Screening of genes involved in epithelial-mesenchymal transition and differential expression of complement-related genes induced by PAX2 in renal tubules

Abstract

Aim: The aim of the present study was to screen and verify downstream genes involved in the epithelial mesenchymal transition (EMT) induced by paired box 2 (PAX2) in NRK-52E cells.

Methods: NRK-52E cells were transfected with lentivirus carrying PAX2 gene or no-load virus respectively. Total RNA was isolated 72 h after transfection from PAX2-overexpressing cells and control cells. Isolated RNA was then hybridized with the Rat OneArray Plus expression profile chip. The chips were examined by Agilent 0.1 XDR to screen for differentially expressed genes, which were further analyzed to investigate complement-related genes as genes of interest.

Results: In NRK-52E cells, PAX2 overexpression promoted EMT followed by upregulation of 298 genes and downregulation of 293 genes. KEGG analysis indicated the differential expression of genes related to cytokines and their receptors, extracellular matrix (ECM), MAPKs, local adhesion, cancer, the complement cascade, and coagulation. Gene oncology analysis screened out genes related to molecular functions (e.g., hydrolase activity, phospholipase activity, components of the ECM) and biological processes (e.g., cell development, signal transduction, phylogeny), and cell components (e.g., cytoplasm, cell membrane, and ECM). Analysis of the complement system revealed upregulation of C3 and downregulation of CD55 and complement regulator factor H (CFH).

Conclusion: PAX2 overexpression upregulates EMT in vitro and may regulate C3, CD55, and CFH.

Keywords: Kyoto Encyclopedia of Genes and Genomes; NRK-52E cells; Paired box 2; complement genes; epithelial-mesenchymal cell transition; gene ontology.

Figure 1

Overexpression of PAX2 in NRK52E cells. (A, B) NRK‐52E cells were transduced with LV‐con for 72 h. Cell morphology was visualized by light or fluorescence microscopy. (C, D) NRK‐52E cells were transduced with LV‐PAX2 for 72 h. Morphology was visualized with light or fluorescence microscopy.

Figure 2

Differential gene expression induced by PAX2 was analyzed using the Rat OneArray® Plus Gene Chip. (A) Clustering was performed to visualize correlations among the replicates and varying sample conditions. Upregulated and downregulated genes are represented in red and green, respectively. A subset of differential genes was selected for clustering analysis. An intensity filter was used to select genes where the difference between the maximum and minimum intensity values >2000 among all microarrays. For this microarray project, the number of genes clustered was 222. (B) The histogram plot shows the distribution of fold‐change for all probes excluding control and flagged probes. Fold change was calculated by Rosetta Resolver 7.2, with the error model adjusted by Amersham Pairwise Ration Builder for signal comparison of samples. (C) Volcano plot of sample OE versus CON. Standard selection criteria to identify differentially expressed genes are established at log2 |fold change| ≧ 1 and P‐value < 0.05. (Blue dots in figure.) (D) PCA plot. The variables for the first three principal components (PC1, PC2, PC3) for this study are 94.86%, 2.56%, and 1.33%, respectively. A subset of differential genes was selected for PCA analysis. Notes: OE represents cells overexpressing PAX2; CON represents control cells. 1, 2 and 3 represent triplicate repetitions.

Figure 3

PAX2 overexpression affects the expression of C3, CD55, CFH (A, C) NRK‐52E cells were transduced with lentivirus containing vector controls or rat PAX2 for 72 h. The expression of PAX2, C3, CD55, and CFH was analyzed by western blot after transduction. GAPDH served as loading control, n = 3, *P < 0.05. (B) mRNA expression was measured by qPCR. mRNA expression levels of PAX2, C3, CD55, and CFH in LV‐PAX2 cells were compared with those in LV‐con cells, n = 3, *P < 0.05.