1. FTO expression was decreased in cardiac fibrotic tissue and hypoxia-induced fibroblasts, which accounted for the excessive m6A modification
To understand the role of m6A modification in cardiac fibrosis, we detected the expression under cardiac fibrosis in vivo and in vitro. RNA m6A dot blot was carried out to detect the levels of m6A modification. The results revealed that the m6A levels were significantly elevated in the cardiac fibrotic tissue of rats (Fig. 1a). The biological process of MI was accompanied by hypoxia. Subsequently, we established the hypoxia chamber (1% O2, 5% CO2, and balanced with N2) to explore the activation of CFs. Then we measured the m6A levels in CFs under hypoxia treatment. Consistent with the expression levels in vivo, hypoxia promoted the m6A modification of CFs (Fig. 1b). The above results demonstrated that MI or hypoxia up-regulated the levels of m6A modification in rats or CFs respectively. Evidence has confirmed that m6A modification levels were adjusted by methylase (METTL3, METTL14, WTAP) and demethylase (FTO, ALKBH5)(15, 16). To find which methylation modification enzyme caused the abnormal m6A modification, the expression levels of these m6A-associated genes were measured in MI rats and hypoxia-treated CFs (Fig. 1c, d). FTO was decreased in both MI tissues and hypoxia-induced CFs. However, the expression of METTL3, METTL14, WTAP, and ALKBH5 remained unchanged (Fig. 1c, d).
To further know the role of aberrant FTO expression in cardiac fibrosis, the cardiac fibrosis rat model was established by ligation of LAD for 3, 7, and 28 days and the hypoxia-induced CFs model was carried out in the hypoxia chamber for 12, 24, and 48 hours. It was observed that fibrotic markers, containing fibronectin (FBN) and α-smooth muscle actin (α-SMA), increased along with MI progression (Fig. 1e). Consistent with the above results in vivo, α-SMA was markedly stimulated by hypoxia for 48 h (Fig. 1f). Western blot and qRT-PCR analysis measured the negative relationship between FTO and fibrotic markers in both the fibrotic tissue of rats and hypoxia-induced CFs, including Collagen type I (COL-1) and Collagen type III (COL-3) (Fig. 1g, i). In the fibrotic tissue of rats, FTO expression was significantly decreased at both mRNA and protein levels 3 days after MI surgery (Fig. 1g, h). Similarly, FTO was downregulated in hypoxia-induced CFs for 24 h at both mRNA and protein levels (Fig. 1i, j).
2. HIF1α bound to FTO promoter and decreased FTO expression
The biological process of MI was accompanied by hypoxia. It was widely known that the HIF1 signal pathway was highly activated under hypoxia conditions (17, 18). Thus, we detected the expression levels of the HIF signal pathway in MI rats and hypoxia-cultured CFs. Western blot and qRT-PCR results showed HIF1α and HIF2α were obviously increased with FTO decreased in MI rat models (Fig. 2a, b). Then, hypoxia accelerated the protein and mRNA levels of HIF1α and HIF2α while inhibiting the expression of FTO (Fig. 2c, d). To identify the biological roles and molecular mechanisms of HIF1α and HIF2α, the specific inhibitors and knockdown of HIF1α and HIF2α were performed. BAY 87-2243, HIF1α inhibitor, significantly promoted FTO mRNA levels by contrast with PT2385, HIF2α inhibitor, remaining FTO unchanged (Fig. 2e). Then, the knockdown of HIF1α and HIF2α of CFs were established. The silencing efficacies of both mRNA and protein levels were definite (Fig. 2f, g). However, only HIF1α knockdown obviously increased the FTO expression level (Fig. 2f, g). Therefore, HIF1α, instead of HIF2α, negatively regulated FTO expression.
CUT&Tag assay was performed to further elucidate the specific molecular mechanisms of HIF1α regulating FTO expression. The results revealed that the binding sites of HIF1α mainly located in the transcriptional start site (TSS) under normoxia and hypoxia conditions (Fig. 2h; Fig. S1). Signal pathways correlated with DNA replication and CF activation were enriched in HIF1α-mediated CUT&Tag samples (Fig. 2i). The CUT&Tag data displayed the peaks of FTO promoter, suggesting the specific binding sites of HIF1α (Fig. 2j). Plenty of evidence have confirmed that HIF-1α could bind to the promoter of target genes via hypoxia transcriptional response elements (HRE) (19, 20). Then, we analyzed the sequences and found two potential HRE sites in the FTO promoter (Fig. 2k). We first conducted a luciferase reporter plasmid controlled by rat FTO promoter (-1034 bp upstream of TSS, FTO-luc). To identify the importance of these HRE sites for HIF1α-regulated FTO expression, site-directed mutagenesis was carried out to mutate the HRE motifs from ACGTG to AAGGA in the FTO-luc construct. Site 1 and Site 2 were shown to significantly decrease the HIF1α-regulated FTO expression, of which Site 1 only had minor effects (Fig. 2k). The following CUT&Tag-qPCR experiments also confirmed more enrichment on Site 2 than that on Site 1 (Fig. 2k).
In all, the above data demonstrated that HIF1α bound to the FTO promoter via HRE in CFs. MI or hypoxia treatment activated HIF1α and inhibited gene FTO transcription. Thus, HIF1α is a transcriptional inhibitor of FTO.
Figure 2 was displayed in PDF.
Figure 2 Negative regulation of FTO mediated by HIF1α binding to its promoter. Western blot (a) and qRT-PCR (b) results of HIF1α, HIF2α, and FTO in MI heart tissues. n = 6. The protein (c) and mRNA (d) levels of HIF1α, HIF2α, and FTO in hypoxia-treated cardiac fibroblasts (CFs). n = 4. e Expression level of FTO after treatment of HIF1α inhibitor BAY 87-2243 and HIF2α inhibitor PT2385. n = 4. Western blot (f) and qRT-PCR (g) results showing the expression of HIF1α, HIF2α, and FTO after HIF1α and HIF2α knockdown in CFs. n = 4. Heatmaps (h) and enrichment maps (i) of KEGG from CUT&Tag-seq reads in CFs. j Representative CUT&Tag-seq signal tracks at the promoter region of FTO. k CUT&Tag-seq predicting the potential binding sites of HIF1α and FTO promoter. Relative luciferase activity after transfection with reporter plasmids showing the binding sites. The ratio of Firefly and Renilla luciferase values calculated the relative luciferase activity. The CUT&Tag-qPCR results showing the degree of enrichment in predicted binding sites of the FTO promoter. n = 3. SCR, scramble sequences. NC, negative control. The data was expressed as mean ± SEM. * P < 0.05 vs NC/SCR, ** P < 0.01 vs NC/SCR, *** P < 0.001 vs NC/SCR, **** P < 0.0001 vs NC/SCR.
3. FTO regulated collagen production and proliferation of CFs in vitro
To determine the function of FTO in CFs, we performed the loss-of-function experiments of FTO in CFs by transfection of small interfering RNA of FTO (si-FTO) and overexpression plasmid of FTO (FTO). The transfection efficiencies of si-FTO and FTO were validated by qRT-PCR (Fig. 3a). RNA m6A dot blot was carried out to detect the levels of m6A modification, which showed that si-FTO increased the m6A levels while FTO decreased the m6A levels in CFs (Fig. 3b). The mRNA (Fig. 3d) and protein (Fig. 3c) levels of COL-1 and COL-3 were markedly increased after transfection of siFTO (100nM) under normal conditions. On the contrary, western blot and qRT-PCR data indicated decreased expression levels of COL-1 and COL-3 with transfection of FTO (1 µg/ml) without any treatment (Fig. 3e, f). EdU fluorescence staining showed that FTO knockdown significantly promoted the proliferation of CFs (Fig. 3g, k), and similar results were obtained from the CCK-8 assay (Fig. 3j). Meanwhile, compared with the scramble sequence (SCR) group, CFs treated with si-FTO markedly increased fibroblast migration indexed by the Transwell assay (Fig. 2h, l) and wound healing assay (Fig. 2i, m).
4. Protective effects of FTO on cardiac fibrosis
Since the above findings indicated that FTO inhibited collagen synthesis under normal conditions, we detected the protective effects of FTO under pathological conditions. We first evaluated the protective effects of FTO on hypoxia-induced CFs. The results indicated that hypoxia stimulation increased collagen expression at mRNA and protein levels. Consistent with the findings in normal conditions, overexpression of FTO ameliorated the mRNA and protein levels of collagen (Fig. 4a, b). In addition, overexpressing FTO reduced cell proliferation mediated by hypoxia using the CCK-8 assay (Fig. 3c) and EdU fluorescence staining (Fig. 3d). Similarly, the migration ability of CFs was weakened as shown by the Transwell assay (Fig. 3e) and wound healing assay (Fig. 3f).
Then, we constructed the adenovirus carrying FTO to investigate the effects on MI rat models. According to the manufacturer's instructions, the dose of 109 titers/rat for overexpressing adenovirus was used in the following study. The efficiency of adenovirus infection was confirmed (Fig. S2). After injection of FTO-overexpressing adenovirus for 7 days, the MI model was established by ligation of LAD. The interstitial fibrosis of MI rats was evaluated 4 weeks after MI surgery. The m6A levels of fibrotic tissues were significantly down-regulated after FTO overexpression treatment (Fig. 4g). We observed that FTO overexpression therapy reduced MI-activated fibrotic markers, including collagen deposition, FBN, and α-SMA (Fig. 4h). Echocardiography was performed to assess the protective effects of FTO on cardiac function. Compared with the vector group, FTO overexpression markedly ameliorated the cardiac function of MI rats, including ejection fraction (EF) and fractional shortening (FS) (Fig. 4i). Meanwhile, overexpressing FTO significantly decreased both mRNA and protein levels of collagen in the infarct border area of rats (Fig. 4j, k).
Collectively, these results manifested that FTO inhibited collagen biosynthesis and activation of CFs. overexpressed FTO could reduce collagen deposition and ameliorate the cardiac function of post-MI rats.
5. FTO targeted EPRS in m6A-seq combined with RNA-seq
RNA-seq was performed to investigate the underlying targets of FTO in cardiac fibrosis. Compared with the vector group, the results identified a total of 2929 differentially expressed genes (1547 upregulated genes, 1382 downregulated genes) in the FTO-overexpressed group. Overexpressed FTO decreased the high mRNA level of collagen-related genes, including EPRS, Col1α1, Col3α1, and FBN (Fig. 5a). Furthermore, gene set enrichment analysis (GSEA) in RNA-seq indicated that collagen, collagen biosynthesis and modifying enzymes, and collagen chain trimerization were significantly negatively enriched in the FTO-overexpressing CFs, suggesting the potential targets involved in collagen synthesis (Fig. 5b). Biological process of Gene Ontology (GO) analysis was performed and suggested that FTO overexpression significantly suppressed cell migration and cell proliferation (Fig. 5c). Based on RNA-seq, pathway analysis was used to determine the significant pathway of the genes according to KEGG database. The MAPK, PI3K-Akt, and Wnt signaling pathways, which were closely associated with the activation of CFs, were negatively enriched in FTO-overexpressed CFs (Fig. S3a). In all, FTO suppressed the activity of CFs according to RNA-seq data.
To verify whether differential genes involved in collagen synthesis were regulated by FTO-mediated m6A modification, m6A-seq was performed. There were 21259 and 17818 m6A peaks identified by m6A-seq, of which 7301 and 3860 were unique in the vector and FTO-overexpressed groups respectively (Fig. 5d). Then, the m6A consensus motif of CCACC was highly enriched in the m6A peaks (Fig. 5e). Consistent with previous studies, m6A modifications were mainly enriched around the initiation and stop codon of the coding region (CDS) in Vector and FTO groups (Fig. 5g). Biological process of GO analysis was also performed and indicated that FTO reduced cell cycle and cell migration in an m6A manner, which was consistent with RNA-seq data (Fig. 5f). Furthermore, KEGG analysis suggested that FTO overexpression remarkably reduced the signal pathways correlated with DNA replication and cell cycle (Fig. S3b). The above sequencing data confirmed that FTO regulated the activation of CFs via m6A modification. Subsequently, m6A-seq and RNA-seq were combined to find out the precise underlying targets of FTO-induced fibrosis. Genes were divided into the m6A group and the non-m6A group, depending on whether they were regulated by FTO-induced m6A modification. The results showed that the transcriptome foldchange of genes in the m6A group was significantly lower and greater than that in the non-m6A group (Fig. 5h). This result suggested that FTO might downregulate the underlying gene expression levels via reducing m6A modification. Therefore, we focused on the decreased mRNA expression of genes with decreased m6A modification levels, which were highlighted in an orange circle (Fig. 5i). Among this circle, m6A-seq uncovered 3703 differential m6A peaks with decreased abundance in the FTO overexpressing group. Meanwhile, RNA-seq data identified corresponding 900 downregulated transcripts in FTO overexpression (Fig. 5i). Based on the above analysis, we focused on the collagen-related genes of which both m6A modification and expression levels were decreased. Then, we screened out 15 genes involved in collagen synthesis from the overlap and discovered that EPRS may be the potential target of FTO-mediated m6A regulation due to promoting collagen translation in CFs (Fig. 5j). The protein level of EPRS was increased in rats after MI (Fig. S4a). qRT-PCR (Fig. 5k) and western blot (Fig. 5l, m; Fig. S4b) and analysis indicated that ERPS was negatively correlated with FTO.
6. FTO regulated EPRS mRNA stability in m6A-dependent manners mediated by IGF2BP3
The m6A modification levels of the target were modified by methylase and demethylase. Then the biological effects of m6A regulation were dependent on selective recognition of m6A sites by “readers”. IGF2BP1-3 were reported to stabilize the mRNA levels (11, 21). To identify whether EPRS expression was regulated by IGF2BPs, the knockdowns of IGF2BP1-3 were conducted. Both western blot and qRT-PCR analysis showed that the knockdown of IGF2BP3, not IGF2BP1/2 (Fig. S5), significantly reduced EPRS in both mRNA and protein levels of CFs (Fig. 6a, b). Then, we further detected whether EPRS expression in FTO-knockdown CFs was affected by IGF2BP3. The IGF2BP3 silencing has remarkably inhibited the protein level of EPRS in CFs with FTO knockdown (Fig. 6c). It was realized that EPRS was the target of IGF2BP3. The following RIP-qPCR experiment confirmed that EPRS mRNA interacted with IGF2BP3 (Fig. 6d).
The m6A -seq data revealed that the m6A peak of EPRS in CDS shrank significantly with FTO overexpression (Fig. 6e). To verify the vital role of m6A modification in regulating EPRS mRNA, the luciferase reporter inserting a wild-type EPRS-CDS sequence (WT) or mutant counterpart (MUT) whose m6A sites were mutated was established (Fig. 6f). While FTO was reduced, the relative luciferase activity in CFs with EPRS-WT plasmid significantly increased whereas that in CFs transfected with mutant plasmid indicated no significance (Fig. 6g). On the contrary, when FTO was overexpressed, the relative luciferase activity with EPRS-WT was downregulated (Fig. 6h). Moreover, the dual luciferase reporter assays demonstrated that knockdown of IGF2BP3 inhibited the luciferase activity of ERPS-WT plasmid through recognition of m6A sites (Fig. 6i, j). These findings suggested that IGF2BP3 regulated EPRS expression level via m6A modification. Next, RNA stability assays were conducted, and the results showed that IGF2BP3 deficiency promoted the degradation of EPRS mRNA (Fig. 6k). RNA degradation curves suggested that the half-life of EPRS mRNA was prolonged with FTO silencing in CFs, while IGF2BP3 knockdown could reverse the increased mRNA stability mediated by FTO silencing (Fig. 6l).
In summary, the upregulation of EPRS induced by FTO silencing can be attributed to the increased stability of EPRS mRNA induced by elevated m6A modification. FTO regulated EPRS mRNA stability in m6A-dependent manners mediated by IGF2BP3.
7. EPRS was required for siFTO-promoted fibrosis in CFs
Based on the above data, we found that knockdown or overexpression of FTO upregulated or downregulated the expression of EPRS at mRNA and protein levels (Fig. 5k-m). To explore the role of EPRS in regulating FTO’s function, we verified the effects of EPRS on CFs. The specific siRNA for EPRS was constructed and the transfection efficiency was validated (Fig. 7a). Knockdown of EPRS significantly reduced the mRNA and protein expression of collagen in CFs (Fig. 7a, b). In addition, EPRS silencing suppressed the proliferation of CFs using EdU fluorescence staining (Fig. 7c) and CCK-8 assays (Fig. 7e). Moreover, the migration speed of CFs was decreased through Transwell assays (Fig. 7d). In all, these findings indicated that EPRS played a significant role in cardiac fibrosis. Subsequently, to further investigate the key role of EPRS as the downstream target of FTO in cardiac fibrosis, rescue experiments were conducted to detect whether EPRS silencing could reverse the effects of FTO silencing. The results showed that EPRS knockdown markedly reduced the synthesis of collagen induced by FTO knockdown at mRNA and protein levels (Fig. 7f, g). Moreover, the high proliferation levels mediated by siFTO were rescued by siEPRS (Fig. 7h, i). Knockdown of EPRS also rescued the abnormal migration speed of CFs caused by siFTO (Fig. 7j, k). These data indicated that EPRS was required for siFTO-induced fibrosis in cardiac fibrosis.