Mechanism of MicroRNA-124-3p Targeting Calpain-1 to Affect the Function of Intervertebral Disc Nucleus Pulposus Cells

Overview

Journal Cytotechnology

Date 2025 Feb 3

PMID 39897108

Authors

Xunan Xu

Yong Liu

Chun Jiang

Peng Jia

Pengfei Cao

Yi He

Yin Zhang

Affiliations

Soon will be listed here.

Abstract

Intervertebral disc degeneration (IVDD) represents a major cause of lower back pain, whose prevalence rises with age. This study probed into the mechanism of microRNA (miR)-124-3p regulating function of nucleus pulposus cells (NPCs) by targeting calpain-1 (CAPN1). Rat IVD NPCs were cultured in vitro and transfected with miR-124-3p mimics, miR-124-3p inhibitor, oe-CAPN1 and their negative controls. The mRNA levels of miR-124-3p and CAPN1 were assessed by RT-qPCR. Cell proliferation, apoptosis and migration were evaluated by CCK-8, flow cytometry and Transwell assays. Levels of CAPN1 protein, apoptosis-related proteins (BAX, Cleaved-Caspase3, BCL-2) and extracellular matrix (ECM) proteins (Collagen II, Aggrecan, Fibronectin, Collagen I, matrix metalloproteinase [MMP]-13) were determined by Western blot. The target binding relationship between miR-124-3p and CAPN1 was verified by dual-luciferase assay. miR-124-3p overexpression facilitated NPC function and the maintenance of ECM homeostasis, as evidenced by increased NPC proliferation and migration, decreased apoptosis, elevated apoptosis-related protein BCL-2 level, diminished BAX and Cleaved-Caspase3 levels, reduced levels of ECM homeostasis-associated factors Collagen I and MMP-13 proteins, as well as raised levels of Collagen II, Aggrecan and Fibronectin proteins. Conversely, miR-124-3p knockdown brought about the opposite results. miR-124-3p targeted CAPN1. Furthermore, overexpression of CAPN1 partially reversed the regulatory effects of miR-124-3p on the ECM homeostasis, proliferation and migration in NPCs, and promoted apoptosis. miR-124-3p contributed to proliferation and migration of IVD NPCs, and reduced their apoptosis by inhibiting CAPN1 expression, thereby modulating ECM homeostasis and maintaining the function of IVD NPCs.

References

Wang Q, Wang J, Gu X, Feng D, Li D, Jiang T . MicroRNA-124-3p inhibits the differentiation of precartilaginous stem cells into nucleus pulposus-like cells via targeting FSTL1. Exp Ther Med. 2021; 22(1):725. PMC: 8120511. DOI: 10.3892/etm.2021.10157. View

Ding F, Shao Z, Xiong L . Cell death in intervertebral disc degeneration. Apoptosis. 2013; 18(7):777-85. DOI: 10.1007/s10495-013-0839-1. View

Dong X, Ni B, Fu J, Yin X, You L, Leng X . Emodin induces apoptosis in human hepatocellular carcinoma HepaRG cells via the mitochondrial caspase‑dependent pathway. Oncol Rep. 2018; 40(4):1985-1993. PMC: 6111625. DOI: 10.3892/or.2018.6620. View

Zhang F, Zhao X, Shen H, Zhang C . Molecular mechanisms of cell death in intervertebral disc degeneration (Review). Int J Mol Med. 2016; 37(6):1439-48. PMC: 4866972. DOI: 10.3892/ijmm.2016.2573. View

Wang T, Li P, Ma X, Tian P, Han C, Zang J . MicroRNA-494 inhibition protects nucleus pulposus cells from TNF-α-induced apoptosis by targeting JunD. Biochimie. 2015; 115:1-7. DOI: 10.1016/j.biochi.2015.04.011. View

Xue X, Xi W, Li W, Xiao J, Wang Z, Zhang Y . Hydrogen-rich saline alleviates cardiomyocyte apoptosis by reducing expression of calpain1 via miR-124-3p. ESC Heart Fail. 2023; 10(5):3077-3090. PMC: 10567641. DOI: 10.1002/ehf2.14492. View

Kanagaraj N, Beiping H, Dheen S, Tay S . Downregulation of miR-124 in MPTP-treated mouse model of Parkinson's disease and MPP iodide-treated MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuroscience. 2014; 272:167-79. DOI: 10.1016/j.neuroscience.2014.04.039. View

Wang C, Cui L, Gu Q, Guo S, Zhu B, Liu X . The Mechanism and Function of miRNA in Intervertebral Disc Degeneration. Orthop Surg. 2022; 14(3):463-471. PMC: 8926997. DOI: 10.1111/os.13204. View

Yue R, Lu S, Luo Y, Wang T, Liang H, Zeng J . Calpain silencing alleviates myocardial ischemia-reperfusion injury through the NLRP3/ASC/Caspase-1 axis in mice. Life Sci. 2019; 233:116631. DOI: 10.1016/j.lfs.2019.116631. View

10.

Yu Q, Chen S, Tang H, Yang H, Zhang J, Shi X . miR‑140‑5p alleviates mouse liver ischemia/reperfusion injury by targeting CAPN1. Mol Med Rep. 2021; 24(3). PMC: 8335737. DOI: 10.3892/mmr.2021.12314. View

11.

Mohd Isa I, Teoh S, Mohd Nor N, Mokhtar S . Discogenic Low Back Pain: Anatomy, Pathophysiology and Treatments of Intervertebral Disc Degeneration. Int J Mol Sci. 2023; 24(1). PMC: 9820240. DOI: 10.3390/ijms24010208. View

12.

Madhu V, Hernandez-Meadows M, Boneski P, Qiu Y, Guntur A, Kurland I . The mitophagy receptor BNIP3 is critical for the regulation of metabolic homeostasis and mitochondrial function in the nucleus pulposus cells of the intervertebral disc. Autophagy. 2023; 19(6):1821-1843. PMC: 10262801. DOI: 10.1080/15548627.2022.2162245. View

13.

Usman K, Hsieh A, Hackett T . The Role of miRNAs in Extracellular Matrix Repair and Chronic Fibrotic Lung Diseases. Cells. 2021; 10(7). PMC: 8304879. DOI: 10.3390/cells10071706. View

14.

Guo W, Mu K, Zhang B, Sun C, Zhao L, Dong Z . The circular RNA FAM169A functions as a competitive endogenous RNA and regulates intervertebral disc degeneration by targeting miR-583 and BTRC. Cell Death Dis. 2020; 11(5):315. PMC: 7198574. DOI: 10.1038/s41419-020-2543-8. View

15.

Liu P, Wilson M . miR-520c and miR-373 upregulate MMP9 expression by targeting mTOR and SIRT1, and activate the Ras/Raf/MEK/Erk signaling pathway and NF-κB factor in human fibrosarcoma cells. J Cell Physiol. 2011; 227(2):867-76. PMC: 3225649. DOI: 10.1002/jcp.22993. View

16.

Miao Z, Miao Z, Teng X, Xu S . Chlorpyrifos triggers epithelioma papulosum cyprini cell pyroptosis via miR-124-3p/CAPN1 axis. J Hazard Mater. 2021; 424(Pt A):127318. DOI: 10.1016/j.jhazmat.2021.127318. View

17.

Vo N, Hartman R, Patil P, Risbud M, Kletsas D, Iatridis J . Molecular mechanisms of biological aging in intervertebral discs. J Orthop Res. 2016; 34(8):1289-306. PMC: 4988945. DOI: 10.1002/jor.23195. View

18.

Li Z, Yu X, Shen J, Chan M, Wu W . MicroRNA in intervertebral disc degeneration. Cell Prolif. 2015; 48(3):278-83. PMC: 6496566. DOI: 10.1111/cpr.12180. View

19.

Shaban M, Yoon S, Ko Y, Ha H, Kim S, So J . Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold. J Biomater Sci Polym Ed. 2008; 19(9):1219-37. DOI: 10.1163/156856208785540163. View

20.

Jing W, Jiang W . MicroRNA-93 regulates collagen loss by targeting MMP3 in human nucleus pulposus cells. Cell Prolif. 2015; 48(3):284-92. PMC: 6495943. DOI: 10.1111/cpr.12176. View