-424-5p in OA. Then the expression of miR-424-5p in sham and OA of rat cartilage tissues and chondrocytes was interrogated. Oppositely, the expression of miR-424-5p in OA was up-regulated compared to sham of rat cartilage tissues (Fig. 1F). and chondrocytes (Fig. 1G). Taken together, our results suggest a plausible role of miR-424-5p in the development of OA.
MiR-424-5p negatively regulated expression of SMAD7
As stated above, negative correlation of miR-424-5p with SMAD7 was established in OA tissues and chondrocytes. We then asked that whether miR-424-5p could target SMAD7 for repression in chondrocytes. We first examined the expression levels of SMAD7 with the transfection of miR-424-5p mimic and anti-miR-424-5p in rat chondrocytes and human C28/I2 cells. After transfection for 48 h, elevated expression of miR-424-5p (Fig. 2A) and reduced expression of SMAD7 (Fig. 2B) in miR-424-5p mimic group were verified by qPCR in rat and human chondrocytes, respectively. Conversely, indicated overtly decreased expression of miR-424-5p (Fig. 2A) and increased expression of SMAD7 (Fig. 2B) in anti-miR-424-5p group was observed. After that, luciferase reporter vectors containing the WT or MUT 3’UTR of SMAD7 (Fig. 2C) were transfected together with miR-424 mimic or anti-miR-424-5p in rat chondrocytes and human C28/I2 cells. Importantly, the luciferase activity of WT-SMAD7 decreased evidently in miR-424-5p mimic group, while elevated luciferase activity was found in anti-miR-424-5p group of rat chondrocytes (Fig. 2D) and human C28/I2 cells (Fig. 2E) in contrast to control group. Nevertheless, there was no difference of the luciferase activity between miR-424-5p mimic and anti-miR-424-5p group with the Mut-SMAD7 vector in rat chondrocytes (Fig. 2D) and human C28/I2 cells (Fig. 2E). To gain detailed orchestration of miR-424-5p on SMAD7, protein levels of SMAD7 was examined with the transfection of miR-424-5p mimic and anti-miR-424-5p. The results showed that SMAD7 protein levels elevated in anti-miR-424-5p group, while it decreased in group of miR-424-5p mimic, all compared to control group in rat chondrocytes (Fig. 2F) and human C28/I2 cells (Fig. 2G). Together, our results support that SMAD7 is a target of miR-424-5p in chondrocytes, further sustaining the putative function of miR-424-5p in OA development.
MiR-424-5p mediated expression of pro-inflammatory cytokines and apoptosis of chondrocytes via a SMDA7-dependent manner
To further clarify the effects of miR-424-5p on SMAD7 expression in chondrocytes cells, we then checked alteration of SMAD7-related inflammatory reaction and cell apoptosis. First, protein levels of secreted IL-6 and TNFα cytokines were detected via ELISA with the transfection of siNC or/and mock, anti-miR-424-5p or/and si-SMAD7 in rat chondrocytes and human C28/I2 cells. The results showed that IL-6 and TNFα levels decreased after the transfection of anti-miR-424-5p, which could be partially rescued by co-knockdown of SMAD7 (Fig. 3A). Consistently, mRNA levels of IL-6 and TNFα reduced with the inhibition of miR-424-5p, which was attenuated by simultaneous depletion of SMAD7 in rat chondrocytes and human C28/I2 cells (Fig. 3B). Morever, western blot results also demonstrated that IL-6 and TNFα levels decreased by virtue of anti-miR-424-5p, which could be offset by further knockdown of SMAD7 in rat chondrocytes (Fig. 3C) and human C28/I2 cells (Fig. 3D). These results suggest that miR-424-5p could regulate inflammatory reaction via a SMAD7-dependent manner.
Given that apoptosis of chondrocytes has been reported to be associated with SMAD7 and OA development, apoptosis of rat chondrocytes and human C28/I2 cells was investigated. These cells were transfected with siNC or/and mock, anti-miR-424-5p or/and si-SMAD7 and the apoptosis was examined by flow cytometry. Intriguingly, the data showed that cell apoptosis rates notably decreased with the treatment of anti-miR-424-5p, while this effect was markedly ameliorated by SMAD7 knockdown (Fig. 3E). Additionally, apoptosis markers were also detected by western blot with antibodies against Bax, Cleaved caspase-3 and TP53. Consistently, protein levels decreased after transfection of anti-miR-424-5p, which could be counteracted by co-knockdown of SMAD7 in rat chondrocytes (Fig. 3F) and human C28/I2 cells (Fig. 3G). Taken together, these results suggest that miR-424-5p might exert function of pro-apoptosis through mediating SMAD7 in chondrocytes.
NEAT1 was verified as a candidate interacting lncRNA of miR-424-5p and regulated SMAD7 expression
Previous researches have shown that NEAT1 is in implicated in occurrence of inflammation, so we reasoned that NEAT1 might be involved in regulation of OA development. Expression levels of NEAT1 between OA and adjacent tissues were tested by qPCR and the data showed that NEAT1 had a higher expression in adjacent compared to OA tissues (Fig. 4A). Given that lncRNA is reported to interact with miRNA to function at the molecular level, we then investigated the association between miR-424-5p and NEAT1. Initially, miR-424-5p and SMAD7 expression levels were examined by qPCR after transfection of NEAT1 or siNEAT1. The results verified that expression levels of miR-425-5p decreased and elevated with the overexpression and knockdown of NEAT1, respectively, in which SMAD7 had an opposite expression profile (Fig. 4B). In addition, it is intriguingly found that NEAT1 had putative binding sites of miR-424-5p by virtue of starBase database (Fig. 4C). To demonstrate the interaction between NEAT1 and miR-424-5p, luciferase reporter assay of WT and MUT-NEAT1 vector was performed in which the luciferase activity was tested with the transfection of anti-miR-424-5p or miR-424-5p into human C28/I2 cells. The results showed that the luciferase activity of WT-NEAT1vector overtly decreased and elevated with the transfection of miR-424-5p mimics and anti-miR-424-5p, respectively, while MUT-NEAT1 vector had rare effects in luciferase activity assay following the similar treatment (Fig. 4D). To further validate the association of NEAT1 and miR-424-5p, RNA immunoprecipitation (RIP) assay with the antibodies against IgG and Argonaute2 protein that is documented to be a crucial part of the RNA induced silencing complex was employed . Significantly, our data found that NEAT1 and miR-424-5p were largely immuoprecipated with anti-Ago2 compared to IgG control group in human C28/I2 cells (Fig. 4E). Together, our results indicate NEAT1 could interact with miR-424-5p and regulate its expression.
To further interrogate the interaction of between NEAT1 and miR-424-5p, luciferase reporter assay of WT and MUT- SMAD7 and its expression with the transfection of pcDNA3.1 and mock, miR-424-5p mimics and pcDNA3.1, miR-424-5p mimics and NEAT1 and mock and NEAT1. Importantly, our data displayed that the luciferase activity of WT-SMAD7 was obviously enhanced by transfection of NEAT1. And the luciferase activity was reduced by overexpression of miR-424-5p reversely, which was then rescued by co-transfection of NEAT1 in human C28/I2 cells (Fig. 4F). However, there was little alteration in MUT-SMAD7 groups. Morever, mRNA and protein levels of SMDA7 were also examined in human C28/I2 cells. The results showed that mRNA and protein levels of SMAD7 decreased after transfection of miR-424-5p mimics, which were counteracted by further overexpression of NEAT1 (Fig. 4G). Taken together, these results propose that NEAT1 is implicated in miR-424-5-mediated SMAD7 regulatory axis and positively regulate SMAD7 expression.
NEAT1 coordinated with miR-424-5p mediating expression of pro-inflammatory cytokines and apoptosis of chondrocytes
To further elucidate the putative effects of NEAT1 on OA development, pro- inflammatory cytokines expression and chondrocytes apoptosis were checked via gain-function of NEAT1 and miR-424-5p. Human C28/I2 cells were transfected with vector of pcDNA3.1 and mock, miR-424-5p mimics and pcDNA3.1, miR-424-5p mimics and NEAT1 and mock and NEAT1, and the secreted IL-6, TNFα cytokines and mRNA of them were detected through ELISA and qPCR. The results showed that IL-6 and TNFα levels increased after the transfection of miR-424-5p mimics, which could be offset by overexpression of NEAT1 together (Fig. 5A). Similarily, mRNA levels of IL-6 and TNFα elevated with the overexpression of miR-424-5p, which was attenuated by simultaneous transfection of NEAT1 (Fig. 5B). Morever, western blot results also proved that IL-6 and TNFα levels increased by virtue of overexpression of miR-424-5p mimics, which could be rescued by further transfection of NEAT1 (Fig. 5C). Importantly, overexpression of NEAT1 induced the decreased expression of IL-6 and TNFα across these experiments (Fig. 5A, 5B, 5C). Additionally, apoptosis rates of these cells were examined and the data showed that cell apoptosis rates apparently elevated treatment of miR-424-5p mimics, of which this effect was markedly rescued by overexpression of NEAT1 (Fig. 5D). In addition, apoptosis markers were also checked by western blot with antibodies against Bax, Cleaved caspase-3 and TP53. Correspondingly, protein levels decreased after transfection of miR-424-5p mimics, which could be offset by further transfection of NEAT1 (Fig. 5E). Significantly, overexpression of NEAT1 induced the elevated apoptosis rates and proteins markers significantly in human C28/I2 cells (Fig. 5D, 5E). Taken together, these results suggest that NEAT1 coordinate with miR-424-5p mediating expression of inflammatory cytokines and cell apoptosis in chondrocytes.