SEMA6D is a down-stream target gene of miR-7 in chondrocytes.
Our previous studies already proved that miR-7 was down-regulated in normal chondrocytes and thereby exacerbated the course of OA through aggravating cartilage degradation (12). Nonetheless, the down-stream regulatory target gene still remains unknown. Further investigation of the regulatory axis of miR-7 could possibly help developing novel therapeutic methods for OA, which therefore encouraged us to continue the lucubration on miR-7. Multiple databases involving TargetScan and starBase were adopted to seek the target gene of miR-7, and SEMA6D was found as a potential regulatory protein of miR-7. Bioinformatics prediction was conducted to discover that there was a direct binding site on SEMA6D sequence of miR-7 (Fig.1A), which suggested the possibility of the existence of interaction between SEMA6D and miR-7. To further confirm this assumption, dual luciferase assay was exerted and found that there was a significant difference between SEMA6D-Mut group and SEMA6D-WT group which were both co-treated with miR-7 mimic (Fig.1B, p<0.01). This result verified that there was a direct interaction between miR-7 and SEMA6D. To identify the exact relation be miR-7 and SEMA6D, miR-7 mimic and miR-7 inhibitor were given to OA chondrocytes, respectively. Then the relative transcription level of SEMA6D was evaluated through qRT-PCR, and the result demonstrated that up-regulation of miR-7 led to transcribed suppression of SEMA6D in OA chondrocytes, while miR-7 inhibitor group presented the opposite phenomenon (Fig.1C, p<0.001), which suggested that SEMA6D was negatively regulated by miR-7 in chondrocytes.
MiR-7 accelerated the development of OA in vivo via down-regulating SEMA6D.
Further understanding the specific role of SEMA6D in vivo was performed in human cartilage tissues and mice cartilage tissues. As depicted in Fig.2A was the immunohistochemistry consequences of SEMA6D in human and mice cartilage tissues, results showed that SEMA6D was in higher expression level both in normal human and mice chondrocytes when compared with OA chondrocytes. Next, to further verified the correlation of miR-7 and SEMA6D in vivo, miR-7 mimics and miR-7 inhibitor were given to normal mice and OA mice to investigate the nuance of SEMA6D expression, and negative controls (mimics-NC, inhibitor-NC) were also given to OA mice. Research consequences manifested that up-regulation of miR-7 would decrease the expression quantities of SEMA6D in vivo when compared with the mimic-NC group, while miR-7 inhibitor in OA mice presented lower existence of SEMA6D, which confirmed the negatively regulatory effect of miR-7 toward SEMA6D. The statistics of SEMA6D expression in normal and OA mice tissues were performed in Fig.2B, which conspicuously indicated the lower-then-usual expression quantities of SEMA6D in OA chondrocytes. These results possibly suggested that SEMA6D might have pathological regulatory function in the course of OA.
SEMA6D was overexpressed in chondrocytes with strong anabolism.
It was reported that the development of OA was closely correlated with the anabolic and catabolic activity of chondrocytes (17, 18). Multiple inflammatory mediators and mechanical stimulation collectively affect the physiological process of chondrocytes and thereby induce excessive catabolic activity and trigger osteoarthritis (19-21). Although previous experiments demonstrated that miR-7 negatively regulated SEMA6D in OA chondrocytes, there is still much confusion about the roles of miR-7 and SEMA6D in mediating the development of OA. We next proposed a hypothesis that SEMA6D might participate in regulating the catabolism and anabolism of chondrocytes. To confirm our assumption, we then constructed chondrocytes with excessive catabolic activity using fibronectin fragment (FN-f)(22), and chondrocytes with excessive anabolic activity by osteogenic protein-1 (OP1)(8). Chondrocytes were given different concentration of FN-f (0μM, 0.5μM, 1μM and 2μM) and OP1 (0ng/ml, 50ng/ml, 100ng/ml and 150ng/ml) to induce catabolism and anabolism, respectively. The status of catabolism and anabolism induced by FN-1 and OP1 presented dose dependent. Subsequently, the transcriptional levels of miR-7 and SEMA6D were detected by qRT-PCR, and the results depicted in Figure.3A and 3B demonstrated miR-7 expressed at a highest level in the chondrocytes with highest catabolic activity level induced by FN-f, which indicated that miR-7 possibly partakes in promoting the catabolism of OA chondrocytes. Contrarily, the expression quantities of SEMA6D presented the lowest in chondrocytes with highest catabolic activity. These results suggested that SEMA6D might dedicate in preventing the catabolism in chondrocytes, and the research consequence further illustrated that the regulatory effect of miR-7 and SEMA6D was opposite in chondrocytes. Subsequently, metalloproteinase 2 (MMP-2) was chosen as a biomarker in this study to examine the injury degree in cartilage cells with catabolism stimulation brought by FN-f, of which MMP-2 has proven up-regulated in OA chondrocytes with high level of catabolism(23). The results of Western blot showed that MMP-2 was significantly up-regulated in 2μM of FN-f treated group (Fig.3C, p<0.001), and SEMA6D was in the lowest expression level in 2μM of FN-f treated group (Fig.3C, p<0.001). These consequences illustrated that SEMA6D was down-regulated in chondrocytes with active catabolism, and SEMA6D might have a pathological correlation with the catabolic activity of OA chondrocytes. From the above research consequence we could conclude that SEMA6D possibly participated in decreasing the catabolism of OA chondrocytes, however, whether SEMA6D is validated in promoting the anabolism of chondrocytes still remains unknown. Therefore, we utilized OP1 to induce active anabolism in chondrocytes and detected the transcriptional level of miR-7 and SEMA6D through qRT-PCR. Results demonstrated that 150ng/ml of OP1 treated group presented the lowest level of miR-7, while the transcriptional quantities of SEMA6D was the highest (Fig.3D-3E, p<0.001). Similarly, the results of western blot showed that MMP-2 was down-regulated when given OP1 treated and presented dose dependent toward OP1. Contrarily, SEMA6D was significantly up-regulated when given OP1 treated, and the expression level also manifested dose dependent (Fig.3F, p<0.001). These consequences collectively illustrated that SEMA6D was pathologically correlated with the catabolism and anabolism in chondrocytes and might act as a favourable factor in activating anabolism.
SEMA6D reduced the catabolism of OA chondrocytes.
According to literature research, the expression quantities of matrix metalloproteinases (MMPs), including MMP-2 and MMP-13, are increased in the cartilage tissues of OA patients(24, 25). To further investigate the functional role of SEMA6D in OA chondrocytes, we constructed and transfected small interfering RNA SEMA6D (si-SEMA6D) and pcDNA3.1-SEMA6D into chondrocytes for SEMA6D knockdown and overexpression, respectively. FN-f was used as inducer to construct OA chondrocytes. Next, we examined the mRNA and proteins expression level of MMP-2, MMP-13 and SEMA6D using qRT-PCR and western blot. After transfection of si-SEMA6D and pcDNA3.1-SEMA6D, we first scrutinized the expression level of SEMA6D. The quantities of SEMA6D in normal chondrocytes was significantly lower in si-SEMA6D transfected group (Fig.4A, p<0.05), while pcDNA3.1-SEMA6D transfected chondrocytes presented significantly higher SEMA6D expression (Fig.4A, p<0.001). Next, the quantities of SEMA6D in FN-f induced OA chondrocytes were also detected using qRT-PCR. The results showed that the transcriptional level of SEMA6D in FN-f induced OA chondrocytes (control+FN-f) was significantly lower than normal chondrocytes (control) (Fig.4A, p<0.05). Also, OA chondrocytes induced by FN-f with si-SEMA6D and pcDNA3.1-SEMA6D transfection presented conspicuously lower and higher level of SEMA6D, respectively. To lucubrate the functional role of SEMA6D in chondrocytes with active catabolism, we then examined the quantities level of MMP-2 and MMP-13. As shown in Figure.4B-4C was the results of qRT-PCR, which depicted that knockdown of SEMA6D significantly increase the transcriptional level of MMP-2 and MMP-13 in normal chondrocytes, while up-regulation of SEMA6D using pcDNA3.1-SEMA6D reduced the quantities of MMP-2 and MMP-13 (p<0.05). While in FN-f induced OA chondrocytes (control+FN-f), the transcriptional quantities of MMP-2 and MMP-13 have significantly increased (p<0.05), which means FN-f has successfully constructed OA chondrocytes. When given si-SEMA6D transfection to silence SEMA6D in OA chondrocytes, the extent of MMP-2 and MMP-13 presented the highest (Fig.4B-4C, p<0.001), while up-regulation of SEMA6D using pcDNA3.1-SEMA6D resulted in decreasing MMP-2 and MMP-13. Subsequently, western blot assay was performed to further confirm the suppressed function of SEMA6D toward MMP-2 and MMP-13. Similarly, silencing of SEMA6D by si-SEMA6D both in normal and OA chondrocytes led to an increasement of MMP-2 and MMP-13 (p<0.01), while up-regulation of SEMA6D demonstrated the opposite effect (Fig.4D, p<0.05). These results gave a joint clarification that SEMA6D might be a functional factor in preventing further damage of OA cartilage.
SEMA6D promoted anabolic activity via inhibiting the activation of p38 pathway.
Although we have discovered that SEMA6D might serve as a favourable factor in preventing catabolism and accelerating anabolism in chondrocytes, the specific regulatory mechanism of SEMA6D is still under exploration. Based on previous literature study, p38 kinase is associated with the exacerbation of OA via accelerating the synthesis of inflammatory factors and MMPs(26, 27), and mediating chondrocytes apoptosis(28). Similarly, over activation of extracellular regulated kinases (ERK) has been reported as a key factor in interfering the remodeling and proliferation of cartilage cells(29), and activation of ERK pathway would further induce the synthesis of MMP-13. Additionally, it was reported that the interaction of ERK1-Smad1 protein could promote the development of OA(30).
For further investigation of the regulatory pathway of SEMA6D, si-SEMA6D and pcDNA3.1-SEMA6D were transfected to normal chondrocytes and OP1 treated chondrocytes, we then estimated that correlated proteins expression of p38, ERK and Smad1 using western blot assay. As depicted in Figure.5 were the results of western blot assay, which indicated that silencing of SEMA6D using si-SEMA6D significantly improved the extent of phosphorylation of p38 in normal chondrocytes (Fig.5, p<0.05), while up-regulation of SEMA6D decreased the quantities of phosphorylation of p38 conspicuously in normal chondrocytes (p<0.05) and OP1 treated chondrocytes (p<0.001). However, no significant difference existed in the expression quantities of ERK, p-ERK, Smad1 and p-Smad1, which might infer that SEMA6D mainly participated in regulating p38 pathway.
Lucubration of SEMA6D in regulating p38 pathway, we further introduced p38 MAPK inhibitor HY-12839 into the research. Firstly, chondrocytes were divided into two groups, of which one group was given 30 nM of HY-12839 to to block the signaling function of p38 pathway; the other group was given same volume of DMSO. Subsequently, si-SEMA6D, pcDNA3.1-SEMA6D, lip-2000-NC were transfected into chondrocytes. Next, we scrutinized the expression extent of anabolic process associated proteins, including Aggrecan, collagen type II A1 (COL2A1) and ID1 proteins, which have been reported at up-regulation level in OA(31-33). As shown in Figure.6A-6C were the results of qRT-PCR, which demonstrated that up-regulation of SEMA6D using pcDNA3.1-SEMA6D could significantly induce the transcriptional quantities of Aggrecan, COL2A1 and ID1 (p<0.01) in chondrocytes without HY-12839 treatment, which suggested that SEMA6D was validated as a promoted factor of chondrocytes anabolism. Additionally, in HY-12839 treated group, the quantities of Aggrecan, COL2A1 and ID1 were up-regulated when compared with control group (p<0.01), while overexpression of SEMA6D through pcDNA3.1-SEMA6D induced even higher quantities of Aggrecan, COL2A1 and ID1 (p<0.001). Moreover, the results of western blot further verified the p38 suppressing effect of SEMA6D (Fig.6D). According to the consequences of western blot, the expression level of phosphorylated p38 has decreased when given OP1 treated (p<0.01), and overexpression of SEMA6D, which suggested that there was lower activation of p38 in chondrocytes with active anabolism. While the expression of Aggrecan, COL2A1 and ID1 significantly increased when there was up-regulation of SEMA6D in HY-12839 treated chondrocytes (p<0.001). From these results we could conclude that SEMA6D was validated in preventing the course of OA via inhibiting the activation of p38.
To confirm the SEMA6D’s effect in improving osteoarthritis, immunofluorescence staining was performed to further examine the expression of COL2A1. As depicted in Figure.7 were the immunofluorescence results, from which we could infer a conclusion, that is, up-regulation of SEMA6D brought by pcDNA3.1-SEMA6D could increase the expression level of COL2A1 in normal chondrocytes. Also, in chondrocytes with active anabolic metabolism brought by OP1, overexpression of SEMA6D improved the quantities of COL2A1 conspicuously. These results further verified that SEMA6D was capable to improve the course of OA.