Silencing KLF6 Alleviates Cigarette Smoke Extract-Induced Mitochondrial Dysfunction in Bronchial Epithelial Cells by SIRT4 Upregulation

Abstract

Background The incidence of chronic obstructive pulmonary disease (COPD) is increasing year by year. Kruppel-like factor 6 (KLF6) plays an important role in inflammatory diseases. However, the regulatory role of KLF6 in COPD has not been reported so far. Methods The viability of human bronchial epithelial cells BEAS-2B induced by cigarette smoke extract (CSE) was detected by CCK-8 assay. The protein expression of KLF6 and sirtuin 4 (SIRT4) was appraised with Western blot. RT-qPCR and Western blot were applied to examine the transfection efficacy of sh-KLF6 and Oe-KLF6. Cell apoptosis was detected using flow cytometry. The levels of inflammatory factors IL-6, TNF-α and IL-1β were assessed with ELISA assay. DCFH-DA staining was employed for the detection of ROS activity and the levels of oxidative stress markers SOD, CAT and MDA were estimated with corresponding assay kits. The mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content and Complex I activity were evaluated with JC-1 staining, ATP colorimetric/fluorometric assay kit and Complex I enzyme activity microplate assay kit. With the application of mitochondrial permeability transition pore detection kit, mPTP opening was measured. Luciferase report assay was employed to evaluate the activity of SIRT4 promoter and chromatin immunoprecipitation (ChIP) to verify the binding ability of KLF6 and SIRT4 promoter. Results KLF6 expression was significantly elevated in CSE-induced cells. KLF6 was confirmed to suppress SIRT4 transcription. Interference with KLF6 expression significantly inhibited cell viability damage, cell apoptosis, inflammatory response, oxidative stress and mitochondrial dysfunction in CSE-induced BEAS-2B cells, which were all reversed by SIRT4 overexpression. Conclusion Silencing KLF6 alleviated CSE-induced mitochondrial dysfunction in bronchial epithelial cells by SIRT4 upregulation.