Methylation of microRNA-129-5P modulates nucleus pulposus cell autophagy by targeting Beclin-1 in intervertebral disc degeneration

Abstract

MicroRNAs play an important role in the etiology and progression of many diseases, including intervertebral disc degeneration (IVDD). The miRNA miR-129-5P regulates autophagy in various cancers, but its role in human nucleus pulposus (NP) cells is unclear. The present study investigated whether miR-129-5p regulates the expression of Beclin-1 which is known to induce autophagy in NP cells by evaluating their levels in normal and degenerative disc tissues and human NP cells transfected with miR-129-5P mimic or inhibitor by quantitative real-time (qRT-)PCR, western blotting, flow cytometry, and immunofluorescence analysis. A bioinformatics analysis was used to predict the relationship between miR-129-5P and Beclin-1, which was confirmed by the dual luciferase assay. DNA methylation status was assessed by methylation-specific PCR, and the effect of demethylation on miR-129-5P level and autophagy was examined by qRT-PCR, western blotting, and flow cytometry. We found that miR-129-5P expression was downregulated while that of Beclin-1 and LC3-II was upregulated in degenerative disc NP cells. Meanwhile, autophagy was reduced in human NP cells transfected with miR-129-5P mimic, whereas the opposite result was observed upon treatment with miR-129-5P inhibitor. Bioinformatics analysis and the luciferase reporter assay revealed that Beclin-1 is a target of and is inhibited by miR-129-5P. We also found that CpG islands in the miR-129-5P promoter region were hypermethylated in degenerative as compared to normal disc tissue. Thus, miR-129-5P blocks NP cell autophagy by directly inhibiting Beclin-1, a process that is dependent on miR-129-5P promoter methylation.

Keywords: Beclin-1; autophagy; intervertebral disc degeneration; methylation; miR-129-5P.

Figure 1. MiR-129-5P and Beclin-1 expression in normal and degenerative NP tissue

(A) Beclin-1 mRNA level was upregulated in degenerative as compared to normal NP tissue. (B) MiR-129-5P level was downregulated in degenerative as compared to normal NP tissue, as determined by qRT-PCR. (C) Correlation between miR-129-5P and Beclin-1 mRNA levels in NP tissue.

Figure 2. MiR-129-5P modulates autophagy in degenerative human NP cells

Cells were transfected with miR-129-5P mimic, miR-129-5P inhibitor, or scrambled mi-129-5P (miR-Scr) for 48 h. (A) MiR-129-5P expression was evaluated by qRT-PCR. (B) LC3 and Beclin-1 mRNA, as determined by qRT-PCR. (C, D) LC3-II, LC3-I, and Beclin-1 protein expression in human NP cells, as determined by western blotting. (E) LC3-II and LC3-I ratio was decreased and increased upon treatment with mimic and inhibitor, respectively. (F, G) LC3 expression was detected by Immunofluorescence analysis in transfected human NP cells. (H) Autophagy in treated NP cells, as determined by flow cytometry. (I) Autophasosomes and autolysosmes in NP cells were determined by electron microscope. Results are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05 vs. miR-Scr.

Figure 3. MiR-129-5P regulates Beclin-1 expression in degenerative human NP cells

(A) Putative miR-129-5P target site in the 3′-UTR of Beclin-1 transcript predicted by bioinformatics analysis. (B) Luciferase activity in human NP cells co-transfected with miR-Scr or miR-129-5P, as measured by a luciferase reporter assay. (C, D) Immunofluorescence analysis of LC3-II expression in human NP cells transfected with miR-129-5P mimic, miR-129-5P, inhibitor, or scrambled mi-129-5P. Results are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05 vs. control.

Figure 4. MiR-129-5P regulates degenerative human NP cell autophagy by directly targeting Beclin-1

Human NP cells were transfected with miR-129-5P and Beclin-1 siRNA. (A) LC3 and Beclin-1 mRNA expression were measured by qRT-PCR. (B, C) The protein of LC3-I, LC3-II, and Beclin-1 in human NP cells, as determined by western blotting. (D) LC3-II and LC3-I protein ratio. (E, F) Apoptosis of transfected NP cells, as analyzed by flow cytometry. (G) Autophagy of treated NP cells, as determined by flow cytometry. Results are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05 vs. miR-Scr+siScr; **P < 0.05 vs. miR-Scr+siScr; #P < 0.05 vs. inhibitor+siScr.

Figure 5. Autophagy inhibits apoptosis by stimulating the cytoplasmic release of cathepsin B in degenerative human NP cells

(A–C) Caspase-3, -8, -9, and cathepsin B protein expression were determined by western blotting. (D–H) Expression of caspases, LC3- II/I, Beclin-1, and cathepsin B protein in NP cells as determined by western blotting following treatment with ammonium chloride, hydroxychloroquine, and 3-MA to block autophagosome and lysosome fusion or autophagosome formation. (I–K) Expression of caspases and cathepsin B protein detected by western blotting in cells treated with the cathepsin B inhibitor CA-074-ME. Results are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05 vs. miR-Scr+siScr; **P < 0.05 vs. miR-Scr+siScr; ***P < 0.05 vs. inhibitor+siScr; #P < 0.05 vs. group A; ##P < 0.05 vs. group B; ###P < 0.05 vs. group B; &P < 0.05 vs. group a; &&P < 0.05 vs. group b.

Figure 6. DNA methylation blocked miR-129-5P expression and induced NP cell autophagy in degenerative human NP cells

NP cells isolated from IVDD tissue were treated with 5-AZA. (A) Methylation status in normal and degenerative NP cells, as detected by MSP. (B, C) MiR-129-5P, LC3, and Beclin-1 mRNA levels were measured by qRT-PCR. (D, E) LC3-I, LC3-II, and Beclin-1 protein expression in treated NP cells, as determined by western blotting. (F) Autophagy in NP cells, as analyzed by flow cytometry. (G) LC3-II and LC3-I protein ratio. Results are shown as mean ± SD. Data are representative of three independent experiments. *P < 0.05 vs. IVDD.