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
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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.
Cite this as
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.27992v2Author 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.
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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.
Additional Information
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Xiaoming Yin conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Fujiang Ma performed the experiments, prepared figures and/or tables.
Xu Fan performed the experiments.
Qi Zhao analyzed the data.
Xin Liu analyzed the data.
Yi Yang conceived and designed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.
Microarray Data Deposition
The following information was supplied regarding the deposition of microarray data:
IPA
Data Deposition
The following information was supplied regarding data availability:
The raw measurements are provided in the supplementary files
Funding
This work was supported by the National Natural Science Foundation of China (No.81571514). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
