MicroRNA-143-5p targeting eEF2 gene mediates intervertebral disc degeneration through the AMPK signaling pathway

Abstract

Background: Intervertebral disc degeneration (IDD) is a major contributor to back, neck, and radicular pain, and the treatment of IDD is costly and relatively ineffective. Dysregulation of microRNAs (miRNAs) has been reported to be involved in IDD. The purpose of our study is to illustrate the potential that miR-143-5p targeting eEF2 gene mediates IDD.

Methods: Following the establishment of the IDD rat models, expression of miR-143-5p, eEF2, Bcl-2, Bax, AMPK, mTOR, cyclinD, COL2, ACAN, and DCN was detected. The NP cells isolated from degenerative intervertebral disc (IVD) were introduced with a series of mimic, inhibitor, or AICAR to explore the functional role of miR-143-5p in IDD and to characterize the relationship between miR-143-5p and eEF2. Cell viability, cell cycle, apoptosis, and senescence were also evaluated.

Results: A reduction in eEF2, an increase in miR-143-5p, and activation of the AMPK signaling pathway were observed in degenerative IVD. Moreover, increased senescent NP cells were observed in degenerative IVD. eEF2 was confirmed as a target gene of miR-143-5p. miR-143-5p was found to activate the AMPK signaling pathway. The restoration of miR-143-5p or the activation of AMPK signaling pathway decreased COL2, ACAN, and DCN expression, coupled with the inhibition of NP cell proliferation and differentiation, and promotion of NP apoptosis and senescence. On the contrary, the inhibition of miR-143-5p led to the reversed results.

Conclusion: The results demonstrated that the inhibition of miR-143-5p may act as a suppressor for the progression of IDD.

Keywords: AMPK signaling pathway; Apoptosis; Differentiation; EEF2; MicroRNA-143-5p; Nucleus pulposus cells; Senescence.

Fig. 1

The induction of IDD in rats exhibits degenerative IVD and shrank NP. a Safranin O staining images of normal IVD tissues and NP (× 100). b Safranin O staining images of degenerative IVD tissues and NP. c HE staining images of normal IVD tissues. d HE staining images of degenerative IVD tissues. e HE staining images of NP in normal IVD tissues. f HE staining images of NP in degenerative IVD tissues. HE, hematoxylin-eosin; IVD, intervertebral disc; IDD, intervertebral disc degeneration; NP, nucleus pulposus

Fig. 2

eEF2 protein is expressed at a low level in degenerative IVD. a Immunohistochemistry images of eEF2 protein expression in normal IVD and degenerative IVD (× 100 in left panel, × 400 in right panel). eEF2-positive particle is stained brownish yellow in notochord cells and chondrocytes. b Positive rate of eEF2 protein expression in normal IVD and degenerative IVD; n = 15; positive rate represents the mean value of the percentage of positively stained cells in total cells; data were expressed by mean ± standard deviation; comparison of data between two groups was conducted using a t-test; *, p < 0.05 vs. normal IVD; IVD, intervertebral disc; IDD, intervertebral disc degeneration; eEF2, eukaryotic elongation factors-2

Fig. 3

Degenerative IVD shares a close association with increased NP cell senescence. a SA-β-gal staining images of senescent NP cells in normal IVD and degenerative IVD under a microscope (× 400); dark blue-stained cells were senescent ones. b The positive rate of SA-β-gal staining in normal IVD and degenerative IVD; the positive rate of SA-β-gal staining represents the percentage of dark blue-stained NP cells in total NP cells; the experiment was repeated three times independently; data were expressed by mean ± standard deviation; comparison of data between two groups was conducted using a t-test; *, p < 0.05 vs. normal IVD; IVD, intervertebral disc; NP, nucleus pulposus; SA-β-gal, senescence-associated β-galactosidase

Fig. 4

IDD rats exhibit upregulated miR-143-5p and activated AMPK signaling pathway yet inhibited NP cell differentiation and eEF2. a miR-143-5p expression and mRNA expression of Bax, AMPK, mTOR, Bcl-2, cyclinD, and eEF2 in normal IVD and degenerative IVD. b, c Protein levels and bands of Bax, AMPK, mTOR, Bcl-2, cyclinD, and eEF2 in normal IVD and degenerative IVD. d Protein levels of COL2, ACAN, and DCN in normal IVD and degenerative IVD; the experiment was repeated three times independently; data were expressed by mean ± standard deviation; comparison of data between two groups was conducted using a t-test; ^*, p < 0.05 vs. normal IVD; miR-143-5p, microRNA-143-5p; IVD, intervertebral disc; eEF2, eukaryotic translation elongation factor 2; AMPK, AMP activated protein kinase; Bax, Bcl-2 Associated X protein; Bcl-2, B cell lymphoma-2; mTOR, mammalian target of rapamycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; COL2, collagen type II; ACAN, aggrecan; DCN, decorin

Fig. 5

miR-143-5p directly targets eEF2. a Putative binding site of miR-143-5p to the 3′UTR of eEF2 predicted using Targetscan. b The luciferase activity detection using vectors constructed with eEF2-3′UTR in the presence of miR-143-5p mimic or NC; three replicates were settled in each group and the experiment was repeated three times independently; data were expressed by mean ± standard deviation; comparison of data between two groups was conducted using a t-test; ^*, p < 0.05 vs. the NC group; miR-143-5p, microRNA-143-5p; eEF2, eukaryotic elongation factors-2; 3′UTR, 3′untranslated regions; Wt, wild-type; Mut, mutant; NC, negative control; ANOVA, analysis of variance

Fig. 6

MiR-143-5p suppresses eEF2 and activates the AMPK signaling pathway in NP cells. a miR-143-5p expression and mRNA expression of cyclinD, eEF2, mTOR, Bcl-2, Bax, and AMPK in NP cells from degenerative IVD treated with miR-143-5p mimic, inhibitor, or AICAR determined by RT-qPCR. b, c Protein levels and bands of cyclinD, eEF2, mTOR, Bcl-2, Bax, and AMPK in NP cells treated with miR-143-5p mimic, inhibitor, or AICAR determined by Western blot analysis; AICAR was the activator of the AMPK signaling pathway; the experiment was repeated three times independently; data were expressed by mean ± standard deviation; comparison of data among multiple groups was analyzed using one-way ANOVA; ^*, p < 0.05 vs. the control group (NP cells from normal IVD); ^#, p < 0.05 vs. the blank group (NP cells from degenerative IVD without transfection) and the NC group (NP cells from degenerative IVD transfected with NC sequence); miR-143-5p mimic group refers to NP cells from degenerative IVD transfected with miR-143-5p mimic; miR-143-5p inhibitor group refers to NP cells from degenerative IVD transfected with miR-143-5p inhibitor; AICAR group refers to NP cells from degenerative IVD treated with AMPK signaling pathway activator, AICAR; miR-143-5p inhibitor + AICAR group refers to NP cells of degenerative IVD treated with AICAR and transfected with miR-143-5p inhibitor. NC, negative control; miR-143-5p, microRNA-143-5p; eEF2, eukaryotic translation elongation factor 2; AMPK, AMP activated protein kinase; Bax, Bcl-2 Associated X protein; Bcl-2, B cell lymphoma-2; mTOR, mammalian target of rapamycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NP, nucleus pulposus; ANOVA, analysis of variance

Fig. 7

Inhibition of miR-143-5p facilitates NP cell differentiation by inactivating the AMPK signaling pathway. a Protein levels of CLO2, ACAN, and DCN in NP cells from degenerative IVD treated with miR-143-5p mimic, inhibitor, or AICAR. b Protein bands of CLO2, ACAN, and DCN in NP cells from degenerative IVD treated with miR-143-5p mimic, inhibitor, or AICAR; AICAR was the activator of the AMPK signaling pathway; the experiment was repeated three times independently; data were expressed by mean ± standard deviation; data among multiple groups were analyzed using one-way ANOVA; ^*, p < 0.05 vs. the control group (NP cells from normal IVD); ^#, p < 0.05 vs. the blank group (NP cells from degenerative IVD without transfection) and the NC group (NP cells from degenerative IVD transfected with NC sequence); miR-143-5p mimic group refers to NP cells from degenerative IVD transfected with miR-143-5p mimic; miR-143-5p inhibitor group refers to NP cells from degenerative IVD transfected with miR-143-5p inhibitor; AICAR group refers to NP cells from degenerative IVD treated with AMPK signaling pathway activator, AICAR; miR-143-5p inhibitor + AICAR group refers to NP cells of degenerative IVD treated with AICAR and transfected with miR-143-5p inhibitor. miR-143-5p, microRNA-143-5p; COL2, collagen type II; ACAN, aggrecan; DCN, decorin; GAPDN, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; AMPK, AMP activated protein kinase; NP, nucleus pulposus; ANOVA, analysis of variance

Fig. 8

Inhibition of miR-143-5p increases NP cell proliferation and cell cycle entry by inactivating the AMPK signaling pathway. a The proliferation of NP cells from degenerative IVD treated with miR-143-5p mimic, inhibitor, or AICAR. b, c Cell cycles following treatment with miR-143-5p mimic, inhibitor or AICAR; AICAR was the activator of the AMPK signaling pathway; the experiment was repeated three times independently; data were expressed by mean ± standard deviation; data among multiple groups were analyzed using one-way ANOVA; *, p < 0.05 vs. the control group (NP cells from normal IVD); #, p < 0.05 vs. the blank and NC groups (NP cells from degenerative IVD without transfection and transfected with NC sequence, respectively); miR-143-5p mimic group refers to NP cells from degenerative IVD transfected with miR-143-5p mimic; miR-143-5p inhibitor group refers to NP cells from degenerative IVD transfected with miR-143-5p inhibitor; AICAR group refers to NP cells from degenerative IVD treated with AMPK signaling pathway activator, AICAR; miR-143-5p inhibitor + AICAR group refers to NP cells of degenerative IVD treated with AICAR and transfected with miR-143-5p inhibitor. NC, negative control; OD value, optical density value; miR-143-5p, microRNA-143-5p; AMPK, AMP activated protein kinase; NP, nucleus pulposus; ANOVA, analysis of variance

Fig. 9

Inhibition of miR-143-5p suppresses NP cell apoptosis and senescence via inactivation of the AMPK signaling pathway. a The apoptosis of NP cells from degenerative IVD treated with miR-143-5p mimic, inhibitor or AICAR. b The percentage of apoptotic cells from degenerative IVD treated with miR-143-5p mimic, inhibitor or AICAR. c Senescent NP cells from degenerative IVD treated with miR-143-5p mimic, inhibitor or AICAR under an optical microscope (× 400). d SA-β-gal staining images of senescent NP cells from degenerative IVD treated with miR-143-5p mimic, inhibitor, or AICAR; the experiment was repeated three times independently; data were expressed by mean ± standard deviation; data among multiple groups were analyzed using one-way ANOVA; *, p < 0.05 vs. the control group (NP cells from normal IVD); #, p < 0.05 vs. blank and NC groups (NP cells from degenerative IVD without transfection and transfected with NC sequence, respectively); miR-143-5p mimic group refers to NP cells from degenerative IVD transfected with miR-143-5p mimic; miR-143-5p inhibitor group refers to NP cells from degenerative IVD transfected with miR-143-5p inhibitor; AICAR group refers to NP cells from degenerative IVD added with 0.5 mmol/L AICAR, AMPK signaling pathway activator; miR-143-5p inhibitor + AICAR group refers to NP cells of degenerative IVD added with 0.5 mmol/L AICAR and transfected with miR-143-5p inhibitor. NC, negative control; miR-143-5p, microRNA-143-5p; FITC, fluorescein isothiocyanate; NP, nucleus pulposus; SA-β-gal, senescence-associated β-galactosidase; ANOVA, analysis of variance