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Heterogeneous nuclear ribonucleoprotein A2/B1 promotes myocardial fibrosis by regulating the miR‐221‐3p/FOXO4‐mediated inflammation

Year: 2024

Type: article

Abstract: Dear Editor, Myocardial fibrosis (MF) is the pathological basis of multiple cardiovascular diseases.1 Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), a member of the hnRNP family, participates in transcription and RNA metabolism by RNA-binding. The process includes reading m6A-marked messenger RNAs (mRNAs) and mediating microRNA (miRNA) maturation.2 Recently, hnRNPA2B1 was shown to be increased in the blood of acute myocardial infarction patients,3, 4 as well as upregulated in MF in an isoproterenol (ISO)-induced model and ISO-treated primary cardiac fibroblasts (Figure S1, S2C–F). In this study, we investigated a novel role for hnRNPA2B1 in MF. In hnRNPA2B1 knockout (eKO) mice, the heart-to-body weight ratio was significantly reduced after ISO injection (Figure 1B). Echocardiography analysis showed a higher LV ejection fraction (%) from 45.93 ± 3.78 to 61.67 ± 1.51 and shortening fraction (FS%, 22.51 ± 2.08 to 32.03 ± 0.99) in the hnRNPA2B1 eKO mice than in the wild-type (WT) mice after ISO stimulation (Figure 1C,D). HE staining and Masson staining results showed that the deletion of hnRNPA2B1 significantly reduced ISO-induced inflammatory infiltration and collagen deposition (Figure 1E,F). Western blotting analysis also confirmed that the loss of hnRNPA2B1 significantly reduced the expression of cardiac fibrosis (CF) biomarkers, including collagen I/III, transforming growth factor beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA), induced by ISO (Figure 1G–L). The above experimental data suggest that the deletion of hnRNPA2B1 improves cardiac function and suppresses CF in ISO-induced mice. Cardiac fibroblasts' proliferation and activation are associated with the development of MF.5 To study whether hnRNPA2B1 is involved in cardiac fibroblast proliferation and activation, we knocked down and overexpressed hnRNPA2B1 in primary cardiac fibroblasts by pretransfection of small interfering RNA (siRNA)-hnRNPA2B1 and hnRNPA2B1 cDNA plasmids (Figure 2A and Figure S3A). The results of the EdU assay (Figure 2B and Figure S3B) showed that hnRNPA2B1 knockdown significantly decreased EdU+ cells after ISO stimulation, while hnRNPA2B1 overexpression increased EdU+ cells. The MTT assay also showed that the growth curve of the siRNA-hnRNPA2B1+ISO group was decreased, however, hnRNPA2B1 overexpression promoted the growth of cardiac fibroblasts after ISO stimulation (Figure 2C and Figure S3C), which indicate that hnRNPA2B1 enhances the proliferation of cardiac fibroblasts. After cardiac fibroblast activation, extracellular matrix proteins such as α-SMA and collagen I/III are highly expressed, thus, we used western blotting to detect their expression after hnRNPA2B1 knockdown or overexpression. HnRNPA2B1 silencing markedly decreased the expression of collagen I/III, TGF-β1 and α-SMA, while hnRNPA2B1 overexpression dramatically increased the expression of these MF-related proteins (Figure 2D–H and Figure S3D–H). These data suggested that hnRNPA2B1 promotes the ISO-stimulated tendency towards cardiac fibroblast fibrosis. Considering that hnRNPA2B1 can read m6A-marked miRNA and mediate miRNA maturation, we collected data on miRNAs regulated by hnRNPA2B12 and miRNAs that were involved in CF (Table S1) from the reported literature. MiR-126, miR-181a, miR-210, miR-99b and miR-221 were screened out (Figure 3A) and determined by quantitative real-time polymerase chain reaction (qRT-PCR), however, only three of them were detected. miR-99b-3p, miR-221-3p and miR-210-5p expression were upregulated in the ISO-induced left ventricle of mice, and miR-221-3p was more significant (Figure 3B). Importantly, in hnRNPA2B1 eKO mice, the elevated miR-99b-3p, miR-210-5p and miRNA-221-3p induced by ISO were blocked compared with that in WT mice (Figure 3C). Moreover, the miR-99b-3p, miR-210-5p and miR-221-3p levels in ISO-induced primary cardiac fibroblasts were significantly decreased after hnRNPA2B1 silencing (Figure 3D). Considering the significant changes in miRNAs at the animal and cell levels regulated by hnRNPA2B1, we selected miR-221-3p for mechanistic discussion. To evaluate the function of miR-221-3p in the activation of CF, transfection of miR-221-3p mimics and inhibitors into primary cardiac fibroblasts was performed. Treatment with miRNA-221-3p mimics enhanced the proliferation of cardiac fibroblasts and increased the expression of CF-related genes (Col1a1, Col3a1, Tgfb1 and Acta2, Figure 3E–G), while miRNA-221-3p inhibitors retrained the proliferation and those genes in ISO-induced primary cardiac fibroblasts (Figure 3H–J). Foxo4, one of the widely expressed forkhead box (Fox) transcription factor O family members, regulates oxidative stress/immune response, metabolism and apoptosis.6 We detected Foxo4 mRNA levels, which are highly expressed in the ISO-induced heart (Figure 4A). Subsequently, two binding sites of miR-221-3p were predicted in the 3′-UTR of Foxo4 using StarBase (Figure 4B). Thus, we speculated that Foxo4 could be a target of miR-221-3p involved in CF. A luciferase reporter assay exhibited that miR-221-3p represses Foxo4 by binding its 3′-UTR. Mutation of the two binding sites of miR-221-3p eliminated the repression (Figure 4C), confirming the direct targeting of Foxo4 by miR-221-3p. Furthermore, qPCR analysis confirmed that treatment with miR-221-3p mimics suppressed Foxo4 mRNA expression in primary cardiac fibroblasts (Figure 4D), while miR-221-3p inhibitors increased Foxo4 expression (Figure 4E). In addition, Foxo4 mRNA expression was significantly elevated in hnRNPA2B1 eKO mice after ISO stimulation (Figure 4F). Consistent with the in vivo results, Foxo4 expression was increased after hnRNPA2B1 silencing in primary cardiac fibroblasts (Figure 4G). Foxo4 is related to the inflammatory response.7, 8 Thus, we evaluated the levels of proinflammatory mediators (Il6, Il1b and Tnfa). Their mRNA levels were increased in the ISO-induced left ventricles of mice and primary cardiac fibroblasts (Figure S4A–C). In addition, the Il6, Il1b and Tnfa mRNA levels markedly declined in hnRNPA2B1 eKO mice under ISO stimulation (Figure 4H–J). Consistent with the in vivo results, Il6, Il1b and Tnfa mRNA levels were also reduced after hnRNPA2B1 silencing in primary cardiac fibroblasts (Figure 4K–M). Interestingly, Foxo4 silencing significantly increased the mRNA level of Il1b but did not affect Il6 and Tnfa (Figure 4N–O and Figure S4D,E). To confirm the role of Foxo4 in hnRNPA2B1-mediated MF, we performed a rescue assay. in hnRNPA2B1-silenced myofibroblasts, Foxo4 knockdown increased EdU+ cells numberAs (Figure 4P,Q). In addition, Foxo4 knockdown blocked the negative regulation of hnRNPA2B1 silencing on MF-related gene expression (Col1a1, Col3a1, Acta2 and Tgf1b, Figure 4R–U). The above results indicated that Foxo4 is a key regulator in hnRNPA2B1-mediated CF. In conclusion, our study indicated that hnRNPA2B1 participated in ISO-induced MF, and the mechanism was associated with regulating the miR-221-3p/Foxo4-mediated inflammatory response and myofibroblast activation (Figure 4V). Xuping Li and Shuotao Shi performed the experiments, analyzed the data and wrote the manuscript; Zipei Li, Ying Wang and Xiaoxiao Qi assisted in animal experiments; Rong Zhang provided technical support and review the manuscript; Zhongqiu Liu and Yuanyuan Cheng supervised the project, and edited the manuscript. All authors read and approved the final manuscript. The authors declare no conflict of interest. We would like to thank the Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine for providing the platform. This work was supported by the National Natural Science Foundation of China (Nos. 82074053, 81930114, 82374070 and U22A20368), the Natural Science Foundation of Guangdong Province (No. 2019A1515010211), Key-Area Research and Development Program of Guangdong Province (No. 2020B1111100004) and Guangzhou Association for Science and Technology Youth Talent Support Program (No. QT-2023-011). The animal expriments were approved by the Guangzhou University of Chinese Medicine Animal Care and Use Committee and the approved number is 20221035 and IITCM-20190149. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Source: Clinical and Translational Medicine

DOI: 10.1002/ctm2.1616