Microarray analysis reveals long non‑coding RNA SOX2OT as a novel candidate regulator in diabetic nephropathy

Abstract

Diabetic nephropathy (DN) is a highly complex syndrome involving multiple dysregulated biological processes. Long non‑coding RNAs (lncRNAs) are now believed to have an important function in various diseases. However, their roles in DN remain largely unknown. Therefore, the present study was performed in order to investigate the lncRNAs that have a crucial role in DN. db/db mice were used as a DN model while db/m mice served as a control to search for lncRNAs which may have important roles in DN. Microarray and bioinformatics analysis gave an overview of the features of differentially expressed genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis demonstrated the typical biological alterations in DN. A co‑expression network of lncRNAs and mRNAs revealed the complex interaction pattern in DN conditions. Further data investigation indicated that SOX2‑overlapping transcript (SOX2OT), which was significantly downregulated in DN mice, may be the potentially functional lncRNA contributing to the onset of DN. The UCSC database demonstrated that SOX2OT was highly conserved in mice and humans. Additionally further study using cultured human podocytes and mesangial cells confirmed the downregulation of SOX2OT using reverse transcription‑quantitative polymerase chain reaction and fluorescence in situ hybridization. However, the cellular location of SOX2OT depended on certain cell types. Taken together, the results of the present study indicated that SOX2OT may act as an important regulator in the pathogenesis of DN by interacting with various mRNAs with critical roles in DN.

Keywords: SOX2-overlapping transcript; bioinformatics analysis; microarray analysis; diabetic nephropathy.

Figure 1.

Microarray analysis revealed DEGs between db/db mice and db/m mice. (A) Representative morphological alterations, including H&E staining, PAS base staining and TEM inspection, in the renal cortex from db/m (n=3) and db/db (n=3) mice. Scale bars are presented in each graph. (B) Hierarchically clustered heat map of DEGs with absolute fold-changes of no less than 2.0. The vertical axis represents each mRNA or long noncoding RNA and the horizontal axis represents different groups. (C) A scatter plot exhibiting the DEGs between db/db mice and db/m mice. (D) A volcano plot exhibiting statistically significant DEGs (fold-change ≥2.0, P<0.05). DEGs, differentially expressed genes; PAS, periodic Acid Schiff; H&E, hematoxylin and eosin; TEM, transmission electron microscopy; db/db, Leprdb/Leprdb.

Figure 2.

GO and KEGG analysis of differentially expressed mRNAs. (A) Major alterations in mRNA expression were categorized into biological processes, cellular component and molecular function by GO enrichment analysis. (B) KEGG analysis demonstrating the most enriched alterations in complex cellular pathways. *1: Sodium-independent organic anion transport. *2: Oxidoreductase activity, acting on the CH-NH2 group of donors. *3: Glycosaminoglycan biosynthesis-heparan sulfate/heparin. *4: Glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate. *5: Endocrine and other factor-regulated calcium reabsorption. GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes.

Figure 3.

lncRNA and mRNA correlation analysis revealed potential connections between them. Hierarchically clustered heat map of (A) mRNA and (B) lncRNA with fold-changes no less than 2.0 between db/db and db/m mice. The vertical axis represents each mRNA or lncRNA and the horizontal axis represents different groups. (C) Interaction network of lncRNA and mRNA were constructed using Pearson Coefficients (P≤0.001). (D) Interaction network of SOX2OT and its associated genes were constructed using the same method. Circles denote lncRNA and rectangles denote mRNA. Red is enriched in the db/db group and green is enriched in the db/m group. Red edges denote positive interactions and green edges denote negative interactions. Lnc, long noncoding; SOX2OT, SOX2-original transcript; db/db, Leprdb/Leprdb.

Figure 4.

Validation of microarray results by RT-qPCR and analysis of SOX2OT function by GO enrichment. (A) Certain mRNAs and highly conserved long noncoding RNAs according to UCSC were chosen for RT-qPCR validation. Results demonstrated consistency between these two methods except for Gm15217 and Pvt1. (B) All the SOX2OT-associated mRNAs were analyzed in GO enrichment. Glutathione metabolic, oxidoreductase activity, flavonoid metabolic and lipid metabolic processes are enriched. RT-qPCR, reverse transcription quantitative polymerase chain reaction; GO, gene ontology; DN, diabetic nephropathy; SOX2OT, SOX2 original transcript.

Figure 5.

Genome structure and conservation analysis of SOX2OT. Genome structure of SOX2OT in mice (A) and humans (B) demonstrating the gene location and conservation sites between species. Data were downloaded from http://genome.ucsc.edu. (C) The structure diagrams of SOX2OT variants according to NCBI database. TSSs, transcription starting sites; Ex, exon; SOX2OT, SOX2 original transcript.

Figure 6.

Expression and location of SOX2OT in HPCs and HMCs. (A) HPCs and HMCs were cultured and stimulated by HG. Expression of SOX2OT was determined by reverse transcription quantitative polymerase chain reaction. Data are presented as the mean relative expression level ± standard deviation. *P=0.0006 vs. Control HPC; #P=0.0043 vs. Control HMC. (n=3). (B) Fluorescence in situ hybridization was performed in cultured HPCs and HMCs. Arrows indicate that SOX2OT was expressed in the nucleus and the arrowhead indicates that it was expressed in the cytoplasm (magnification, ×200). HPCs, human podocyte cells; HMCs, human mesangial cells; HG, high glucose treatment; SOX2OT, SOX2 original transcript.