Microarray analysis of long non‑coding RNA expression profiles in Marfan syndrome

Figures

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  Figure 1. H&E and VG staining of aortic tissue. (A) H&E staining of normal aortic tissue. (B) H&E staining of MFS aortic tissue. (C) VG staining of normal aortic tissue. (D) VG staining of MFS aortic tissue. Collagen and muscle fibers were stained red and yellow, respectively. Magnification, x200. H&E, hematoxylin and eosin; MFS, Marfan syndrome, VG, Verhoeff-Van Gieson.

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  Figure 2. Immunohistochemistry staining of aortic tissue. (A) CD3 T cells in aortic tissue of NA and MFS samples. (B) CD68 macrophages in the aortic tissue of NA and MFS samples. Immunohistochemical staining indicated that inflammatory cells (CD3 and CD68) were present diffusely in the media and adventitia of the aortas from patients with MFS, but were rarely found in control aortas. (C) SMA immunostaining in the aortic tissue of NA and MFS samples. (D) Desmin immunostaining in aortic tissue of NA and MFS samples. SMCs were both strongly positive in all aortic tissues, but the structure was degenerative and disintegrative in MFS samples compared with NAs. Magnification x100. MFS, Marfan syndrome; NA, normal aorta; SMA, smooth muscle actin; SMCs, smooth muscle cells.

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  Figure 3. Microarray profiling of lncRNAs in the MFS and NA tissue specimens. (A) Volcano plots, (B) scatter plots and (C) hierarchical clustering showing the lncRNA expression profiling (P<0.05 and fold change ≥1.5). Upregulated lncRNAs are denoted in red and downregulated in green. lncRNA, long noncoding RNA; MFS, Marfan syndrome; NA, normal aorta.

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  Figure 4. GO and KEGG pathway analysis of differentially expressed mRNAs in Marfan syndrome compared with normal aortas. The top 10 GO terms that were associated with the gene function of (A) upregulated mRNAs and (B) downregulated mRNAs are listed. The top 10 pathways that were associated with the (C) upregulated mRNAs and (D) downregulated mRNAs are listed. ARVC, Arrhythmogenic right ventricular cardiomyopathy; ECM, extracellular matrix; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; TNF, tumor necrosis factor; NOD, nucleotide binding oligomerization domain containing.

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  Figure 5. The lncRNAs and mRNA co-expression network. The interaction network of differentially expressed genes (lncRNA: uc003jka.1, uc003jox.1, XIST, linc-LPA-1, linc-PPWD1). Round nodes represent protein-coding genes and square nodes represent lncRNAs. Blue nodes represent upregulated genes or lncRNAs, red nodes represent downregulated genes or lncRNAs. The node size represents the connectivity, with larger node showing that more genes or lncRNAs are co-expressed with this gene or lncRNA. Solid lines represent positive correlation and dotted lines negative correlation. lncRNA, long noncoding RNA; XIST, X-inactive specific transcript; linc-LPA-1, linc-lysophosphatidic acid receptor 1; linc-PPWD1, linc-peptidylprolyl isomerase domain and wd repeat containing 1.

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  Figure 6. Reverse transcription-quantitative PCR validation. Compared to healthy controls, 5 long noncoding RNAs (uc003jka.1, uc003jox.1, XIST, linc-LPA-1, linc-PPWD1) with highest degree were selected. Results were consistent with the findings obtained from the microarray chip analysis (n=6). Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. NA samples. MFS, Marfan syndrome; NA, normal aorta; XIST, X-inactive specific transcript; linc-LPA-1, linc-lysophosphatidic acid receptor 1; linc-PPWD1, linc-peptidylprolyl isomerase domain and WD repeat containing 1.