Identifying the Growth Factors for Improving Neointestinal Regeneration in Rats through Transcriptome Analysis Using RNA-Seq Data

Abstract

Using our novel surgical model of simultaneous intestinal adaptation "A" and neointestinal regeneration "N" conditions in individual rats to determine feasibility for research and clinical application, we further utilized next generation RNA sequencing (RNA-Seq) here in normal control tissue and both conditions ("A" and "N") across time to decipher transcriptome changes in neoregeneration and adaptation of intestinal tissue at weeks 1, 4, and 12. We also performed bioinformatics analyses to identify key growth factors for improving intestinal adaptation and neointestinal regeneration. Our analyses indicate several interesting phenomena. First, Gene Ontology and pathway analyses indicate that cell cycle and DNA replication processes are enhanced in week 1 "A"; however, in week 1 "N", many immune-related processes are involved. Second, we found some growth factors upregulated or downregulated especially in week 1 "N" versus "A". Third, based on each condition and time point versus normal control tissue, we found in week 1 "N" BMP2, BMP3, and NTF3 are significantly and specifically downregulated, indicating that the regenerative process may be inhibited in the absence of these growth factors. This study reveals complex growth factor regulation in small neointestinal regeneration and intestinal adaptation and provides potential applications in tissue engineering by introducing key growth factors identified here into the injury site.

Figure 1

Histological analysis of regenerative constructs under various conditions. Hematoxylin and eosin staining of longitudinal sections of adapted intestine (a, b, c), regenerated neointestine (d, e, f), and the normal control (g, h, i) from weeks 1, 4, and 12 after surgery regenerative constructs. (a–c) Villi and crypts of tissues undergoing intestinal adaptation. (d–f) The regeneration process of neointestine was time-dependent. (g–i) No histological evidence of significant difference in normal control was identified at any time point. Muc, mucosa layer; Mus, muscle layer; DI, donor intestine; Junction; start point of regeneration; NI, neointestine. Magnification, 40X; bar, 500 μm.

Figure 2

Global expression pattern of each condition and time point. (a) Principle component analysis (PCA) plot of each sample on whole transcriptome. Red: control; blue: week 1, green: week 4, purple: week 12. Octahedron: “N”, Icosahedron: “A”. Light green octahedron: centroid of “N”; pink icosahedron: centroid of “A”; yellow sphere: centroid of control. Blue oval: region of “N”; magenta oval: region of “A”. (b) Heat map of stage specific upregulated genes of “A”. (c) Heat map of stage specific upregulated gene of “N”.

Figure 3

Gene expression pattern of each time point. (a) Heat map of the top 50 most significant genes of week 1 after surgical treatment for both “N” and “A” conditions. (b) Week 4. (c) Week 12.

Figure 4

Growth factor expression patterns of each time point. (a) Tile chart demonstrated the significant upregulated growth factors in “N”. X-axis: “N” vs. “A” at week 1, week 4, and week 12. (b) Upper panel: heat map of differential expressed growth factors of both “N” and “A” at week 1. Lower panel: quantitative PCR validation of some growth factors. ∗∗∗: P value < 0.001, ∗∗: P value < 0.01, ∗: P value < 0.05. (c) Week 4. (d) Week 12. (e) Tile chart for significant downregulated growth factors in “N”. (f) Upper panel: heat map of differential expressed growth factors of both “N” and “A” at week 1. Lower panel: quantitative PCR validation of some growth factors. (g) Week 4. (h) Week 12.

Figure 5

Stage specific growth factor expression patterns on A and N conditions. (a) Tile chart demonstrated the significant (P_adj < 0.05) upregulated growth factors in each time point and condition compared to control. (b) Tile chart demonstrated the significant (P_adj < 0.05) downregulated growth factors in each time point and condition compared to control. (c) Quantitative PCR confirmation of selected growth factors. (d) Heatmap of week 1 “A” and “N” compared to control. (e) Heatmap of week 1 “A” and “N” compared to control. (f) Quantitative PCR confirmation of selected growth factors.