The Effect of CircTMOD3 on LPS-Induced Alveolar Epithelial Cell Injuryby Regulating the miR-139-5p/ROCK2 Axis
YANG Xiaojun1*, OUYANG Yunping2, CHEN Tao2, LI Peng2, ZHAO Bo2
The aim of this study was to investigate the impact of CircTMOD3 on LPS-induced alveolar epithelial cell injury by regulating the miR-139-5p/ROCK2 (Rho associated coiled coil containing proteinkinase 2) axis. Human alveolar epithelial cells A549 were cultured in vitro and grouped into control group, LPSgroup, LPS+si-NC group, LPS+si-CircTMOD3 group, LPS+si-CircTMOD3+inhibitor NC group, and LPS+siCircTMOD3+miR-139-5p inhibitor group; qRT-PCR was applied to detect the expression levels of CircTMOD3, miR139-5p, and ROCK2 of cells in each group; CCK-8 and EdU method were applied to detect cell proliferation; flowcytometry was applied to detect cell apoptosis rate; ELISA kits were applied to detect TNF-α, IL-6, and IL-1β levels;the commercial reagent kits were applied to analyze MDA, SOD, and CAT levels; Western blot was applied to detectthe expression levels of Bax, Bcl-2, and ROCK2 proteins in cells; the relationship of miR-139-5p with CircTMOD3and ROCK2 was verified by double luciferase reporter gene experiment. Compared with the control group, the levelsof CircTMOD3, ROCK2 mRNAs, TNF-α, IL-6, IL-1β, MDA, apoptosis rate, Bax and ROCK2 proteins of A549 cellsin the LPS group were obviously increased, and the expression of miR-139-5p, proliferative activity, CAT, SOD, Bcl2 protein were obviously reduced (P<0.05); compared with the LPS group and the LPS+si-NC group, the levels ofCircTMOD3 and ROCK2 mRNAs, TNF-α, IL-6, IL-1β and MDA, apoptosis rate, Bax and ROCK2 proteins of A549cells in the LPS+si-CircTMOD3 group were obviously reduced, and the levels of miR-139-5p, proliferative activity,CAT, SOD, and Bcl-2 protein were obviously increased (P<0.05); compared with the LPS+si-CircTMOD3+inhibitorNC group, the levels of ROCK2 mRNA, TNF-α, IL-6, IL-1β, MDA, apoptosis rate, Bax and ROCK2 proteins of A549cells in the LPS+si-CircTMOD3+miR-139-5p inhibitor group were obviously increased, and the levels of miR-139-5p, proliferative activity, CAT, SOD, and Bcl-2 protein were obviously reduced (P<0.05). Dual luciferase reportergene experiment confirmed that miR-139-5p had a targeted regulatory relationship with TMOD3 and ROCK2. Thestudy concluded that interference with CircTMOD3 expression could up-regulate miR-139-5p expression and inhibitROCK2 expression, alleviating LPS-induced alveolar epithelial cell injury.
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