Knockdown of AMP-activated protein kinase α2 impairs 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
- Published
- Accepted
- Subject Areas
- Cell Biology, Urology
- Keywords
- AMPKα2 , Epithelial mesenchymal transition , ETS1 , Ingenuity Pathway Analysis , RPS6KA1 , Ureteropelvic junction obstruction , Gene Microarray , renal fibrosis , ERK/MAPK pathway , TGF-β1
- Copyright
- © 2019 Yin et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. Knockdown of AMP-activated protein kinase α2 impairs 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. PeerJ Preprints 7:e27992v2 https://doi.org/10.7287/peerj.preprints.27992v2
Abstract
Background. Epithelial mesenchymal transition (EMT) plays an important regulatory role in obstructive nephropathy and renal fibrosis. As an intracellular energy sensor, AMP-activated 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.
Author Comment
We measured the gray scale of protein bands again by 3 times for each band (measured by 3 different researchers). One-way ANOVA statistical analysis showed that control virus had no independent effect on the expression of three proteins. Furthermore, we choose more representative Western blot images of E-cadherin and Vimentin (Figure 2 has been modified and uploaded). mRNA expression of ETS1, rps6ka1, CREB1 and HRAS were tested by using qPCR again, and one-way ANOVA statistical analysis showed that genes changed without regulating by control virus.
Supplemental Information
Rawdata of Immunofluorescence Staining
Rawdata of Immunofluorescence Staining
Quality Control of Gene chip
Including Pearson_s_Correlation,Principal_Component_Analysis,Relative_Signal_Box_Plot and Signal_Histogram.
Significant difference analysis of Gene chip
Including Different Gene, Heat map,Scatter Plot and Volcano Plot.
Ingenuity Pathway Analysis of Gene chip
Including Canonical Pathway,Canonical Pathway Histogram and Canonical Pathway MAP.