Knockdown of AMP-activated protein kinase α2 affects the epithelial-mesenchymal transition in rat renal tubular epithelial cells by downregulating v-ets erythroblastosis virus E26 oncogene homolog-1 and ribosomal protein S6 kinase A1

Abstract

Background. Epithelial mesenchymal transition (EMT) plays an important regulatory role in obstructive nephropathy and renal fibrosis. As an intracellular energy sensor, AMPactivated protein kinase (AMPK) is essential in the process of EMT. The aim of this study was to reveal changes in the expression of AMPKα2 and to elucidate which AMPKα2 genes play a role during EMT. Methods. In this study, TGF-β1 was used to induce EMT in normal rat renal tubular epithelial (NRK-52E) cells. The shAMPKα2 lentivirus was used to interfere with AMPKα2 expression in EMT-derived NRK-52E cells, where AMPKα2 expression and EMT were detected. Differential gene expression after the AMPKα2 knockdown in EMT-derived NRK-52E cells was examined using a gene microarray. Possible regulatory pathways were analyzed using ingenuity pathway analysis (IPA) and differentially expressed genes were partially verified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Results. It was found that AMPKα2 was upregulated in TGF-β1-induced EMT-derived NRK-52E cells. EMT progression was significantly inhibited after the expression of AMPKα2 was downregulated by the shAMPKα2 lentivirus. A total of 1,588 differentially expressed genes were detected after the AMPKα2 knockdown in NRK-52E cells in which EMT occurred. The ERK/MAPK pathway was significantly impaired after the AMPKα2 knockdown, as indicated by the IPA analysis. Furthermore, qRT-PCR and western blot results revealed that the expression of AMPKα2, v-ets erythroblastosis virus E26 oncogene homolog-1 (ETS1), and ribosomal protein S6 kinase A1 (RPS6KA1) was upregulated after EMT in NRK-52E cells, while expression of ETS1 and RPS6KA1 was downregulated after the AMPKα2 knockdown. Conclusions. AMPKα2 plays an important role in the regulation of rat renal tubular EMT, which may be achieved by modulating ETS1 and RPS6KA1 in the ERK/MAPK pathway. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27992v2 | CC BY 4.0 Open Access | rec: 2 Dec 2019, publ: 2 Dec 2019 40 Abstract 41 Background. Epithelial mesenchymal transition (EMT) plays an important regulatory role in 42 obstructive nephropathy and renal fibrosis. As an intracellular energy sensor, AMP-activated 43 protein kinase (AMPK) is essential in the process of EMT. The aim of this study was to reveal 44 changes in the expression of AMPKα2 and to elucidate which AMPKα2 genes play a role during 45 EMT. 46 Methods. In this study, TGF-β1 was used to induce EMT in normal rat renal tubular epithelial 47 (NRK-52E) cells. The shAMPKα2 lentivirus was used to interfere with AMPKα2 expression in 48 EMT-derived NRK-52E cells, where AMPKα2 expression and EMT were detected. Differential 49 gene expression after the AMPKα2 knockdown in EMT-derived NRK-52E cells was examined 50 using a gene microarray. Possible regulatory pathways were analyzed using ingenuity pathway 51 analysis (IPA) and differentially expressed genes were partially verified by quantitative real-time 52 polymerase chain reaction (qRT-PCR) and western blotting.53R...