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

Figure 1 Identification of rat intervertebral disc annulus fibrosus cells and detection of miR-887-3p expression levelsNote： 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^***###

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 mimicsNote： Compared to the normal group， ***P<0.001； Compared to the control group， ###P<0.001.

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 mimicsNote： Compared to the normal group， ***P<0.001； Compared to the control group， #P<0.05， ##P<0.01.

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 mimicsNote： 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.

References 30

1	罗卓荆, 杨柳, 王迪. 我国椎间盘退变的生物学研究成就及展望[J]. 空军军医大学学报, 2023, 44(6):481-485, 489. DOI:10.13276/j.issn.2097-1656.2023.06.001 .
	LUO Z J, YANG L, WANG D. Achievements and prospects of biological research of intervertebral disc degeneration in China[J]. J Air Force Med Univ, 2023, 44(6):481-485, 489. DOI: 10.13276/j.issn.2097-1656.2023.06.001 .
2	冷佳俐, 汪振宇, 刘芳. MiR-335-3p靶向调控CCL5在TNF-α诱导椎间盘退变中相关机制[J]. 中国老年学杂志, 2023, 43(12):2956-2961. DOI:10.3969/j.issn.1005-9202.2023.12.038 .
	LENG J L, WANG Z Y, LIU F. Mechanism of miR-335-3p targeted regulation of CCL5 in TNF-α-induced intervertebral disc degeneration[J]. Chin J Gerontol, 2023, 43(12):2956-2961. DOI: 10.3969/j.issn.1005-9202.2023.12.038 .
3	FENG C C, LIU H, YANG M H, et al. Disc cell senescence in intervertebral disc degeneration: causes and molecular pathways[J]. Cell Cycle, 2016, 15(13):1674-1684. DOI: 10.1080/15384101.2016.1152433 .
4	肖冰, 齐军强, 王浩田, 等. 微小RNA在椎间盘退变中的作用机制及治疗研究进展[J]. 中国脊柱脊髓杂志, 2023, 33(3):274-280. DOI: 10.3969/j.issn.1004-406X.2023.03.13 .
	XIAO B, QI J Q, WANG H T,et al. Research progress in mechanism and treatment of microRNA in intervertebral disc degeneration[J]. Chin J Spine Spinal Cord, 2023, 33(3):274-280. DOI:10.3969/j.issn.1004-406X.2023.03.13 .
5	KEPLER C K, PONNAPPAN R K, TANNOURY C A, et al. The molecular basis of intervertebral disc degeneration[J]. Spine J, 2013, 13(3):318-330. DOI: 10.1016/j.spinee.2012.12.003 .
6	许刚, 张长春, 朱坤, 等. MiR-141-3p对腰椎间盘突出症大鼠背根神经节炎症及下肢疼痛的抑制和改善作用[J]. 中国组织工程研究, 2024, 10(16):2593-2598. DOI: 10.12307/2024.277 .
	XU G, ZHANG C C, ZHU K, et al. Effects of miR-141-3p on dorsal root ganglion inflammation and lower limb pain in rats with lumbar disc herniation[J]. Chin J Tissue EngRes, 2024, 10(16):2593-2598. DOI: 10.12307/2024.277 .
7	黄皆和, 王茜, 郏舜杰, 等. 微小RNA-103a-3p通过肿瘤蛋白53调控凋亡抑制剂1/P53对骨质疏松症的影响[J]. 解剖学报, 2024, 55(2):174-180. DOI:10.16098/j.issn.0529-1356.2024.02.007 .
	HUANG J H, WANG Q, JIA S J, et al. Effects of microRNA-103a-3p on osteoporosis through tumor protein 53-regulated inhibitor of apoptosis 1/P53[J]. Acta Anat Sin, 2024, 55(2):174-180. DOI: 10.16098/j.issn.0529-1356.2024.02.007 .
8	SHARMA B, TORRES M M, RODRIGUEZ S, et al. MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons[J]. Neural Regen Res, 2024, 19(12):2698-2707. DOI: 10.4103/NRR.NRR-D-23-01064 .
9	应璞, 许岳, 路通, 等. MiR-34a-5p/PLCD3轴调控骨关节炎进展的机制[J]. 中国组织工程研究, 2024, 28(33):5320-5325. DOI: 10.12307/2024.653 .
	YING P, XU Y, LU T, et al. Mechanism by which miR-34a-5p/PLCD3 axis regulates osteoarthritis progression[J]. Chin J Tissue Eng Res, 2024, 28(33):5320-5325. DOI: 10.12307/2024.653 .
10	王晓玲, 孟莉丹, 王学敏, 等. HOTTIP通过竞争性结合miR-506调节Vimentin基因的表达调控肾透明细胞癌细胞迁移[J]. 现代肿瘤医学, 2024, 32(7):1200-1207. DOI:10.3969/j.issn.1672-4992.2024.07.004 .
	WANG X L, MENG L D, WANG X M, et al. HOTTIP regulates the expression of Vimentin gene through competitively binding miR-506 to regulate the migration of clear cell renal cell carcinoma cells[J]. J Mod Oncol, 2024, 32(7):1200-1207. DOI:10.3969/j.issn.1672-4992.2024.07.004 .
11	陈胜乐, 米盼盼, 许雅芳, 等. 微小RNA-375对腰椎间盘突出大鼠JAK2/STAT3信号通路的影响[J]. 临床和实验医学杂志, 2021, 20(19):2036-2040. DOI:10.3969/j.issn.1671-4695.2021.19.006 .
	CHEN S L, MI P P, XU Y F, et al. Effect of microRNA-375 on JAK2/STAT3 signaling pathway in lumbar disc herniation[J]. J Clin Exp Med, 2021, 20(19):2036-2040. DOI: 10.3969/j.issn.1671-4695.2021.19.006 .
12	张海英, 郑晨颖. 膝骨关节炎关节液和滑膜中微小RNA-140-5p的表达及临床意义[J]. 安徽医药, 2022, 26(6):1179-1182. DOI:10.3969/j.issn.1009-6469.2022.06.028 .
	ZHANG H Y, ZHENG C Y. Expression and clinical significance of miR-140-5p in synovial fluid and synovium of knee osteoarthritis[J]. Anhui Med Pharm J, 2022, 26(6):1179-1182. DOI: 10.3969/j.issn.1009-6469.2022.06.028 .
13	XU X B, ZHENG S S. MiR-887-3p negatively regulates STARD13 and promotes pancreatic cancer progression[J]. Cancer Manag Res, 2020, 12:6137-6147. DOI: 10.2147/CMAR.S260542 .
14	GOODWIN A J, LI P F, HALUSHKA P V, et al. Circulating miRNA 887 is differentially expressed in ARDS and modulates endothelial function[J]. Am J Physiol Lung Cell Mol Physiol, 2020, 318(6): L1261-L1269. DOI: 10.1152/ajplung. 00494.2019 .
15	IWAMOTO N, FUKUI S, KOGA T, et al. FRI0067 Microrna profiling of mtx-treated fibroblast-like synovial cells in rheumatoid arthritis revealed a possibility of microrna-887-3p as novel therapeutic target of ra[J]. Ann Rheum Dis, 2017, 76():503.DOI: 10.1136/annrheumdis-2017-eular.3327 .
16	ALMEIDA G M, CASTILHO A C, ADAMOSKI D, et al. MDM4: what do we know about the association between its polymorphisms and cancer?[J]. Med Oncol, 2022, 40(1):61. DOI: 10.1007/s12032-022-01929-z .
17	卢今, 张颖, 潘学营, 等. 2020版美国兽医协会动物安乐死指南解析[J]. 实验动物与比较医学, 2021, 41(3):195-206. DOI:10.12300/j.issn.1674-5817.2021.086 .
	LU J, ZHANG Y, PAN X Y, et al. A brief interpretation of AVMA guide lines on euthanasia of animals: 2020 edition[J]. Lab Anim Comp Med, 2021, 41(3):195-206. DOI: 10.12300/j.issn.1674-5817.2021.086 .
18	YANG X, WANG L, YUANZQ, et al. Interleukin-1β induces apoptosis in annulus fibrosus cells through the extracellular signal-regulated kinase pathway[J]. Connect Tissue Res, 2018, 59(6):593-600. DOI: 10.1080/03008207.2018.1442445 .
19	LI Z Q, KONG L, LIU C, et al. Human bone marrow mesenchymal stem cell-derived exosomes attenuate IL-1β- induced annulus fibrosus cell damage[J]. Am J Med Sci, 2020, 360(6):693-700. DOI: 10.1016/j.amjms.2020.07.025 .
20	GONÇALVES R M, SAGGESE T, YONG Z Y, et al. Interleukin-1β more than mechanical loading induces a degenerative phenotype in human annulus fibrosus cells, partially impaired by anti-proteolytic activity of mesenchymal stem cell secretome[J]. Front Bioeng Biotechnol, 2022, 9:802789. DOI: 10.3389/fbioe.2021.802789 .
21	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method[J]. Methods, 2001, 25(4):402-408. DOI:10.1006/meth.2001.1262 .
22	HAWKINS S F C, GUEST P C. Multiplex analyses using real-time quantitative PCR[J]. Methods Mol Biol, 2017, 1546:125-133. DOI: 10.1007/978-1-4939-6730-8_8 .
23	TSIRIMONAKI E, FEDONIDIS C, PNEUMATICOS S G, et al. PKCε signalling activates ERK1/2, and regulates aggrecan, ADAMTS5, and miR377 gene expression in human nucleus pulposus cells[J]. PLoS One, 2013, 8(11): e82045. DOI: 10.1371/journal.pone.0082045 .
24	李小川, 阮狄克. MicroRNAs在退变椎间盘中表达与调控机制的研究进展[J]. 中国脊柱脊髓杂志, 2015, 25(10):942-945. DOI:10.3969/j.issn.1004-406X.2015.10.13 .
	LI X C, RUAN D K. The research progress on expression and regulation mechanism of microRNAs in degenerative intervertabral disc diseases[J]. Chin J Spine Spinal Cord, 2015, 25(10):942-945. DOI: 10.3969/j.issn.1004-406X.2015.10.13 .
25	YAN J, WU L G, ZHANG M, et al. MiR-328-5p induces human intervertebral disc degeneration by targeting WWP2[J]. Oxid Med Cell Longev, 2022, 2022:3511967. DOI: 10.1155/2022/3511967 .
26	ASHKENAZI A, FAIRBROTHER W J, LEVERSON J D, et al. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors [J]. Nat Rev Drug Discov, 2017, 16(4):273-284. DOI: 10.1038/nrd.2016.253 .
27	ASADI M, TAGHIZADEH S, KAVIANI E, et al. Caspase-3: structure, function, and biotechnological aspects[J]. Biotech And App Biochem, 2022, 69(4):1633-1645. DOI: 10.1002/bab.2233 .
28	GAO F, XIONG X Y, PAN W T, et al. A regulatory MDM4 genetic variant locating in the binding sequence of multiple microRNAs contributes to susceptibility of small cell lung cancer[J]. PLoS One, 2015, 10(8): e0135647. DOI: 10.1371/journal.pone.0135647 .
29	刘磊, 傅立国, 隋鑫. MicroRNA miR-34a-5p通过靶向MDM4抑制巨噬细胞氧化应激损伤的研究[J]. 智慧健康, 2023, 9(18):158-161, 165. DOI:10.19335/j.cnki.2096-1219.2023.18.037 .
	LIU L, FU L K, SUI X. Microrna mir-34a-5p inhibiting oxidative stress damage of macrophages by targeting MDM4[J]. Smart Healthc, 2023, 9(18):158-161, 165. DOI: 10.19335/j.cnki.2096-1219.2023.18.037 .
30	梁舒, 左淑飞, 崔玉荣, 等. 槲皮素通过调控miR-431-5p/鼠双微基因4信号通路抑制类风湿关节炎成纤维样滑膜细胞侵袭和促进凋亡[J]. 实用临床医药杂志, 2023, 27(12):50-56. DOI:10.7619/jcmp.20231319 .
	LIANG S, ZUO S F, CUI Y R, et al. Quercetin inhibits invasion and promotes apoptosis of rheumatoid arthritis fibroblast-like synoviocytes by regulating miR-431-5p/mouse double minute 4 signaling pathway[J]. J Clin Med Pract, 2023, 27(12):50-56. DOI: 10.7619/jcmp.20231319 .

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