Alteration of the m⁶A Methylation Landscape in a Mouse Model of Scleroderma

Figures & data

Figure 1. A mouse model of scleroderma induced by bleomycin was established.

(A) Skin specimens were stained with hematoxylin and eosin (top) and Masson’s trichrome (bottom). Original magnification ×100. Scale bar = 100 μm. (B) Dermal thickness and (C) collagen content were quantified from the hematoxylin and eosin and Masson’s trichrome staining data, respectively (n = 8 per group). (D)Col1α1 and (E)Col3α1 mRNA levels were examined by quantitative PCR (n = 4 per group). (F) Changes in body weight of mice in the control group and BLM-treated group within 4 weeks. Mean ± standard deviation.

*p < 0.05; **p < 0.01; ****p < 0.001 compared with the control group.

^#No significance compared with the control group.

BLM: Bleomycin.

Figure 2. The change of N⁶-methyladenosine-methylated mRNAs in the bleomycin-induced mouse model of scleroderma.

(A) PC analysis was conducted to classify the differentially methylated mRNAs of the control group and the BLM group. (B) Amplification of positive control primer set (Pos) and negative control primer set (Neg) by quantitative PCR assay indicated comparable m⁶A-RNA-immunoprecipitation enrichment % (m⁶A-RNA-immunoprecipitation/Input) between the control group and the BLM group. (C) The heat map shows opposite m⁶A-methylated mRNAs between the control group and the BLM group. (D) The volcano plot shows the differently m⁶A-methylated mRNAs between the control and BLM groups with fold change ≥1.5 and p < 0.05. (E) Chromosomal distribution of m⁶A-methylated mRNAs. n = 4, each group. Mean ± standard deviation.

^#No significance compared with the control group.

BLM: Bleomycin; Chr: Chromosome; m⁶A: N⁶-methyladenosine; Neg: Negative; PC: Principal componant; Pos: Positive.

Table 1. Specific gene expression associated with scleroderma.

Gene	Fold change	p-value	Chromosome	Association
Tgfb3	3.5257147	2.35E-03	12	The most important signal transduction pathway
Tgfb1	1.7898921	4.99E-03	7	The most important signal transduction pathway
Col2a1	3.1446831	1.89E-03	15	Skin fibrosis (collagen deposition)
Col1a1	2.9964116	1.91E-02	11	Skin fibrosis (collagen deposition)
Col3a1	2.5785271	2.93E-02	1	Skin fibrosis (collagen deposition)
Acta2	3.6369475	1.85E-03	19	Skin fibrosis (epithelial–mesenchymal transition)
Vim	2.4594609	2.82E-02	2	Skin fibrosis (epithelial–mesenchymal transition)
Pecam1	1.688203	4.03E-02	11	Vascular damage
Vcam1	5.104913	2.63E-05	3	Vascular damage
Il1b	4.037706	2.04E-03	2	Inflammatory reaction
Il6	9.747919	1.88E-04	5	Inflammatory reaction
Il33	1.8198779	3.26E-02	19	Inflammatory reaction

Table 2. The top 20 differentially expressed mRNAs in the mouse model of scleroderma.

Gene	Regulation	Fold change	p-value	Chromosome
Clec4d	Up	130.39764	1.04E-06	6
Mmp13	Up	98.831288	5.35E-06	9
Sprr2d	Up	88.734418	2.72E-06	3
Sprr2e	Up	86.931675	5.94E-05	3
Ccl3	Up	81.853399	1.67E-06	11
Ccl4	Up	76.467386	4.66E-06	11
Wfdc12	Up	71.755784	1.30E-06	2
Cenpn	Up	49.673087	1.14E-05	8
Ankrd1	Up	48.647211	1.09E-04	19
Il1b	Up	46.46236	1.88E-05	2
Lce3f	Up	41.530177	1.51E-05	3
Nmu	Up	37.086461	3.82E-07	5
Myh7	Up	27.048769	1.71E-02	14
Spink12	Down	0.060961	4.51E-04	18
Krt72	Down	0.0627622	3.79E-05	15
Inpp5a	Down	0.0712282	3.53E-02	7
Asphd2	Down	0.0741685	3.49E-02	5
Tmc8	Down	0.0828253	1.14E-03	11
Txndc5	Down	0.0828767	1.12E-04	13
Crisp3	Down	0.0828781	1.22E-04	17

Figure 3. Association analysis of N⁶-methyladenosine-methylated and expressed mRNAs in the bleomycin-induced mouse model of scleroderma.

(A) The four-quadrant and Venn diagram showing the N⁶-methyladenosine-methylated and expressed mRNAs (fold change ≥1.5). (B) The protein–protein interaction network showed the connection between mRNAs in the combined analysis (fold change ≥2).

BLM: Bleomycin.

Figure 4. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of differentially N⁶-methyladenosine-methylated mRNAs.

(A) The top ten Gene Ontology terms of hypermethylated mRNAs, ranked by enrichment score. (B) The top ten Gene Ontology terms of hypomethylated mRNAs, ranked by enrichment score. (C) The top ten significantly enriched pathways of hypermethylated mRNAs, ranked by enrichment score. (D) The top ten significantly enriched pathways of hypomethylated mRNAs, ranked by enrichment score.

Table 3. Review for the selected genes in scleroderma.

Gene	Chromosome	Regulation	Regulatory roles on fibrosis	Ref.
Hras	7	Down	Accelerating fibroblast proliferation rates	[42]
Lama3	18	Up	Destroying the balance of epithelial–mesenchymal transition	[43]
Tnc	4	Up	Inducing collagen deposition and myofibroblast differentiation; a leading-edge gene of the PI3K-Akt pathway in skin fibrosis	[44–46]
Stat3	11	Up	A transcription factor activated downstream of TGF-β	[47,48]
Ccl3	11	Up	A critical chemokine for fibrosis and inflammation in scleroderma	[49]
Ccl9	11	Up	Mediates macrophage regulation of extracellular matrix	[50]
Saa1	7	Up	Promotes macrophage-to-myofibroblast transition	[51]
Il1b	2	Up	An important proinflammatory factor in scleroderma	[52]

Table 4. Differentially hypermethylated mRNAs involved in the extracellular matrix–receptor interaction pathway.

Transcript_ID	Gene	Fold change	p-value
ENSMUST00000030056	Tnc	2.906998354	0.001655676
ENSMUST00000208095	Col4a1	2.752693479	0.00095491
uc008ecg.1	Lama3	1.935457924	0.00273348
uc008kia.1	Itgav	1.900846576	0.019797529
ENSMUST00000166905	Dag1	1.726454757	0.039187672
ENSMUST00000042729	Npnt	1.584309017	0.005438113
ENSMUST00000191899	Dag1	1.466675764	0.017589962
ENSMUST00000141725	Itgav	1.226947816	0.016770676

Table 5. Differentially hypermethylated mRNAs involved in the growth hormone synthesis, secretion and action pathway.

Transcript_ID	Gene	Fold change	p-value
ENSMUST00000026572	Hras	2.113980372	0.032686597
ENSMUST00000105689	Pik3cd	1.906831767	0.040208284
ENSMUST00000112064	Cacna1s	1.803270257	0.041289197
ENSMUST00000154685	Pik3r2	1.725025645	0.049359786
ENSMUST00000204163	Ghrl	1.719122443	0.020129527
uc012aqj.2	Atf6b	1.710327299	0.046871714
ENSMUST00000112016	Atf2	1.677316698	0.009778686
ENSMUST00000127638	Stat3	1.587988361	0.041871594
ENSMUST00000020215	Socs2	1.536058126	0.009307881
NM_001256001	Cacna1c	1.528964604	0.003969553
ENSMUST00000205120	Creb5	1.52871572	0.028358205
ENSMUST00000112793	Cacna1c	1.52271918	0.014032103

Table 6. Differentially hypomethylated mRNAs involved in cytokine–cytokine receptor interaction pathways.

Transcript_ID	Gene	Fold change	p-value
ENSMUST00000028361	Il1f6	0.331783132	0.00016362
uc008mhu.2	Il1b	0.474644948	0.01262423
ENSMUST00000001008	Ccl3	0.488395453	0.017125028
ENSMUST00000108648	Tnfsf13	0.549612429	0.005544923
ENSMUST00000019266	Ccl9	0.579709293	0.041642398

Table 7. Differentially hypomethylated mRNAs involved in inflammatory response (biological process).

Transcript_ID	Gene	Fold change	p-value
ENSMUST00000028361	Il1f6	0.331783132	0.00016362
uc008mhu.2	Il1b	0.474644948	0.01262423
ENSMUST00000001008	Ccl3	0.488395453	0.017125028
ENSMUST00000128088	Saa1	0.499300978	0.030405502
ENSMUST00000069927	S100a8	0.499945891	0.020490792
ENSMUST00000088041	Ptgis	0.578128978	0.039478702
ENSMUST00000019266	Ccl9	0.579709293	0.041642398
ENSMUST00000026881	Ly96	0.593689188	0.033149311

Figure 5. N⁶-methyladenosine enrichment and mRNA expression in the candidate mRNAs.

(A) The relative m⁶A level of the selected mRNAs was detected using MeRIP-qPCR. (B) The comparison between microarray data and MeRIP-qPCR of the selected mRNAs. (C) The six mRNAs were analyzed using qPCR. (D) The comparison between microarray data and qPCR of the selected mRNAs. (E) Identification of RNA binding proteins to the validated m⁶A-methylated mRNAs differentially. n = 4, each group. Mean ± standard deviation.

^#No significance.

*p < 0.05; **p < 0.01; ***p < 0.001 compared with the control group.

BLM: Bleomycin; m⁶A: N⁶-methyladenosine; MeRIP: m⁶A-RNA-immunoprecipitation; qPCR: Quantitative PCR.

Figure 6. Analysis of N⁶-methyladenosine methylation regulators in the bleomycin-induced mouse model of scleroderma.

(A) The relative expression of writers, erasers and readers in microarray data. (B)Mettl3 and Alkbh5 were analyzed using quantitative PCR. n = 4, each group. Mean ± standard deviation.

BLM: Bleomycin.

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