The TBX1/miR-193a-3p/TGF-β2 Axis Mediates CHD by Promoting Ferroptosis

Congenital heart disease (CHD) is the most common noninfectious cause of death during the neonatal stage. T-box transcription factor 1 (TBX1) is the main genetic determinant of 22q11.2 deletion syndrome (22q11.2DS), which is a common cause of CHD. Moreover, ferroptosis is a newly discovered kind of programmed cell death. In this study, the interaction among TBX1, miR-193a-3p, and TGF-β2 was tested using quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and dual-luciferase reporter assays. TBX1 silencing was found to promote TGF-β2 messenger ribonucleic acid (mRNA) and protein expression by downregulating the miR-193a-3p levels in H9c2 cells. In addition, the TBX1/miR-193a-3p/TGF-β2 axis was found to promote ferroptosis based on assessments of lipid reactive oxygen species (ROS) levels, Fe2+ concentrations, mitochondrial ROS levels, and malondialdehyde (MDA) contents; Cell Counting Kit-8 (CCK-8) assays and transmission electron microscopy; and Western blotting analysis of glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), NADPH oxidase 4 (NOX4), and acyl-CoA synthase long-chain family member 4 (ACSL4) protein expression. The protein expression of NRF2, GPX4, HO-1, NOX4, and ACSL4 and the level of MDA in human CHD specimens were also detected. In addition, TBX1 and miR-193a-3p expression was significantly downregulated and TGF-β2 levels were high in human embryonic CHD tissues, as indicated by the H9c2 cell experiments. In summary, the TBX1/miR-193a-3p/TGF-β2 axis mediates CHD by inducing ferroptosis in cardiomyocytes. TGF-β2 may be a target gene for CHD diagnosis and treatment in children.


Introduction
Congenital heart disease (CHD), a congenital malformation caused by abnormal growth of the heart and great vessels during the fetal period, is the main noninfectious cause of death during the neonatal stage. Currently, the average global prevalence of CHD is 9.410‰. In China, the prevalence of CHD has reached 4.905‰ [1,2]. For every 1000 live births, 8 infants suffer from CHD. A Norwegian study showed that the survival rate of patients with complex coronary heart disease by the age of 16 was 87% from 1990 to 2011; that is, the mortality of patients with CHD decreased significantly. However, less than 50% of the causes of CHD have been identified thus far, and 67% of adult patients with CHD still die of heart disease [3][4][5]. This research was aimed at offering new ideas for CHD diagnosis and treatment in children.
MicroRNAs (miRNAs), which are approximately 22 nucleotides long, are single-stranded endogenous noncoding RNAs and play an important role in the progression of CHD [11]. The expression of miRNAs exhibits strict temporal and spatial specificity, and miRNAs function by reducing target mRNA stability or interfering with mRNA translation. Abnormal expression of miRNAs can lead to heart failure, myocardial infarction, myocardial hypertrophy, and other cardiovascular diseases [12][13][14][15] and is involved in CHD occurrence [16,17]. miR-219-5p was found to be involved in the development of cyanotic CHD [18]. Overexpression of miR-29c inhibits zebrafish embryonic heart development [19]. The host gene of miR-193a, which is highly evolutionarily conserved, is located in the transcriptionally active region of chromosome 17q11.2. miR-193a-3p is the transcriptional product of its 3 ′ end. In normal cells cultured in vitro, miR-193a-3p overexpression appears to reduce cell proliferation and arrest cell cycle progression [20,21], but it is unclear whether this molecule is important for the development of CHD.
Programmed cell death (PCD) plays a significant role in animal development and tissue homeostasis. Ferroptosis, which is characterized by iron dependence and excessive intracellular lipid ROS accumulation, is a kind of PCD. In mammals, there are three subtypes of TGF-β, namely, TGF-β1, TGF-β2, and TGF-β3 [22]. TGF-β1 promotes ultrastructural variation in mitochondria, similar to ferroptosis, accompanied by increased ROS and MDA levels [23,24]. In addition, Fer-1 can alleviate TGF-β1-induced ferroptosis [25,26]. Therefore, we speculate that TGF-β2 may promote ferroptosis. Iron, lipid ROS, and GPX4 are three important factors affecting ferroptosis. GPX4 can reduce toxic phospholipid hydroperoxides to generate nontoxic phospholipid alcohols. The reduction in GPX4 levels leads to an imbalance in intracellular lipid ROS production and clearance, thus promoting ferroptosis [27]. NRF2 protects the body from ferroptosis through its downstream target GPX4 and cystine/glutamate transporter (XC/XCT) system [28]. In addition, NRF2 induces HO-1 as the main transcriptional regulatory factor [29]. Research has found that ferroptosis can be promoted in HO-1 RNA interference hepatocellular carcinoma (HCC) cells [30]. High levels of ACSL4 and NOX4 are biomarkers associated with enhanced sensitivity to ferroptosis. ACSL4 catalyses the conversion of polyunsaturated fatty acids of phospholipids (PUFA-PL) into lipid peroxides, and NOX4 induces an increase in reactive oxygen species levels in mitochondria [28,31]. Overexpression of TGF-β2 can inhibit the protein expression of NRF2 in fibroblasts [32]. In addition, TGF-β2 increases the expression of NOX4 [33]. Therefore, we speculate that TGF-β2 may affect the expression of GPX4, NRF2, HO-1, NOX4, and ACSL4 to promote ferroptosis. Current research on cardiovascular ferroptosis focuses on the occurrence of atherosclerosis and myocardial ischaemia-reperfusion injury and the cardiotoxic effects of anthracyclines [34][35][36]. However, whether ferroptosis plays a role in CHD development has not been reported.
Here, we confirmed the existence of the TBX1/miR-193a-3p/TGF-β2 axis, which mediates CHD occurrence by regulating ferroptosis in human fetal CHD samples and rat H9c2 embryonic cardiomyocytes cultured in vitro. Our research may provide a new direction for the expansion and improvement of prenatal CHD diagnosis and treatment strategies.

Patients and
Tissues. This research study was performed according to the Declaration of Helsinki, and the protocol conforms to the provisions of the China Medical University ethics committee (the ethical permit number is AF-SOP-07-1.1-01). Specimens were used after obtaining the informed and signed consent of the subject's guardians. Myocardial tissue samples were obtained from fetuses with CHD (n = 7) and normal heart structure (n = 6), as diagnosed by prenatal ultrasound and autopsy (the clinical manifestations of the CHD group and the control group are shown in Table 1). Termination of pregnancy due to CHD or other major birth defects was performed at Shengjing Hospital Affiliated with China Medical University. After the termination of pregnancy, we immediately washed the right ventricular outflow tract of the fetal myocardial tissue with normal saline three times and transferred the tissue to freezer tubes. The samples were registered, numbered, and then stored in a freezer at -80°C.  3 Oxidative Medicine and Cellular Longevity 2.9. Flow Cytometry-Based Lipid Peroxidation Assay. H9c2 cardiomyocytes were collected 72 h after transfection as described above, 5 μM C11-BODIPY581/591 (Invitrogen; USA) was added in the dark, and the cells were incubated in a 37°C incubator for 30 min. The fluorescence intensity was detected by flow cytometry (BD LSRFortessa; USA). The fluorescence of lipid ROS is inspired at 488 nm, and the emission is collected at 525 nm.

Transmission Electron Microscopy.
H9c2 cells were fixed with 2.5% glutaraldehyde for at least 2 hours at 4°C. The cells were washed in 0.1 M sodium dimethylarsenate buffer (pH 7.4), fixed with 1% osmium tetroxide at 4°C for 90 minutes, rinsed with water, and embedded in epoxy resin. Next, 70-90 nm sections were cut using an EM UC7 instrument (Leica, Germany). After double staining with uranyl acetate and lead citrate, the sections were observed under a transmission electron microscope (Hitachi H-7650, Japan).
2.11. Measurement of Fe 2+ Concentrations. H9c2 cardiomyocytes were inoculated in 24-well culture plates at 2000 cells/ well and tested 48 h after transfection. The plates were washed 3× in phosphate-buffered saline (PBS). Next, the cells were stained in PBS with 1 μM FerroOrange (Dojindo, Japan) at 37°C for 30 min and immediately imaged using a DMI3000B fluorescence microscope (Leica, Germany). Fluorescence was quantified using ImageJ analysis software.
2.12. Mitochondrial ROS Measurements. H9c2 cardiomyocytes were inoculated in 24-well culture plates at 2000 cells/well and tested 48 h after transfection. The plates were washed 1× in PBS. Next, the cells were stained in PBS with 2 μM MitoSOX (Invitrogen, M36008) at 37°C for 30 min. The cells were washed 3× in PBS and fixed in 4% paraformaldehyde at 37°C for 10 min. The cells were washed 3× in PBS and incubated with 10 μg/ml DAPI for 5 min at 37°C. After washing 3× in PBS, the stained cells were imaged on a DMI3000B fluorescence microscope (Leica, Germany). Fluorescence was quantified using ImageJ analysis software.
2.13. Statistical Analysis. IBM SPSS Statistics 19.0 was used for statistical analyses, and GraphPad Prism 5.0 was used for descriptive analyses. The data represent 3 independent experiments and are expressed as the mean ± standard deviation. A t-test was used to analyse the significant differences between two groups, and one-way analysis of variance (ANOVA) was used to analyse the data among three or more groups. P < 0:05 indicates statistical significance.

TBX1 Negatively Regulates the Expression of TGF-β2 in
H9c2 Cells. In a previous study, we found by microarray analysis that TGF-β2 expression in TBX1-silenced cardiomyocytes was significantly upregulated (Table S5). We used TBX1 siRNA to silence TBX1 gene expression in H9c2 cells. The TBX1 mRNA and protein levels were apparently inhibited (P < 0:05) in the TBX1 siRNA group. The mRNA and protein expression levels of TGF-β2 were raised in the TBX1 siRNA group (P < 0:05) (Figures 1(a) and 1(b)). The experimental results show that TBX1 negatively regulates TGF-β2 expression in H9c2 cells.
3.5. Effect of miR-193a-3p on Ferroptosis in H9c2 Cells. The NRF2, GPX4, and HO-1 protein expression was downregulated in the miR-193a-3p inhibitor group. In addition, the NOX4 and ACSL4 protein levels were upregulated in the miR-193a-3p inhibitor group (Figure 5(a)). The intracellular lipid ROS and MDA levels, Fe 2+ concentrations, and These results show that miR-193a-3p inhibition can promote ferroptosis in H9c2 cells.

Ferroptosis in CHD Samples.
The mRNA levels of NRF2 and GPX4 in the CHD group were increased (P < 0:05) (Figures 7(a) and 7(b)), while the protein expression of NRF2 and GPX4 was decreased (P < 0:05) (Figures 7(c) and 7(d)). The protein levels of HO-1 were downregulated in the CHD group (Figure 7(e)), and the protein levels of ACSL4 and NOX4 were upregulated in the CHD group (Figures 7(f) and 7(g)). The level of MDA in the tissue was detected, and the results indicated that the level of MDA in the CHD group was increased (P < 0:05) (Figure 7(h)). These results are similar to those of the cell culture experiments. Together, the results demonstrate that ferroptosis plays important roles in the development of CHD.

Discussion
CHD is one of the most well-known congenital deficiencies. The occurrence of CHD is associated with complex genetic factors and teratogenic factors in the environment during the first 3 months of pregnancy and during the second to seventh weeks of gestation [40][41][42]. However, less than 50% of the causes of CHD have been identified thus far [5], and it is still of great clinical value to investigate the aetiology and pathogenesis of CHD. TBX1 belongs to the T-box gene family, and its mutation can lead to double-outlet right ventricle and ventricular septal defects [43]. In addition, TBX1 haploinsufficiency can lead to aortic arch defects in mice [44]. Our group previously In all panels, the data are representative of three independent experiments. The data are presented as the mean ± SD. Statistical significance is shown as * P < 0:05 vs. controls, * * P < 0:01 vs. controls, and * * * P < 0:001 vs. controls. 7 Oxidative Medicine and Cellular Longevity performed targeted knockdown of TBX1 expression in H9c2 cells and found significant upregulation of TGF-β2 expression by chip microarray analysis (Table S5). TGF-β2 belongs to the highly conserved TGF family and plays a meaningful role in cardiac morphogenesis [45]. In a mouse model, elevated TGF-β2 levels promote abnormal outflow tract morphogenesis and interfere with the normal development of the aortopulmonary septum [10]. In this study, we found that TBX1 was expressed at low levels, while TGF-β2 expression was elevated in human CHD specimens (Figures 6(a)-6(d)). Moreover, in TBX1-silenced H9c2 cells, TGF-β2 expression was significantly upregulated (Figures 1(a) and 1(b)).
miRNAs regulate target gene expression by specifically binding to the 3 ′ -UTRs of target mRNAs, participate in many biological processes, such as apoptosis, migration, and proliferation, and are associated with cardiovascular diseases [46]. As the dual-luciferase reporter assay shows, miR-193a-3p negatively targets TGF-β2 in H9c2 cells, which indicates an association between TBX1 and TGF-β2, suggesting the existence of a TBX1/miR-193a-3p/TGF-β2 axis (Table S6). In addition, the expression of TGF-β2 was upregulated when miR-193a-3p expression was inhibited, and miR-193a-3p expression was downregulated when TBX1 expression was silenced (Figures 2(a)-2(e)). These results show that TBX1 negatively regulates TGF-β2 expression by positively regulating miR-193a-3p expression in H9c2 cells. In this study, we also found that miR-193a-3p was expressed at low levels ( Figure 6(e)). This indicates that the TBX1/miR-193a-3p/TGF-β2 signalling axis is involved in the occurrence of CHD.
As a new kind of PCD, ferroptosis is involved in the development of atherosclerosis and myocardial ischaemiareperfusion [34,35]. Ferroptosis is also involved in the cardiotoxic response caused by doxorubicin [36]. However, whether ferroptosis is related to the development of CHD is still unknown [47]. In Tenon's capsule fibroblasts, TGF-β2 overexpression can inhibit NRF2 protein expression In all panels, the data are representative of three independent experiments. The data are presented as the mean ± SD. Statistical significance is shown as * P < 0:05 vs. controls, * * P < 0:01 vs. controls, and * * * P < 0:001 vs. controls. 8 Oxidative Medicine and Cellular Longevity [32]. NRF2 has been demonstrated to inhibit ferroptosis by upregulating the expression of GPX4 [48]. In addition, pretreatment with TGF-β1 for 2 days enhanced the reduction in cell viability induced by a GPX4 inhibitor [49]. Osimertinib-resistant NCI-H1975/OSIR cells exhibited upregulated TGFβ2 expression, and the cells were sensitive to GPX4 inhibitor-induced ferroptosis [50]. Therefore, we tested some classical indexes of ferroptosis to examine whether the TBX1/miR-193a-3p/axis mediated CHD by promoting cardiomyocyte ferroptosis during heart In all panels, the data were obtained from fetuses with CHD (n = 7) and normal heart structure (n = 6). The data are presented as the mean ± SD. Statistical significance is shown as * P < 0:05 vs. controls, * * P < 0:01 vs. controls, and * * * P < 0:001 vs. controls. In all panels, the data were obtained from fetuses with CHD (n = 7) and normal heart structure (n = 6). The data are presented as the mean ± SD. Statistical significance is shown as * P < 0:05 vs. controls, * * P < 0:01 vs. controls, and * * * P < 0:001 vs. controls. 9 Oxidative Medicine and Cellular Longevity development. In the CHD specimens and the TBX1silenced, miR-193a-3p-inhibited, and TGF-β2-overexpressing H9c2 cells, the downregulated protein expression of NRF2, GPX4, and HO-1 and the upregulated protein expression of NOX4 and ACSL4, as well as the elevated lipid ROS levels, MDA levels, Fe 2+ concentrations, and mitochondrial ROS levels, confirm that ferroptosis is involved in the occurrence of CHD (Figures 3(d)-3(f), 3(h)-3(i), 4(a)-4(e), 5(a)-5(e), and 7(a)-7(h)). Fer-1 is a powerful drug that inhibits ferroptosis in cancer cells and can protect ventricular function by inhibiting ferroptosis in a myocardial ischaemiareperfusion injury model [19]. In our cell models, we found that Fer-1 could reverse the elevated levels of MDA and the decreased cell survival caused by TGF-β2 overexpression (Figures 3(e) and 3(g)). Based on the results described above, we infer that ferroptosis is involved in the occurrence of CHD. Currently, there are no studies that have confirmed the relationship between CHD and ferroptosis, which makes our findings more meaningful.
Transcriptome and proteome profiles are not completely consistent [51]. This may explain the difference between the mRNA and protein expression of GPX4 and NRF2. Gene expression includes four basic processes: transcription, mRNA degradation, translation, and protein degradation. Researchers have found that mRNA levels can explain 40% of the difference in protein levels, but protein levels are mainly controlled by translation efficiency [52]. Another study of embryonic stem cells also showed that changes in protein levels are not accompanied by changes in the corresponding mRNA levels [53]. The TBX1/miR-193a-3p/TGF-β2 axis may control the protein abundance of NRF2 and GPX4 through posttranscriptional, translational, and posttranslational regulatory mechanisms.
The TBX1, miR-193a-3p, and TGF-β2 genes may be used as diagnostic biomolecules and potential therapeutic targets for CHD. In addition, the link between miR-193a-3p and ferroptosis has never been reported before, which may provide references for later research. Unexpectedly, according to transmission electron microscopy, we found that there were a large number of autophagosomes in TGF-β2-overexpressing cells ( Figure S2), indicating that autophagy may be involved in the occurrence of CHD. Whether this process is also mediated by the TBX1/miR-193a-3p/TGF-β2 axis will be explored in the future. We used the rat H9c2 cell line and the right ventricular outflow tract of human CHD fetal myocardial tissue in this experiment because these cells and tissues are widely applied in multitudinous studies and can accurately represent the process of heart development. One way in which our study is unique is the use of the precious human CHD fetal myocardial specimens. Considering the integrity and reliability of the experiments, laboratory CHD animal models, primary myocardial cells, or other cell lines may also be included in our future studies to increase the credibility of the results.

Conclusion
Based on the experimental results described above together with previous studies performed by our team, we  Figure 8: Schematic of the working mechanisms by which the TBX1/miR-193a-3p/TGF-β2 signalling axis participates in CHD progression by promoting ferroptosis. In H9c2 cells, the TBX1/miR-193a-3p/TGF-β2 signalling axis inhibits the expression of NRF2, GPX4, and HO-1, thereby increasing the lipid ROS levels and Fe 2+ concentrations to induce ferroptosis. In addition, the TBX1/miR-193a-3p/TGF-β2 signalling axis induces ferroptosis by upregulating the expression of NOX4 and ACSL4. The upregulated expression of NOX4 and ACSL4 can increase the mitochondrial ROS levels and lipid ROS levels. Table S1: grouping of the dual-luciferase reporter assay. Table S2: the sequences of TBX1 siRNA and control siRNA. Table S3: the sequences of miRNA mimics and inhibitors. Table S4: primer sequences. Table S5: GO analysis of TBX1-silenced cells. Table S6: predicted binding sites of miR-193a-3p, miR-200a-3p, and miR-141-3p on the TGF-β2 3 ′ -UTR. Figure S1: the vector map of GV141. Figure  S2: TGF-β2 induces autophagy in H9c2 cells. Ultrastructural morphology of H9c2 cells was detected by transmission electron microscopy. The yellow arrow shows autophagosomes.