Differential analysis of non-coding RNA in NP tissues of IDD patients
Among the 2549 miRNAs detected in the miRNAs microarray (GSE116726 dataset), 56 miRNAs in degenerated NP tissue were up-regulated (logFC>2.0 and adj.P.Val<0.01), and 8 miRNAs were down-regulated ( logFC< -2.0 and adj.P.Val<0.01) (Figure 1A). In addition, we found that these 8 miRNAs were not only down-regulated in the NP tissues of IDD patients (Figure 1B), but also significantly down-regulated in hNPCs induced by 5ng/ml TNF-α+IL-1β (Figure 1C).
Among the 2894 circRNAs detected in the circRNAs microarray (GSE67566 dataset), 48 circRNAs in the degenerated NP tissue were up-regulated (logFC>2.0 and adj.P.Val<0.01) and 58 circRNAs were down-regulated (logFC<-2.0 and adj.P.Val<0.01) (Figure 1D).
Hsa-miR-181c-5p silence or hsa_circ_0001658 over-expression inhibited the proliferation and promoted apoptosis of hNPCs
The results of starBase (version 2.0) analysis showed that among the down-regulated miRNAs, hsa-miR-181c-5p has the binding site of hsa_circ_0001658 (Figure 2A). Hsa_circ_0001658 was significantly up-regulated in the NP tissues of IDD patients (logFC>2.0 and adj.P.Val<0.01), so we chose hsa-miR-181c-5p and hsa_circ_0001658 for research.
In 5ng/ml TNF-α+IL-1β treated hNPCs, the expression of hsa-miR-181c-5p was significantly down-regulated, while hsa-miR-181c-5p mimic or si-hsa_circ_0001658 significantly reversed the expression of hsa-miR-181c-5p (Figure 2B). In addition, we found that hsa-miR-181c-5p inhibitor or hsa_circ_0001658 significantly inhibited the proliferation of hNPCs (Figure 2C) and promoted the apoptosis of hNPCs (Figure 2D), up-regulated the expression of MMP-3 and MMP-13, inhibited the expression of collagen II and aggrecan, and all these effects could be reversed by hsa-miR-181c-5p mimic or hsa_circ_0001658 (Figure 2E).
These results indicated that hsa-miR-181c-5p silence or hsa_circ_0001658 over-expression inhibited the proliferation of hNPCs and the metabolic function of extra-cellular matrix (ECM), but promoted the apoptosis of hNPCs.
Hsa_circ_0001658 acted as a sponge of hsa-miR-181c-5p
We tried to study the mechanism of hsa_circ_0001658 and hsa-miR-181c-5p in the development of IDD. According to research reports, the function of circRNAs related to IDD were mainly as a sponge of miRNAs to combine with functional miRNAs[17, 18], and then regulated the proliferation and apoptosis of NP cells. Therefore, starBase was used to predict the relationship between hsa_circ_0001658 and hsa-miR-181c-5p. The results showed that there was a binding site of hsa_circ_0001658 on hsa-miR-181c-5p (Figure 2A). We found that the expression level of hsa-miR-181c-5p in the NP tissue cells of IDD patients was low, but hsa_circ_0001658 was highly expressed. In 5ng/ml TNF-α+IL-1β treated hNPCs, hsa-miR-181c-5p was low expression, while hsa_circ_0001658 was high, and there was a negative correlation between them (R2=0.8967, P=0.0146, Figure 3A). The results suggested that hsa_circ_0001658 may regulate the expression of hsa-miR-181c-5p by directly sponging hsa-miR-181c-5p.
To verify this hypothesis, we performed luciferase reporter assay. Considering that there were two hsa-miR-181c-5p binding sites on hsa_circ_0001658, we set mutation sequences for these two binding sites respectively. Hsa_circ_0001658 WT1, hsa_circ_0001658 WT2, hsa_circ_0001658 Mut1 and hsa_circ_0001658 Mut2 sequences were inserted to the downstream of the luciferase reporter molecule. Then the hsa-miR-181c-5p mimic and luciferase reporter gene were co-transfected into hNPCs. Compared with the control group (NC), hsa-miR-181c-5p mimic significantly reduced the luciferase activity in the hsa_circ_0001658 WT1 and hsa_circ_0001658 WT2 groups (P<0.05), but not in the hsa_circ_0001658 Mut2 and hsa_circ_0001658 Mut2 groups(P>0.05) (Figure 3B, 3C). These results indicated that hsa-miR-181c-5p bound to two predicted sites on hsa_circ_0001658.
To determine whether hsa_circ_0001658 affected the proliferation and apoptosis of hNPCs by sponging hsa-miR-181c-5p, we used hsa_circ_0001658 overexpression vector to transfect hNPCs, and qRT-PCR results showed that hsa_circ_0001658 overexpressed hNPCs were successfully constructed (Figure 3D). The expression level of hsa_circ_0001658 in the NP tissues of IDD patients was significantly increased (Figure 3E). In this study, we found that, compared with the control, hsa_circ_0001658 over-expression inhibited the proliferation and promoted the apoptosis of hNPCs, and these effects were reversed by hsa-miR-181c-5p mimic (Figure 3F, 3G).
These results showed that hsa_circ_0001658 acted as a sponge of hsa-miR-181c-5p.
Hsa-miR-181c-5p down-regulated the expression of FAS, promoted the proliferation and inhibited the apoptosis of hNPCs
As a member of the TNF receptor super-family, the protein encoded by FAS plays a central role in the physiological regulation of programmed cell death, and has been involved in the onset of various malignant tumors and immune system diseases[19-21]. The bioinformatics prediction results showed that FAS was a potential target of hsa-miR-181c-5p (Figure 4A). The results of dual luciferase reporter gene detection showed that the luciferase signal of wild-type FAS reporter gene was significantly inhibited by hsa-miR-181c-5p, however, the luciferase signal of the mutant FAS reporter gene was not significantly affected by hsa-miR-181c-5p (Figure 4A). The results of loss-of-function and gain-of-function experiments showed that hsa-miR-181c-5p mimic significantly inhibited the expression of FAS in hNPCs, while the expression of FAS in hNPCs increased significantly after treatment with hsa-miR-181c-5p inhibitor (Figure 4B). After treated with 5ng/ml TNF-α and IL-1β, the FAS expression level in hNPCs was significantly increased, and hsa-miR-181c-5p mimic treatment could significantly reverse the decrease in FAS expression level (Figure 4C). In addition, after overexpression of FAS, the proliferation ability of hNPCs was significantly decreased, the apoptosis rate was significantly increased, the expression levels of MMP-3 and MMP-13 proteins were up-regulated, the expressions of collagen II and aggrecan were down-regulated, while the hsa-miR-181c-5p mimic transfection reversed the effects of FAS overexpression on hNPCs proliferation, apoptosis and expression of MMP-3, MMP-13, collagen II and aggrecan (Figure 4D, 4E, 4F).
These results demonstrated that hsa-miR-181c-5p down-regulated the expression of FAS, promoted the proliferation and inhibited the apoptosis of hNPCs, and at the same time inhibited catabolic reactions and promoted the expression of ECM compositions.
Hsa_circ_0001658 functioned in hNPCs through targeting hsa-miR-181c-5p/FAS
Adenovirus carrying hsa_circ_0001658, hsa_circ_0001658 siRNA or blank control (si-NC) was transfected into hNPCs, the results of qRT-PCR showed that when the exogenous hsa_circ_0001658 overexpression vector was transfected, the hsa_circ_0001658 level in hNPCs was significantly increased. On the contrary, the hsa_circ_0001658 expression level was significantly inhibited after hsa_circ_0001658 siRNA transfection (Figure 5A). The results of western blot analysis showed that overexpression of hsa_circ_0001658 caused an increase in the expression level of FAS protein in hNPCs, and the change in FAS expression level was reversed by hsa-miR-181c-5p mimic (Figure 5B). When hNPCs were treated with 5ng/ml TNF-α and IL-1β, the expression levels of hsa_circ_0001658 and FAS increased significantly, while the expression of hsa-miR-181c-5p decreased, these effects were reversed after the transfection with si-hsa_circ_0001658 (Figure 5C, 5D). Next, we studied whether FAS acted as a downstream mediator of hsa_circ_0001658 in 5ng/ml TNF-α and IL-1β treated hNPCs. The results showed that si-hsa_circ_0001658 and FAS knockdown significantly promoted the proliferation and inhibited the apoptosis of 5ng/ml TNF-α and IL-1β treated hNPCs (Figure 5E, 5F).
Based on the above results, we confirmed that hsa_circ_0001658 functioned in hNPCs through targeting hsa-miR-181c-5p/FAS.