MicroRNA-887-3p Inhibited MDM4 Expression and Proliferation but Promoted Apoptosis of Intervertebral Disc Annulus Fibrosus Cells in Rats

doi:10.12300/j.issn.1674-5817.2024.003

Abstract

Abstract:

Objective To investigate the effects of microRNA (miRNA, miR)-887-3p on the proliferation and apoptosis of rat intervertebral disc annulus fibrosus cells and its underlying molecular mechanism. Methods Annulus fibrosus tissues were obtained from 8-week-old SPF-grade SD male rats, centrifuged to prepare and identify annulus fibrosus cells. Rats in the experiment were randomly divided into four groups: a Normal group consisting of primary annulus fibrosus cells without any treatment; a Control group treated with 10 ng/mL interleukin-1β (IL-1β) for 24 hours to establish a degenerative cell model; an interference group (miR-887-3p inhibitor) transfected with miR-887-3p inhibitor using Lipo3000 based on the Control group; and an overexpression group (miR-887-3p mimics) transfected with miR-887-3p mimics using Lipo3000 based on the Control group. CCK-8 assay was used to assess cell viability; flow cytometry was used to measure cell apoptosis rates; real-time fluorescence quantitative PCR (qPCR) was used to detect the expression levels of miR-887-3p and murine double minute 4 (MDM4) mRNA; Western blotting was used to measure the protein expression levels of MDM4, Bcl-2, and Caspase-3. Results Immunofluorescence staining of isolated and cultured cells revealed a Collagen I positive rate of over 90% in rat intervertebral disc annulus fibrosus cells, indicating a cell purity level greater than 90%. Real-time fluorescence qPCR results showed that after establishing an annulus fibrosus degenerative cell model using IL-1β, the expression level of miR-887-3p significantly increased compared to the Normal group (P<0.001). Compared to the Control group, transfection with miR-887-3p inhibitor resulted in a significant decrease in its expression level (P<0.001). The CCK-8 assay showed that compared to the Normal group, cell viability significantly decreased in the Control group (P<0.001). Compared to the Control group, cell proliferation ability significantly increased after miR-887-3p inhibition, and significantly decreased after overexpression of miR-887-3p. Flow cytometry results revealed that compared to the Normal group, the apoptosis rate in the Control group significantly increased (P<0.001). Compared to the Control group, the cell apoptosis rate significantly decreased in the miR-887-3p interference group (P<0.001) and increased in the overexpression group (P<0.001). Western blotting analysis showed that compared to the Normal group, Bcl-2 expression level significantly decreased (P<0.001) and Caspase-3 expression level significantly increased (P<0.001) in the Control group. Compared to the Control group, Bcl-2 and MDM4 expression levels significantly increased (P<0.01), and Caspase-3 expression level significantly decreased (P<0.01) in the miR-887-3p interference group; whereas in the overexpression group, Bcl?2 and MDM4 expression levels significantly decreased (P<0.05), and Caspase-3 levels significantly increased (P<0.05). Real-time fluorescence qPCR and protein immunoblotting results showed that after interfering with miR-887-3p, the expression of MDM4 protein and mRNA increased (P<0.001); after overexpressing miR-887-3p, their expression decreased (protein, P<0.01; mRNA, P<0.001). Conclusion MiR-887-3p may modulate the cell proliferation and apoptosis of rat intervertebral disc annulus fibrosus cells by regulating MDM4 expression, thereby influencing the development and progression of disc degeneration.

Key words: miR-887-3p, Intervertebral disc annulus fibrosus cells, Degenerative model, Cell proliferation, Apoptosis, Rats

CLC Number:

R681.5

Xiaoyu ZHU,Hantao YUAN,Sibo LI. MicroRNA-887-3p Inhibited MDM4 Expression and Proliferation but Promoted Apoptosis of Intervertebral Disc Annulus Fibrosus Cells in Rats[J]. Laboratory Animal and Comparative Medicine, 2024, 44(3): 270-278. DOI: 10.12300/j.issn.1674-5817.2024.003.

Figures/Tables 5

Note： A, Immunofluorescence staining was used to identify rat intervertebral disc annulus fibrosus cells (Collagen I expression marked by Cy3 fluorescent antibody appeared red; cell nuclei stained with DAPI appeared blue; Merged, the overlay of A and B; Scale bar: 100 μm). B, Relative expression of miR-887-3p after transfection with miR-887-3p inhibitor and mimics detected by real-time fluorescence quantitative PCR. Compared to the normal group, ^***P<0.001; Compared to the control group, ^###P<0.001.
">

Figure 1 Identification of rat intervertebral disc annulus fibrosus cells and detection of miR-887-3p expression levels
Note： A, Immunofluorescence staining was used to identify rat intervertebral disc annulus fibrosus cells (Collagen I expression marked by Cy3 fluorescent antibody appeared red; cell nuclei stained with DAPI appeared blue; Merged, the overlay of A and B; Scale bar: 100 μm). B, Relative expression of miR-887-3p after transfection with miR-887-3p inhibitor and mimics detected by real-time fluorescence quantitative PCR. Compared to the normal group, ^***P<0.001; Compared to the control group, ^###P<0.001.

Table 1 CCK-8 assay used to test cell proliferation ability of rat intervertebral disc annulus fibrosus cells after transfection with miR-887-3p inhibitor and mimics

组别 Groups	细胞活力 /% Cell viability /%
组别 Groups	12 h	24 h	48 h	72 h
正常组（Normal group）	100.00±3.24	100.00±2.65	100.0±3.02	100.00±1.24
对照组（Control group）	98.65±2.54	75.25±6.26^**	62.40±6.86^***	36.54±2.04^***
miR-887-3p inhibitor	96.56±2.23	95.46±1.25	94.54±3.01	95.23±2.25
miR-887-3p mimics	95.71±1.62	62.71±7.62^**##	40.74±5.13^***###	23.15±3.63^***###

Note： Compared to the normal group， ^***P<0.001； Compared to the control group， ^###P<0.001.
">

Figure 2 Flow cytometry measurement of apoptosis rates in rat intervertebral disc annulus fibrosus cells 48 h after transfection with miR-887-3p inhibitor and mimics
Note： Compared to the normal group， ^***P<0.001； Compared to the control group， ^###P<0.001.

Note： Compared to the normal group， ^***P<0.001； Compared to the control group， ^#P<0.05， ^##P<0.01.
">

Figure 3 Western blotting analysis of Bcl-2（A） and Caspase-3（B） protein expression in rat intervertebral disc annulus fibrosus cells 48 h after transient transfection with miR-887-3p inhibitor and mimics
Note： Compared to the normal group， ^***P<0.001； Compared to the control group， ^#P<0.05， ^##P<0.01.

Note： Expression of murine double minute 4 (MDM4) was detected by Western blotting (A) and real-time fluorescence quantitative PCR (B). Compared to the normal group, ^**P<0.01, ^***P<0.001; Compared to the control group, ^###P<0.001.
">

Figure 4 Changes in MDM4 mRNA and protein expression in rat intervertebral disc annulus fibrosus cells 48 h after transient transfection with miR-887-3p inhibitor and mimics
Note： Expression of murine double minute 4 (MDM4) was detected by Western blotting (A) and real-time fluorescence quantitative PCR (B). Compared to the normal group, ^**P<0.01, ^***P<0.001; Compared to the control group, ^###P<0.001.

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