Inhibited microRNA-494-5p promotes proliferation and suppresses senescence of nucleus pulposus cells in mice with intervertebral disc degeneration by elevating TIMP3

Abstract

It has been unraveled that microRNAs (miRNAs) played crucial roles in processes of human diseases, while the role of miR-494-5p in intervertebral disc degeneration (IDD) remains scarcely studied. We aimed to investigate the mechanisms of miR-494-5p in IDD with the involvement of tissue inhibitor of metalloproteinase 3 (TIMP3). Expression of miR-494-5p and TIMP3 in IDD clinical specimens was assessed. The IDD models were established by needle punching, which were then injected with low expression of miR-494-5p or TIMP3 overexpression lentivirus to observe their effects on pathology and apoptosis in IDD mice. The nucleus pulposus cells were isolated and, respectively, treated with miR-494-5p inhibitor or TIMP3 overexpression plasmid to determine the viability, apoptosis, and senescence in vitro. Furthermore, the expression of Aggrecan, Col-2, Caveolin-1, and SA-β-gal in nucleus pulposus cells in vitro were measured. The target relation between miR-494-5p and TIMP3 was determined. An increased expression of miR-494-5p and a decreased expression of TIMP3 were found in IDD. Downregulation of miR-494-5p or overexpression of TIMP3 could relieve pathology and suppress nucleus pulposus cell apoptosis in IDD mice, as well as promote the viability and attenuate the apoptosis and senescence of nucleus pulposus cells from IDD mice. Moreover, inhibition of miR-494-5p or overexpression of TIMP3 upregulated Aggrecan and Col-2 expression while downregulated Caveolin-1 and SA-β-gal expression, and TIMP3 was the target gene of miR-494-5p. Results of this study indicated that the degradation of miR-494-5p ameliorates the development of IDD by elevating TIPM3, which may provide new targets for IDD treatment.

Keywords: Intervertebral disc degeneration; MicroRNA-494-5p; cellular senescence; differentiation; nucleus pulposus cells; proliferation; tissue inhibitor of metalloproteinase 3.

Figure 1.

An increased expression of miR-494-5p and a decreased expression of TIMP3 appear in IDD clinical samples. (a), representative images of HE staining in nucleus pulposus tissues of the IDD group and the control group; (b), miR-494-5p expression in nucleus pulposus tissues of the IDD group and the control group; (c), TIMP3 mRNA expression in nucleus pulposus tissues of the IDD group and the control group; (d), protein expression of TIMP3 in nucleus pulposus tissues of the IDD group and the control group; n = 20. Data are expressed as mean ± standard deviation, and the unpaired t-test was performed for comparisons between two groups

Figure 2.

Inhibited miR-494-5p or elevated TIMP3 alleviates pathology and decelerates cell apoptosis in nucleus pulposus tissues of IDD mice. (a), miR-494-5p expression in mice’s nucleus pulposus tissues of each group; (b), TIMP3 mRNA expression in mice’ nucleus pulposus tissues of each group; (c), protein expression of TIMP3 in mice’ nucleus pulposus tissues of each group; (d), representative images of HE staining in nucleus pulposus tissues of mice in each group; (e), representative images of TUNEL staining in nucleus pulposus tissues of mice in each group; (f), statistical results of TUNEL staining in nucleus pulposus tissues of mice in each group; n = 8, a P < 0.05 vs the normal group, b P < 0.05 vs the NC group, c P < 0.05 vs the anti-miR-494-5p group. Data are expressed as mean ± standard deviation and one-way ANOVA was used for comparisons among multiple groups

Figure 3.

The 3ʹUTR of TIMP3 mRNA is a direct target of miR-494-5p. (a), binding sites of miR-494-5p and TIMP3 that predicted by http://www.targetscan.org/vert_72/; (b), results of dual-luciferase reporter gene assay; (c), miR-494-5p expression in transfected mice’ nucleus pulposus cells of each group; (d), TIMP3 mRNA expression in transfected mice’ nucleus pulposus cells of each group; (e), protein expression of TIMP3 in transfected mice’ nucleus pulposus cells of each group; N = 3, a P < 0.05 vs the normal group, b P < 0.05 vs the NC group, c P < 0.05 vs the miR-494-5p inhibitor group. Data are expressed as mean ± standard deviation, and one-way ANOVA was used for comparisons among multiple groups. The experiment was independently repeated for 3 times, and the unpaired t-test was performed for comparisons between two groups

Figure 4.

Inhibited miR-494-5p or elevated TIMP3 attenuates senescence of nucleus pulposus cells from IDD mice. (a), expression of Aggrecan mRNA in transfected mice’ nucleus pulposus cells of each group; (b), expression of Col-2 mRNA in transfected mice’ nucleus pulposus cells of each group; (c), expression of Caveolin-1 mRNA in transfected mice’ nucleus pulposus cells of each group; (d), expression of SA-β-gal mRNA in transfected mice’ nucleus pulposus cells of each group; (e), representative images and statistical results of SA-β-gal staining in transfected mice’ nucleus pulposus cells of each group; N = 3, a P < 0.05 vs the normal group, b P < 0.05 vs the NC group, c P < 0.05 vs the miR-494-5p inhibitor group. Data are expressed as mean ± standard deviation and one-way ANOVA was used for comparisons among multiple groups

Figure 5.

Inhibited miR-494-5p or elevated TIMP3 promotes proliferation and abates apoptosis of nucleus pulposus cells from IDD mice. (a), the viability of transfected mice’ nucleus pulposus cells in each group was detected using CCK-8 assay; (b), cell cycle distribution of transfected mice’ nucleus pulposus cells in each group was assessed by flow cytometry; (c), numbers of mice’ nucleus pulposus cells that arrested in the G0/G1, S, and G2/M phases in each group; (d), apoptosis of transfected mice’ nucleus pulposus cells in each group was measured by flow cytometry; (e), comparisons of apoptosis rate of mice’ nucleus pulposus cells among the groups; N = 3, a P < 0.05 vs the normal group, b P < 0.05 vs the NC group, c P < 0.05 vs the miR-494-5p inhibitor group. Data are expressed as mean ± standard deviation and one-way ANOVA was used for comparisons among multiple groups