TGFB2-AS1 binding to MED1 promotes doxorubicin-induced cardiomyocyte apoptosis via BMP7 pathway

doi:10.21203/rs.3.rs-4616701/v1

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TGFB2-AS1 binding to MED1 promotes doxorubicin-induced cardiomyocyte apoptosis via BMP7 pathway

https://doi.org/10.21203/rs.3.rs-4616701/v1

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Doxorubicin-induced cardiotoxicity (DIC) is similar to dilated cardiomyopathy (DCM) in morphological and functional defects, eventually progressing to heart failure. Recently, intensive investigation showed that specific expression profiles of lncRNA have been closely related to cardiovascular disease, but many gaps remain, including the emerging roles of lncRNA in DIC. We identified TGFB2-AS1 as a highly conserved regulator of DCM by reanalyzing publicly available RNA sequencing datasets from GEO and producing conservation scores of lncRNAs using PHAST software. TGFB2-AS1 expression is dramatically increased in murine and cell models, and TGFB2-AS1 has a pro-apoptotic effect in vitro. Moreover, TGFB2-AS1 mediated apoptosis via the BMP7 pathway by activating the Smad1/5/9 phosphorylation to upregulate the target gene expression Id2. Recombinant human bone morphogenetic protein (rhBMP-7) aggravates doxorubicin-induced cardiomyocyte apoptosis, and knockdown of BMP7 significantly reverses the pro-apoptotic effect of TGFB2-AS1 overexpression in vitro. Mechanistically, we found that TGFB2-AS1 combines with transcriptional co-activator MED1, promoting H3K27 acetylation modification level in the promoter of the BMP7 gene and then facilitating BMP7 transcription. Collectively, this study illuminates that TGFB2-AS1 is an evolutionarily conserved long noncoding RNA with a previously unappreciated role in promoting the apoptotic phenotype of DIC and sheds light on the more effective clinical application of doxorubicin.

Doxorubicin

TGFB2-AS1

Apoptosis

BMP7

MED1

Doxorubicin (DOX) is an anti-cancer anthracycline that can be used to treat diverse kinds of cancer, containing acute lymphocytic and myelogenous leukemia, Hodgkin’s and non-Hodgkin’s lymphomas, bladder, gastric and breast cancers and so on, but its clinical use is a double-edged sword and one of the most severe side effects in clinical application is doxorubicin-induced cardiotoxicity (DIC), which eventually contributes to heart failure with dilated cardiomyopathy [1–3]. Therefore, ascertaining key molecules and elucidating the mechanism of DIC are of great worth for the clinical use of doxorubicin.

Long noncoding RNA (lncRNA) is generally defined as a sort of transcript larger than 200 nucleotides and cannot code protein[4]. Nuclear lncRNA modulates nuclear processes like chromatin organization and RNA transcription and splicing, and cytoplasmic lncRNA regulates mRNA transport, stability, translation, protein stability, post-translational modification, and function[5, 6]. Specific expression profiles of lncRNA have been closely related to cardiovascular disease, and dysregulation of lncRNA participates in the regulation of physiological and pathological processes, including cardiac pathological hypertrophy, cell differentiation, apoptosis, and fibrosis[7–10]. However, lncRNA's emerging and pivotal roles in DIC remain elusive and deserve further in-depth studies.

The mechanisms of DIC are still perplexing, and most studies support the view that cardiomyocyte apoptosis is bound up with DIC [11] [12]. Besides, cardiomyocyte apoptosis is a critical point in the transition between compensatory cardiac hypertrophy and heart failure[13]. Bone morphogenetic proteins (BMPs), a subfamily of the transforming growth factor (TGF)-β family, acting on a tetrameric receptor complex, transduce signals to the canonical Smad-dependent and non-canonical Smad-independent signaling pathways to regulate bone formation, bone homeostasis, cell proliferation, apoptosis, and vascular homeostasis[14, 15]. However, few studies have explored the regulatory relationship between lncRNA and BMP signaling in DIC.

In this study, we confirmed the increased expression of transforming growth factor beta 2 antisense RNA 1 (TGFB2-AS1) in heart failure patients with dilated cardiomyopathy using bioinformatics analysis and discovered that highly conserved TGFB2-AS1 is upregulated in various models. We also found that TGFB2-AS1 is critical for doxorubicin-induced cell apoptosis in vitro by directly binding to MED1, potentiating acetylation of H3K27, and then activating the BMP7 pathway. Taken together, these findings suggest that TGFB2-AS1 is a crucial regulator of DIC and represents a potential therapeutic target for treating DIC.

Establishment of mouse DIC model

Eight-week-old male C57BL/6 mice (SPF Biotechnology Co., Ltd) were divided into a doxorubicin-induced group and a control group. Doxorubicin-induced mice were performed intraperitoneal injection with 5 mg/kg doxorubicin dose of each one once a week for four weeks[16]. Cardiac ultrasonography monitored cardiac function, and the mice were anesthetized to death with sodium pentobarbital on the sixth weekend. After being isolated, washed, and dried, the hearts were used for pathology experiments and RNA and protein extraction, respectively. The Ethics Committee of Jinshan Hospital, Fudan University approved animal ethics.

Ultrasonography of the mouse heart

The mice were depilated in the anterior thoracic region with depilatory cream before the examination. Cardiac ultrasonography was performed using a VEVO LAZR multimodal small animal ultrasound/photo-acoustic imaging system from Fujifilm VisualSonics, Inc., and mice were anesthetized with 4% isoflurane. Ultrasound pictures were acquired in parasternal short-axis view and parasternal long-axis view when the heart rate of mice stabilized to about 400 beats/min with 1%-2% continuous isoflurane anesthesia inhalation. Relevant indexes of overall left ventricular systolic function, including ejection fraction (EF), fraction shortening (FS), left ventricular end-diastolic diameter (LVEDD), and left ventricular end-systolic diameter (LVESD), were measured with ultrasound analysis software.

Flow cytometry

After 24 hours of doxorubicin induction of cardiomyocytes, cells were collected, and the cell sediments were retained by centrifugation after cold PBS washing twice. The cells were resuspended with 500 µL of 1×Binding Buffer, 5 µL of Annexin V-FITC (BD Bioscience, USA), and 2.5 µL of DAPI (Sangon biotech, China), and were gently blown to mix well, and incubated for 15–20 minutes at room temperature in the dark. Unstained cells with doxorubicin treatment, unstained cells without doxorubicin treatment, cells stained with Annexin V-FITC alone (no DAPI), cells stained with DAPI alone (no Annexin V-FITC) were used to set up compensation and quadrants. The results were detected by CytoFLEX flow cytometry within 1 hour (Beckman Coulter, USA) and analyzed with FlowJo (Version 10).

RNA extraction and qRT-PCR

Total RNA was extracted according to the instructions of TRIzol (Invitrogen, America), and qRT-PCR was performed according to the SYBR Green PCR kit (Yeasen, China). GAPDH was used as an internal reference for normalizing the results, and the primer sequences are given in Table S1.

Protein extraction and western blotting

The samples were lysed with RIPA lysate containing phosphatase inhibitor (Beyotime Biotechnology, China), and the supernatants acquired by centrifugation and ultrasound of lysates and the added loading buffer were then boiled for the following Western blotting. Antibodies and concentrations used in this experiment: Bax (Proteintech, USA, 1:2500); BCL-2 (ABclonal, China, 1:2500); Caspase3 (Abmart, China, 1:1000); GAPDH (Abmart, China, 1:5000); β-ACTIN (Abmart, China, 1:5000); BMP7 (Proteintech, USA, 1:500); SMAD1 (ABclonal, China, 1:2500); P-SMAD1/5/8 (Cell Signaling Technology, USA, 1:2500); ID2 (ABclonal, China, 1:1000); ID1 (ABclonal, China,1:1000); Mouse secondary antibody (Proteintech, USA, 1:5000); Rabbit secondary antibody (Abmart, China,1:5000).

Immunofluorescence and cytoskeleton staining

The cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature and then were penetrated with 0.1% TritonX-100 for 10 minutes. Block the cells with QuickBlock™ Blocking Buffer (Beyotime Biotechnology, China) at room temperature for 30min-60min before antibody incubation. Incubate the cells overnight at 4°C with the primary antibody, BMP7 (Proteintech, USA, 1:100), followed by the incubation of the secondary antibody, CoraLite488-conjugated Goat Anti-Rabbit lgG(H + L) (Proteintech, USA, 1:500) at room temperature for 2 hours in the dark. Observe and take pictures under the fluorescence microscope (Olympus, Japan).

Cytoskeleton staining

After fixation and permeabilization of the cells, the cells were incubated with FITC-phalloidin (Yeasen, China) for 30 min at room temperature, washed three times with PBS, and then photographed with the fluorescence microscope (Olympus, Japan) in high magnification versus low magnification fields in a light-avoiding environment.

Cell culture and cell transfection

The mouse myocardial-originated HL-1 cells and the human myocardial-originated AC16 cells were cultured in Dulbecco's Modified Eagle Medium with High Glucose (DMEM-HG), which was enriched with 10% fetal bovine serum (FBS, Gibco, USA). Doxorubicin (MedChemExpress, China) with a concentration of 0.5µM was used to induce apoptosis, and rhBMP7 (MedChemExpress, China) with 100 ng/ml concentration was used to study the effect on cell apoptosis. For overexpression analyses, AC16 and HL-1 cells were transfected for 48 h with Lipofectamine 3000 according to the manufacturer’s instructions (Thermo Fisher Scientific, USA) with the GV712-TGFB2-AS1 plasmid (Genechem, China) and GV712-Gm29491 plasmid (Genechem, China) respectively. SiRNA-mediated TGFB2-AS1 and BMP7 gene silencing was performed by transfecting AC16 cells with human TGFB2-AS1 siRNA and human BMP7 siRNA (Sangon biotech, China)), using scrambled siRNA as a control. The BMP7 siRNA and TGFB2-AS1 siRNA sequences are shown in Table S2.

RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (CHIP)

The RNA immunoprecipitation experiment was performed according to the instructions of the reagent RNA Immunoprecipitation Kit (Bersinbio, China). After immunoprecipitation using MED1 antibody (Active Motif, USA, 1:1000) and IgG, RNA was extracted with Trizol and then subjected to reverse transcription and qRT-PCR. After cross-linking the cells using formaldehyde and glycine, chromatin immunoprecipitation used H3K27ac antibody (Active Motif, USA, 1:2500) and IgG following the procedure of Chromatin Immunoprecipitation (ChIP) Kit (Bersinbio, China), and eventually enrichment efficiency was verified by qPCR and agarose gel electrophoresis.

RNA nucleoplasmic separation test and RNA fluorescence in situ hybridization (RNA FISH)

qRT-PCR experiments were performed after nucleoplasmic separation using an RNA nucleoplasmic separation kit (Norgen Biotek, Canada). Follow the instructions of the RNA Fluorescence In Situ Hybridisation Kit (Genepharma, China) to perform the experiments and analyze the fluorescence position and intensity by Image J after observing the samples with a fluorescence microscope. The sequences of the TGFB2-AS1 probe, U6 probe, and 18S probe are given in Table S3.

Bioinformatics analysis

Two eligible datasets were acquired from the GEO database (https://www.ncbi.nlm.nih.gov/geo/) by retrieving keywords "heart failure" and " doxorubicin", and two high-throughput sequencing datasets of myocardial tissues from normal subjects and heart failure patients with dilated cardiomyopathy were extracted. Dataset GSE141910 was used as the training set, with 166 cases of heart failure and 166 normal cases. The two sets of gene expression data for different races were analyzed using the limma package in RStudio to identify genes with differential expression, and differentially expressed genes were filtered out using the thresholds of Pvalue < 0.05 and |logFC| ≥ 1.5. Volcano plots and heatmaps were created using the ggplot and pheatmap packages, respectively. The key lncRNAs were identified by finding the intersection of the results from the two analyses and visualized using Venn diagrams. The dataset GSE116250 (heart failure n = 39, Normal n = 13) was used as a validation set. Aspera Connect software was used to download the SRA raw data in the Linux system. FastQC was utilized to analyze the reads for quality, and Trimmomatic software was employed to filter them. HiSat2 program was used to compare the reads to the reference genome, and Hiseq software was used to compare and quantify the results. Subsequently, the results were annotated in RStudio, and the counts were normalized to RPKM, FPKM, or TPM. The LncRNA expression matrices were filtered, and the key lncRNAs obtained from the intersections were validated in the LncRNA expression matrices of the GSE116250 dataset.

Statistical analysis

Each experiment was repeated three times independently, and data analysis and statistical chart drawing were done using GraphPad Prism 9. The results were expressed as Mean ± SEM. Comparisons between groups were made using independent samples t-test, and comparisons between multiple groups were made using one-way ANOVA, with p < 0.05 indicating a statistical difference.

TGFB2-AS1 was a conserved lncRNA and upregulated in DCM/HF Patients

To identify key lncRNA linked to HF patients with dilated cardiomyopathy, we reanalyze 2 high-throughput sequencing datasets. The expression profile of LncRNA in datasets GSE141910 was divided into two parts according to ethnicity because of principal component analysis (PCA) showing chaotic cluster (Fig.S1), and then we performed analysis respectively. We filtered out 30 differentially expressed Caucasian-specific lncRNA expression profiles, and 29 differentially expressed lncRNAs in African American-specific lncRNA expression profile lncRNAs with cut-off criteria (|logFC|≥1.5) between DCM/HF patients and controls (Fig. 1A,1B, Table S3 and S4). There are 22 up-expressed and 8 downregulated lncRNAs involved in 30 differentially expressed lncRNAs (Fig. 1C), and 20 upregulated and 9 downregulated genes involved in 29 differentially expressed lncRNAs (Fig. 1D). Next, 19 overlapped lncRNAs were used as hub genes discovered by the intersection of 30 and 29 differentially expressed lncRNAs (Fig. 1E).In dataset GSE116250, we verified the expression of 19 lncRNAs in DCM/HF patients, and the four lncRNAs, including TGFB2-AS1, CEROX1, CCDC144NL-AS1, DNAAF3-AS1, significantly increased(Fig. 1F, bottom). While lncRNAs tend to be less conserved than protein-coding genes, and most do not exhibit marked sequence conservation, a subset of conserved lncRNAs does exist[17]. A computer program called phastCons was based on a two-state phylogenetic hidden Markov model (phylo-HMM), and phastCons scores were applied to identify conserved elements [18]. Therefore, we measured the level of conservation of the best 200-bp window of the 19 lncRNAs, and TGFB2-AS1 exhibited the second-highest degree of base-wise conservation (Fig. 1F, top). Therefore, we focused on the potential roles of TGFB2-AS1 in DCM in the following work.

Doxorubicin-induced cardiotoxicity in vitro and in vivo model

DIC mice model was induced by intraperitoneal injection of doxorubicin once a week for four weeks, and echocardiography was applied to monitor the cardiac function at the end of the sixth week (Fig. 2A). The obtained data show that the ejection fraction (EF) and fraction shortening (FS) of mice in the Dox group were both significantly reduced, left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) was both enlarged in the hearts of mice in the Dox group as compared with the control group (Fig. 2C). Histological analysis demonstrated that doxorubicin could induce disruption of cardiomyocytes, infiltration of inflammatory cells, and increase myocardial fibrosis (Fig. 2B). Cardiac hypertrophy in the Dox group was verified by enlarged cardiomyocytes through observing WGA and H&E staining and the increased mRNA levels of cardiac hypertrophic genes ANP, BNP, and MYH7 (Fig. 2B,2D). The results of TUNEL staining showed that TUNEL-positive CM was increased in mice that received doxorubicin injection compared with controls, revealing DOX-induced myocardial apoptosis (Fig. 2E). Therefore, DIC is characterized pathologically by myocardial hypertrophy, fibrosis, apoptosis and remodeling, and cardiac dysfunction and heart failure in mice with doxorubicin injection. In conclusion, morphological and functional defects of the heart in doxorubicin-induced cardiotoxicity are similar to those of DCM[19, 20]. Meanwhile, according to the previous study[21], the HL-1 and AC16 cells were respectively administrated with Dox (0.5µmol/L) for 24 h. As shown in Fig. 2F-2H, we found that doxorubicin administration promoted the enlargement of cardiac cells and enhanced the mRNA levels of cardiac hypertrophic genes. Flow cytometry using Annexin V/DAPI double staining was carried out to measure doxorubicin-induced cell apoptosis in vitro[22, 23], and the assay results uncovered that the number of apoptotic cells in the Dox group was significantly higher than that of the control group (Fig. 2I,2J). The expression of apoptosis-associated proteins (including Bax, Bcl-2, and Cleaved Caspase-3) in HL-1 and AC16 cells were detected by Western blotting(Fig. 2K,2L) and the results of Western blot revealed that doxorubicin treatment led to upregulation of Bax and cleaved-caspase 3 as well as the downregulation of Bcl-2 and the ratio of Bcl-2 and Bax (Fig. 2M,2N).

TGFB2-AS1 was upregulated in hearts and cardiomyocytes upon doxorubicin stimulation

By searching the UCSC Genome Browser, we identified that the mouse Tgfb2 locus encompasses a non-coding RNA, Gm29491, and the position of Gm29491 in the genome is shown in Fig. 3A. Mouse Gm29491 is located at the positive strand of chromosome 1:186,437,892 − 186,439,230. Its corresponding transcript is ENSMUST00000189481, which has a length of 506 bp. Homo sapiens TGFB2 antisense RNA 1 (head to head) (TGFB2-AS1) is located at the negative strand of chromosome 1:218,344,195–218,346,036 with a genomic length of 1,842 nt, and its corresponding transcript is ENST00000689961 with a length of 557 bp, located in the antisense strand of TGFB2. The conservation of human TGFB2-AS1 extends to exons 1,2 and 3, which are 3-exon homologs in mouse Gm29491 (Fig. 3B), and more details about the conserved position, and conservation scores (PhastCons and PhyloP) visualized by UCSC Genome Browser (Supplemental Fig. 2C,2D). The results of alignment identified by MAFFT software between the mouse Gm29491 and the human TGFB2-AS1 were shown in Supplemental Fig. 2A. The RNA secondary structure of the human TGFB2-AS1 and mouse Gm29491 was predicted by RNA fold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) and visualized by Forna (http://rna.tbi.univie.ac.at/forna/) (Supplemental Fig. 2B). The coding potential of TGFB2-AS1 was analyzed by bioinformatics tools[24]. The low coding potential scores calculated by coding potential calculator2(CPC2) and coding potential assessment tool (CPAT) indicated that human TGFB2-AS1 and mouse Gm29491 both have low protein-coding capacity (Fig. 3C). To investigate the nucleoplasmic expression of TGFB2-AS1, we carried out cell fractionation experiments to extract nuclear and cytoplasmic RNA from AC16 cells. The results demonstrated that TGFB2-AS1 was mainly distributed in the nucleus (73%) (Fig. 3D). Afterward, our observation shows that Cy3 marking TGFB2-AS1 probe (red fluorescence) was primarily located in the nucleus using RNA FISH (Fig. 3E), manifesting that TGFB2-AS1 may primarily regulate chromatin organization or RNA transcription in the nucleus. Most importantly, we assessed the expression levels of TGFB2-AS1 in mice, AC16, and HL-1 cells with doxorubicin induction. Quantitative RT-PCR analysis found that in the doxorubicin induction group, TGFB2-AS1 expression was remarkably elevated compared to the control group (Fig. 3F,3G, and 3H). These results showed that TGFB2-AS1 may play a pivotal role in DIC.

TGFB2-AS1 modulated the doxorubicin-induced cellular hypertrophy and apoptosis in vitro

To determine the functional role of TGFB2-AS1, we evaluated the effect of TGFB2-AS1 loss- and gain-of-function on cardiomyocytes with doxorubicin stimulation. First of all, plasmid DNA was used to transfect cardiomyocyte to overexpress TGFB2-AS1 or Gm29491, and Quantitative RT-PCR confirmed the successful overexpression of TGFB2-AS1 and Gm29491(Fig. 4A,4B). We designed 3 siRNAs specific-targeting TGFB2-AS1 to silence the expression of TGFB2-AS1 in AC16 cells and the third of three siRNAs (siRNA 3), which decreased 71% of the expression of TGFB2-AS1 compared with the negative control, was used in the following work (Fig. 4C). The overexpression of TGFB2-AS1 in AC16 cells promoted doxorubicin-induced cellular hypertrophy; however, the silencing of TGFB2-AS1 reversed cellular hypertrophy (Fig. 4D,4E). Compared with the Dox–treated NC group, TGFB2-AS1 overexpression followed by doxorubicin stimulation for 24 hours increased the apoptosis rate, and the knockdown of TGFB2-AS1 resulted in a partial reduction in apoptosis rate (Fig. 4F-4I). As expected, the same proapoptotic effects of TGFB2-AS1 overexpression were observed by western blot in HL-1 and AC16 cells with doxorubicin stimulation (Fig. 5A,5C). In contrast to the Dox–treated NC group, TGFB2-AS1 overexpression with doxorubicin stimulation increased Bax and cleaved-caspase 3 expression and decreased the ratio of Bcl-2 and Bax in AC16 cells, while Bcl-2 protein abundance did not differ significantly between two groups (Fig. 5B). Gm29491 overexpression with doxorubicin exposure exerted higher expression levels of Bax and cleaved-caspase 3, lower expression levels of Bcl-2, and non-differential ratio of Bcl-2 and Bax in HL-1 cells as opposed to the Dox–treated NC group (Fig. 5D). The expression of apoptosis-associated proteins in the Dox-treated NC group was reversed by the downregulation of TGFB2-AS1 (Fig. 5E,6F). These data provide in vitro evidence that TGFB2-AS1 overexpression in the HL-1 and AC16 cells can result in the promotion of cellular hypertrophy and apoptosis.

TGFB2-AS1 activated BMP7 signaling pathway

To further explore the potential function of TGFB2-AS1, we explore the regulatory possibility of TGFB2-AS1 of downstream signaling pathways by conducting a gene set enrichment analysis (GSEA). Some significantly activated pathways, including TGF-β signaling pathways, apoptosis, Wnt signaling pathways, p53 signaling pathways, mitophagy, oxidative phosphorylation, BMP signaling pathway, and ferroptosis, were noteworthy (Fig. 6A). We focused on the BMP signaling pathway among these pathways, and the enrichment scores of the genes of the BMP signaling pathway gene set are shown in Fig. 6B. Based on the cis-regulatory mechanism of action of lncRNAs, we investigated the possibility of TGFB2-AS1 directly regulating TGFB2 transcription. Neither TGFB2-AS1 overexpression nor TGFB2-AS1 inhibition changed the expression of TGFB2 at mRNA levels (Supplemental Fig. 3A,3B). However, immunofluorescence and western blot indicated that the protein level of BMP7 was elevated with doxorubicin treatment (Fig. 6C-6E). Besides, BMP receptor-phosphorylated Smad1/5/8 levels, BMP-7 triggered Id2 level and phosphorylated Smad1/5/8 to Smad1 ratio was enhanced upon overexpressing TGFB2-AS1 (Fig. 6D, 6F-6I). These results prove that TGFB2-AS1 activates the BMP7 signaling pathway. To Fig. out the effect of BMP-7 on doxorubicin-induced cell apoptosis, AC16 cells were treated with recombinant BMP-7. As expected, doxorubicin-induced cell apoptosis was significantly enhanced in the presence of BMP-7 by flow cytometric analysis (Fig. 6J-6K). Furthermore, rhBMP-7 significantly increased cleaved-caspase 3 protein level while decreasing Bcl-2 protein level and Bcl-2 to Bax ratio, and Bax levels did not show appreciable changes between Dox-treated group and Dox-treated with BMP-7 group, suggesting that BMP-7 might mainly function by promoting doxorubicin-induced cell apoptosis (Fig. 6L-6M).

TGFB2-AS1 mediated cardiomyocyte apoptosis through BMP7/ID2 pathway

Rescue experiments were performed to confirm that BMP-7 and the BM7 pathway were the mediators of TGFB2-AS1 mediated promotion of doxorubicin-induced cell apoptosis. Flow cytometric analysis indicated that the apoptotic rate decreased in doxorubicin-treated cardiomyocytes with knockdown of BMP-7 and overexpression of TGFB2-AS1 (Fig. 7A,7B). Similarly, the protein level of Bax, cleaved-caspase 3, phosphorylated smad1/5/8, and Id2 decreased in cells transfected with siBMP-7 and OE-TGFB2-AS1 while the protein level of Bcl-2 increased (Fig. 7D, 7E,7H). In conclusion, TGFB2-AS1 mediated promotion of doxorubicin-induced cell apoptosis and activation of the BMP7 pathway was partly abolished with BMP-7 knockdown. Oppositely, the rhBMP7 treatment in TGFB2-AS1 downregulation cells exerted a higher apoptotic rate (Fig. 7A,7C), in comparison to the TGFB2-AS1 downregulation doxorubicin-treated group. The influence on cardiomyocytes with rhBMP7 and TGFB2-AS1 silencing was that the protein level of Bax cleaved-caspase 3, phosphorylated smad1/5/8 and Id2 elevated, while the protein level of Bcl-2 reduces (Fig. 7F,7G,7I).

TGFB2-AS1 bonded to MED1 and activated H3K27ac to promote BMP7 transcription

In the previous study, RNA pull-down assay combined with unbiased mass spectrometry analysis of proteins showed that one of the top-scoring proteins showing specific interaction with TGFB2-AS1 was mediator complex subunit 1(MED1), which is a subunit of the mediator complex essential for the pre-initiation complex of transcription[25, 26]. Therefore, we performed RNA-binding protein immunoprecipitation (RIP) followed by qRT-PCR to detect the endogenous association between TGFB2-AS1 and MED1. MED1-specific endogenous immunoprecipitation significantly enriched for TGFB2-AS1 compared with control immunoglobulin confirmed a direct interaction between TGFB2-AS1 and MED1 in AC16 cells (Fig. 8A,8B). Furthermore, H3K27ac- and MED1-specific ChIP-qPCR analysis showed cells overexpressing TGFB2-AS1 acquired much higher H3K27ac and MED1 levels to the BMP7 promoter (Fig. 8D–8F). Our findings supported the notion that TGFB2-AS1 enhances the recruitment of MED1 and promotes the acetylation of H3K27 on BMP7.

The current study provides insight into the proapoptotic effect, and mechanistic action of a novel, highly conserved lncRNA, TGFB2-AS1, in DIC. Evidence is provided that TGFB2-AS1 upregulation promotes doxorubicin-induced cell apoptosis and hypertrophy, and TGFB2-AS1 downregulation partly reverses these effects. Our study not only elucidates a previously unknown function of BMP7 governing doxorubicin-induced cell apoptosis but also uncovers TGFB2-AS1 activating the BMP7 pathway to potentiate its proapoptotic role.

Recent studies found the role of long non-coding RNA in doxorubicin-induced cardiomyopathy, lncRNA NONMMUT015745 inhibits doxorubicin-mediated cardiomyocytes apoptosis by regulating the Rab2A-p53 axis [27], METTL14 silencing suppresses the ferroptosis of cardiomyocyte in doxorubicin-induced mice[28].TGFB2-AS1 has been reported in breast cancer, glioma, lung adenocarcinoma, gastric cancer, and other tumor diseases, and more importantly, the limited molecular biological role of TGFB2-AS1 in some diseases has been illustrated [29]. Studies have shown that mutations in the TGFBR1 and TGFB2-AS1 genes were associated with myopia in preschoolers, mediated by the TGF-β signaling pathway[30]; loss of TGFB2-AS1 significantly promoted Apoptosis of HepG2 liver cancer cells and inhibited migration and invasion[31], and TGFB2-AS1 interacted with the Core subunit of SWI/SNF complex, SMARCA4 to inhibit the expression of target genes TGFB2 and SOX2, ultimately inhibiting the breast Cancer progression[32]. Evolutionary conservation is widely used as an indicator of the functional significance of newly discovered lncRNAs and the evolutionary conservation of lncRNAs is multidimensional including Sequence, structure, function, and syntenic expression, and different from the conserved analysis of coding gene proteins [33]. In our study, by bioinformatics analysis and biological validations, we verified that TGFB2-AS1 was highly conserved between mice and humans, and the expression of TGFB2-AS1 increased in the mice, human myocardial cell lines and mouse myocardial cell lines treated with doxorubicin. In addition, we found that silencing of TGFB2-AS1 improved doxorubicin induction of cardiomyocyte hypertrophy and apoptosis.

We lucubrate the mechanism of the proapoptotic effect of TGFB2-AS1 in DIC. GSEA revealed that TGFB2-AS1 is associated with TGF-β/BMP signaling. we found that that TGFB2-AS1 did not influence TGFB2 expression which was inconsistent with Cixiang Zhou’s research[32], although TGFB2-AS1 is located on the strand opposite that of the coding gene, TGFB2. Interestingly, our further studies have demonstrated that TGFB2-AS1 could upregulate BMP-7 expression in cardiomyocytes treated with doxorubicin. BMP-7, a secretory protein, mediates the canonical signal transduction of the BMP family by phosphorylating Smad1/5/8, and BMP signal transduction targeting a class gene is the Id (Inhibitors of DNA binding/Differentiation) family, which consists of four members, Id1 to Id4[34–36]. BMP7 exerted a different role in cell death, especially apoptosis. BMP7 could strongly inhibit serum starvation-induced apoptosis in C4-2B cell line; while BMP-7 induced apoptosis in human myeloma cells [37, 38]. We first showed that BMP7 protein levels were significantly upregulated in TGFB2-AS1 overexpression cells treated with doxorubicin, and rhBMP7 treatment enhanced doxorubicin-induced apoptosis. However, as the targeted gene of the BMP7, the proapoptotic effect of the Id protein has been proven in many studies[39–41]. In line with these findings, TGFB2-AS1 functions with a proapoptotic effect by activating BMP7 signal transduction, ultimately triggering the downstream effector molecules, Id2.

Emerging studies demonstrate that lncRNAs can regulate epigenetic modifications in the nucleus by regulating histone modifications, mainly methylation and acetylation, thereby promoting or inhibiting the transcription of genes at the transcriptional level, and the lncRNAs work either by regulating histone methylation or acetylation alone or by acting as scaffolding for interactions with enzymes or regulatory complexes[42]. At present, the acetylation sites of histones are mainly H3K4ac, H3K9ac, H3K27ac, H3K56ac, and H4K16ac and acetylation is generally associated with the activation or opening of chromatin[43, 44]. RNA transcription enhancement is marked by histone modifications such as H3K4m1/2 and H3K27ac and is rich in transcriptional regulators such as LDTF, P300, CBP, BRD4, and MED1[45]. MED1, the mediator complex subunit 1, bridges transcription factors or nuclear receptors that bind to promoter enhancer elements through RNA polymerase II and general transcription mechanisms; therefore, it acts as a co-activator to increase the target transcription of genes[46].Heart-specific loss of MED1 leads to dilated cardiomyopathy, and decreased heart function, in addition, MED1 deletion also disrupts cardiac mitochondrial and metabolic gene expression patterns[47].MED1 is an important epigenetic regulator of TGF-β/BMP signal transduction in pulmonary hypertension [26]. In a recent study, MED1 was confirmed to bind with TGFB2-AS1 by RNA pull-down combined with mass spectrometry[25]. Consistent with the indication of RNA transcription enhancement, in our works, it was worth noting that TGFB2-AS1 overexpression led to an increase in MED1 binding and H3K27 acetylation on the BMP7 promoter, which means TGFB2-AS1 overexpression resulted in enhanced BMP7 transcription.

There are several limitations to the present works. First, whether the expression of TGFB2-AS1 is regulated by the BMP7 signaling pathway in AC16 and HL-1 cells or not is still elusive, and the existence of negative or positive feedback between TGFB2-AS1 and BMP7 signaling pathway deserves further attention and further study. Papoutsoglou et al. show that TGFB2-AS, which can be induced by TGF-β through Smad and protein kinase pathways, in return, exerts inhibitory functions on TGF-β/BMP signaling output by interacting with the EED adaptor of the Polycomb repressor complex 2 in Human HaCaT keratinocytes [25]. Besides, given the robust activation of TGFB2-AS1 gene expression in response to doxorubicin treatment, some cis-elements may be responsible for TGFB2-AS1 transcription. Second, although overexpression of TGFB2-AS1 promoted doxorubicin-induced cell apoptosis and hypertrophy, only the mechanism underlying the doxorubicin-induced cell apoptosis had been demonstrated clearly. In future studies, it is necessary to clarify the regulatory mechanism between TGFB2-AS1 and doxorubicin-induced cell hypertrophy. Third, we pored over whether the effect of TGFB2-AS1 on cell apoptosis was attributable to its impact on several nearby gene expressions because lncRNAs have been reported to exert cis-regulatory effects on neighboring genes[48]. We also detected the expression of nearby genes, including RRP15, LYPLAL1, SPATA17, and GPATCH2, with a distance within 50kb from TGFB2-AS1. Only GPATCH2 expression approximately decreased 70% after TGFB2-AS1 knocking-down(Supplemental Fig. 3D). However, we didn’t lucubrate the role of GPATCH2 in cell apoptosis. Fourth, the functional and mechanical study of TGFB2-AS1 was in AC16 and HL-1 cells rather than the animal model. SiRNA-mediated TGFB2-AS1 knockdown may have potential off-target effects, and especially we designed 5 oligonucleotides targeted Gm29491, which were confirmed an invalid knockdown of Gm29491 in HL-1 cells (Fig.S3C). Other powerful knocking-down methods, including ASO and CRISPR/CAS9 approaches, can serve to downregulate TGFB2-AS1 in AC16 and HL-1 cells. Furthermore, using CM-specific TGFB2-AS1-KO mice can elucidate the function of TGFB2-AS1 in cardiac dysfunction and myocardial injury. Fifth, our study only makes a point of doxorubicin-induced cardiomyopathy, although acknowledged causes of DCM were compounded of heritable genetic mutation, alcohol excess, chemotherapy drugs, and pregnancy[49]. Thus, different models of DCM, such as MLP (muscle LIM protein)-deficient mice,cΔex3 mice, and chronic myocarditis mice model [50–52], are required to validate whether the function of TGFB2-AS1 is specific in DCM or not.

In conclusion, our work reveals that a novel identified lncRNA TGFB2-AS1 which is highly evolutionarily conserved enhances cell apoptosis and hypertrophy induced by doxorubicin and the lncRNA TGFB2-AS1 mediates cardiomyocyte apoptosis via mechanisms involving activating BMP7 transcription and BMP7 pathway signal transduction. The lncRNA TGFB2-AS1 might serve as a target for the treatment of DIC and shed light on the more effective clinical application of doxorubicin.

Data availability

The RNA sequencing datasets used in this study can be accessed at NCBI-GEO: GSE141910 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141910),and GSE116250(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE116250).

Acknowledgments

Not applicable.

Funding

This work was supported by grants from the Youth Scientific Research Project of Jinshan Hospital of Fudan University (Grant Number: JYQN-LC-202204).

Contributions

HL G designed this study, carried out the whole experiment, analyzed the data, and drafted the manuscript. K L and JX L assisted in drawing figures and diagrams. K L participated in statistical analysis. F P and K L checked the manuscript in detail, and XY L and H G provided suggestions and instructions throughout the research. All the authors read and revised the ultimate manuscript.

Ethics declarations

Ethics committee approval

All animal experiments in this study were approved by the Ethics Committee of Jinshan Hospital, Fudan University and were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Competing interests

The authors declare no competing interests related to this work.

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TGFB2-AS1 binding to MED1 promotes doxorubicin-induced cardiomyocyte apoptosis via BMP7 pathway

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Abstract

Figures

Introduction

Materials and methods

Establishment of mouse DIC model

Ultrasonography of the mouse heart

Flow cytometry

RNA extraction and qRT-PCR

Protein extraction and western blotting

Immunofluorescence and cytoskeleton staining

Cytoskeleton staining

Cell culture and cell transfection

RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (CHIP)

RNA nucleoplasmic separation test and RNA fluorescence in situ hybridization (RNA FISH)

Bioinformatics analysis

Statistical analysis

Results

TGFB2-AS1 was a conserved lncRNA and upregulated in DCM/HF Patients

Doxorubicin-induced cardiotoxicity in vitro and in vivo model

TGFB2-AS1 was upregulated in hearts and cardiomyocytes upon doxorubicin stimulation

TGFB2-AS1 modulated the doxorubicin-induced cellular hypertrophy and apoptosis in vitro

TGFB2-AS1 activated BMP7 signaling pathway

TGFB2-AS1 mediated cardiomyocyte apoptosis through BMP7/ID2 pathway

TGFB2-AS1 bonded to MED1 and activated H3K27ac to promote BMP7 transcription

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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