In the first place, we observed significant miR-195 up-regulation and Bcl-2 down-regulation in NP tissues of IVDD patients. The elevated miR-195-5p was also observed in age-related macular degeneration (AMD) by using miRNA microarray chip detection, which was regarded as a potential biomarker for AMD diagnosis, as indicated by Chengda Ren et al. (17). On the other aspect, Bcl-2 expression was found to be dramatically down-regulated, whereas Bax expression was remarkably up-regulated in the rabbit IVDD model (18). Notably, Ping Cai et al. identified the increased expression of miR-15a, which belonged to the same family as miR-195, in NP tissues of IVDD, and in particular, over-expressed miR-15a can inhibit the Bcl-2 expression to promote the apoptosis of NP cells (19). Although miR-195 demonstrated different targets and functions in different tissues or diseases, according to the published data, many studies pointed out a pathogenic role of miR-195 in inhibiting cell proliferation and promoting cell apoptosis (20–22). More importantly, miR-195 expression was found negatively correlated with the Bcl-2 expression in our research, and the dual-luciferase reporter gene assay confirmed Bcl-2 to be a target gene of miR-195. At the same time, miR-195 can play its regulatory role by targeting Bcl-2 in many cells, such as tumor cells (23) and cardiomyocytes (24). All these findings suggested that miR-195 can regulate the expression of its target gene Bcl-2 to promote the progression of IVDD.
The presence of a significant number of senescent cells were shown in degenerative nucleus pulposus, which can induce the expression of multiple inflammatory cytokines and matrix degrading enzymes to aggravate the living environment of NP cells, thereby affecting the growth and function of NP cells and further triggering the apoptosis of more cells (25). In fact, TNF-α is a multifunctional pro-inflammatory cytokine and considered to be a key factor in IVDD (26). As such, we constructed TNF-α-induced NP cell apoptosis model by referring to a previous study (27), aiming to observe the mechanism of miR-195 in inducing NP cell apoptosis in vitro. Firstly, we found that the proliferation of TNF-α-induced NP cells was dramatically decreased, while the cell apoptosis was apparently increased, which was in consistency with the previous findings (28, 29). Besides, inhibiting miR-195 effectively enhanced the proliferation and limited the apoptosis of TNF-α-induced NP cells, whereas inhibiting Bcl-2 promoted NP cell apoptosis. Similarly, miR-199 was exhibited by Wei Wang et al. to mitigate TNF-α-induced NP cell apoptosis by targeted down-regulation of MAP3K5, and thereby playing its protective role for NP cells (30). Also, HOTAIR can inhibit miR-34a expression to up-regulate the expression of its target gene Bcl-2 to further attenuate TNF-α-induced NP cell apoptosis (31). Here in this study, Bcl-2 siRNA can reverse the protective effect of miR-195 inhibitor on NP cells. Not surprisingly, Huaqing Zhu et al. noted that miR-195 may reduce Sirt1 and Bcl-2 expression to enhance the reactive oxygen species production and promote the apoptosis of palmitate-induced cardiomyocytes (32). Taken together, inhibiting miR-195 can inhibit TNF-α-induced NP cell apoptosis by targeted regulation of its target gene Bcl-2.
As for cell apoptosis in IVDD, it can occur through death receptor pathway, mitochondrial pathway and endoplasmic reticulum signaling pathway (33), while its apoptosis induced by endogenous pathway was initially found in mitochondria, namely mitochondrial pathway, which mainly exert functions via Bcl-2 protein family (34). As a major anti-apoptotic member, Bcl-2 can maintain the outer mitochondrial membrane integrity (35). On the contrary, Bax, a pro-apoptotic member of the Bcl-2 family of proteins, mainly plays its regulatory role by destroying the integrity of mitochondrial membrane (36). Bcl-2 protein family can change the permeability of mitochondrial membrane and induce the opening of mitochondrial pore, thus allowing the apoptosis inducing factor, cytochrome C, and pro-apoptotic protein into the cytoplasm, and finally inducing cell apoptosis by activating caspase (37, 38). In agreement, we also found down-regulated miR-195 can improve MMP of TNF-α-induced NP cells, elevate Bcl-2 protein, and reduce Bax and cleaved caspase 3 proteins, which however can be reversed by silencing Bcl-2. Besides, inhibition of miR-494 can effectively reduce Caspase-3 and Bax, and elevate Bcl-2, thus promoting the proliferation and hindering the apoptosis of NP cells (39). As reported by Ping Liu et al., miRNA-125a can up-regulate anti-apoptotic protein Bcl-2 and inhibit Caspase-3 and Bax proteins by down-regulating BAK1, thus inhibiting the apoptosis of NP cells (40). Moreover, Chang-Kui Gao et al. demonstrated that down-expressed miR-195 can elevate Bcl-2 but reduce Bax, Cyt-c and caspase-3 to increase the MMP of cardiomyocytes and mitigate hypoxia re-oxygenation-induced cardiomyocyte apoptosis (41). Given the above, inhibiting miR-195 can specifically up-regulate Bcl-2 to mediate mitochondrial apoptosis pathway, thus playing a protective role to reduce TNF-α-induced NP cell apoptosis.
To sum up, we observed the increased miR-195 in NP tissues of IVDD patients, and inhibiting miR-195 can attenuate TNF-α-induced NP cell apoptosis by up-regulating Bcl-2 expression. This study provides a new pathway and scientific basis for the clinical gene-based diagnosis, prevention and treatment of IVDD.