LncRNA TM1‐3P Regulates Proliferation, Apoptosis and Inflammation of Fibroblasts in Osteoarthritis through miR‐144‐3p/ONECUT2 Axis
Authors: Yi Yangfei , Yang Ningyin , Yang Zirui , Tao Xiaojun , Li Yufei 
Published: September 2022

Objective

This study explores LncRNA TM1‐3P effects on the proliferation, apoptosis, and inflammatory response of fibroblasts in osteoarthritis (OA) and its underlying mechanism.

Methods

Bioinformatics was performed to analyze OA disease‐related genes, miRNA profiles, and function. The targeted regulation of LncRNA TM1‐3P and miR‐144‐3p, ONECUT2 and miR‐144‐3p were analyzed by dual luciferase reporter gene assay, RNA Binding Protein Immunoprecipitation (RIP), and RNA pull down. Histopathological morphology of the knee joint was observed by hematoxylin–eosin (HE) and Annona Red O/Fast Green. The expressions of mRNAs and proteins were detected by RT‐qPCR, Western blot, and immunohistochemistry. Unpaired T test was used between groups, and the one‐way analysis of variance of repeated measurement data was applied for multi‐group comparison, following Tukey's post‐test.

Results

ONECUT2 and Smurf2 genes were significantly elevated in the osteoarthritis group compared with the normal group (P < 0.001, P < 0.001). Expressions of ONECUT2 and LncRNA TM1‐3P were increased, and expression of miR‐144‐3p was decreased in interleukin (IL)‐1β‐induced human fibroblast synovial cells (hFSCs) (mRNA: 1.06 ± 0.24 vs. 3.29 ± 0.73, proteins: 0.22 ± 0.03 vs. 0.46 ± 0.22, 1.23 ± 0.22 vs. 3.76 ± 0.73, 1.06 ± 0.25 vs. 0.37 ± 0.13, P < 0.01, P < 0.001, P < 0.01, P < 0.05). Overexpression of miR‐144‐3p down‐regulated the ONECUT2 expression, reduced cell proliferation, promoted apoptosis in hFSCs induced by IL‐1β (mRNA: 0.89 ± 0.14 vs. 0.15 ± 0.01, P < 0.05; proteins: 0.46 ± 0.01 vs. 0.23 ± 0.01, P < 0.001; CCK8: 1.88 ± 0.07 vs. 1.65 ± 0.07; P < 0.05; EDU: 55.82 ± 1.44 vs 40.57 ± 2.24, P < 0.05; apoptosis: 10.57 ± 0.79 vs 16.36 ± 0.35, P < 0.0001). Overexpression of LncRNA TM1‐3P up‐regulated the expression of ONECUT2, promoted cell proliferation, and inhibited apoptosis (mRNA: 0.9 ± 0.09 vs 1.94 ± 0.12, P < 0.05; proteins: 0.61 ± 0.05 vs 0.76 ± 0.03, P > 0.05; CCK8: 2.07 ± 0.05 vs 2.47 ± 0.06; P < 0.01; EDU: 52.67 ± 1.17 vs 60.06 ± 3.24, P < 0.05; apoptosis: 10.57 ± 0.79 vs 16.36 ± 0.35, P < 0.001), which were reversed by the overexpression of miR‐144‐3p treatment (mRNA: 1.82 ± 0.07 vs 0.31 ± 0.07, P < 0.0001; proteins: 0.74 ± 0.02 vs 0.35 ± 0.01, P < 0.01; CCK8: 2.41 ± 0.01 vs 1.67 ± 0.02; P < 0.0001; EDU: 66.85 ± 2.86 vs 44.68 ± 1.97, P < 0.0001; apoptosis: 7.19 ± 0.19 vs 13.36 ± 0.53, P < 0.0001). Silencing LncRNA TM1‐3P attenuated the injury of knee joint tissue, down‐regulated the expression of ONECUT2, Smurf2, IL‐1β, IL‐6, TNF‐α, and improved the expression of Rap1 in rats (0.71 ± 0.04 vs 0.48 ± 0.02, 0.68 ± 0.06 vs 0.36 ± 0.02, 0.74 ± 0.03 vs 0.49 ± 0.04, 0.78 ± 0.01 vs 0.54 ± 0.03, 0.68 ± 0.02 vs 0.4 ± 0.04, 0.24 ± 0.01 vs 0.4 ± 0.03, P < 0.05, P < 0.05, P < 0.05, P < 0.01, P < 0.01, P < 0.05).

Conclusion

LncRNA TM1‐3P improved inflammation and damage of knee joints in OA rats through miR‐144‐3p/ONECUT2 axis, providing a new theoretical basis for gene therapy of OA.