LncRNA PART1 Attenuates Myocardial Ischemia-Reperfusion Injury by Regulating TFAP2C/DUSP5 Axis via miR-302a-3p

Ethical statement

All the animal procedures were performed in accordance with the Guiding Principles in the Use and Care of Animals and was authorized by the Animal Ethics Committee of Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University) (Med-Eth-Re[2022] 750).

Cell culture and treatment

Human AC16 cells were purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences (SIBS, Shanghai, China) and cultured in Dulbecco’s Modified Eagle Medium (Corning, Bedford, MA, USA) with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% Penicillin-Streptomycin (Sigma, St. Louis, MO, USA) at 37°C and 5% CO₂. For H/R treatment, cells (1×10⁶/well) were plated on 6-well plates and incubated in a hypoxic incubator (5% CO₂, 94% N₂, 1% O₂) for 1, 2, 4, 6 hours. Subsequently, cells were put into an atmosphere of 21% O₂ and 5% CO₂ for 6 hours. Control cells were cultured normally (in vitro experimental cell processing timeline can be seen in Supplementary Figure 1).

Cell transfection

Short hairpin RNAs against TFAP2C (sh-TFAP2C), DUSP5 (sh-DUSP5), miR-302a-3p (mimics and inhibitor) and negative controls (NCs) were purchased from GeneChem (Shanghai, China). The empty vector and plasmids containing TFAP2C (overexpression plasmids of TFAP2C; oe-TFAP2C) were synthesized by Sangon Biotech (Shanghai, China). Cells were transfected with the above vectors utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and harvested after 24 hours. The lentivirus for overexpressing PART1 and the control lentivirus were obtained from GenePharma Co., Ltd. (Shanghai, China). AC16 cells were infected with oe-PART1 or oe-NC in enhanced infection solution supplemented with polybrene according to the manufacturer’s instructions. A stable cell line of AC16 cells with PART1 overexpression was constructed after screening with puromycin for 2 weeks.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Cells (1×10³/well) were inoculated into 96-well plates, followed by designated treatment for 24 hours. Later, every well was introduced with 5 mg/mL MTT solution (10 μL; Sigma) for 4 hours incubation under 37°C, followed by adding dimethyl sulfoxide (150 μL/well, 10 minutes). Optical density (OD) values were identified with the microplate reader (Perkin Elmer, Waltham, MA, USA) at 570 nm.

Flow cytometry

Cells were resuspended within binding buffer at 1×10⁶ cells/mL, followed by 15 minutes staining using Annexin V-fluorescein isothiocyanate (FITC) (5 μL) according to the cell apoptosis assay kit (BD Biosciences, San Jose, CA, USA). Later, cell suspension was introduced with propidium iodide (10 μL) in dark for 15 minutes. The FACScan flow cytometry (BD Biosciences) was conducted to examine stained cells. FlowJo 7.6 software (TreeStar, Ashland, OR, USA) was adopted for data analysis.

Immunofluorescence

Cells on coverslips were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin for 1 hour, followed by incubation with LC3 antibody (#4108, 1:500; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing with phosphate-buffered saline, cells were stained with FITC-labeled goat anti-rabbit IgG (ab6717, 1:1,000; Abcam, Cambridge, MA, USA) at 37°C for 1 hour. Cells were observed utilizing a confocal microscopy (Olympus, Tokyo, Japan).

Quantitative real-time polymerase chain reaction (RT-qPCR)

TRIzol reagent (Invitrogen) was utilized to separate total RNAs from cells. For mRNA, RNA was reverse transcribed to cDNA with PrimeScript RT Reagent Kit (Invitrogen). For miRNA, cDNA was synthesized with PrimeScript miRNA cDNA Synthesis Kit (Takara, Dalian, China). qPCR was conducted on ABI 7500 system (Applied Biosystems, Foster City, CA, USA) with SYBR Premix ExTaq II (Takara). The following specific primers were used: PART1 F: 5′-GGACTCGT GCTTCTCGTACGCTGG-3′, R: 5′-GCCTGCCCTTTGGTTTCTGGGAC-3′; miR-302a-3p F: 5′-ACACUCCAGCUGGGAGUGGUUUUGUACCUUC-3′, R: 5′-CUCAACUGGUGUCGUGGAGUCGGCAAUUCAGUUGAGUCGUGAAU-3′; TFAP2C F: 5'-ACAGGATCCATGTTGTGGAAAATAACCGAT-3', R: 5'-ATACTCGAGTTTCCTGTGTTTCTCCATTTT-3'; DUSP5 F: 5′-TCAGCCAGTGTGGAAAACCAG-3′, R: 5′-AGGCACTTCCAAGGTAGAGGA-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) F: 5′-CCAGGTGGTCTCCTCTGA-3′, R: 5′-GCTGTAGCCAAATCGTTGT-3′; U6 F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-AACGCTTCACGAATTTGCGT-3′. GAPDH or U6 was used as endogenous controls. Data were calculated utilizing 2^−∆∆Ct method.

Western blot

We prepared protein lysates using RIPA lysis buffer. Protein concentration was tested with BCA Protein Quantitation Kit (Beyotime, Shanghai, China). Protein aliquots were separated through 10–12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Boston, MA, USA), which were blocked using 5% defatted milk (Bio-Rad, Hercules, CA, USA) under ambient temperature for 1 hour and incubated using primary antibodies overnight under 4°C: TFAP2C (AV38284, 1:1,000; Sigma), DUSP5 (ab200708; 1:1,000; Abcam), p62 (ab56416, 1:1,000; Abcam), LC3 (#4108, 1:1,000; Cell Signaling Technology), Beclin1 (ab210498, 1:1,000; Abcam), Bax (ab182733, 1:2,000; Abcam), Bcl-2 (MA5-11757, 1:1,000; Invitrogen), GAPDH (ab9485, 1:1,000; Abcam), followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody (#7074, 1:1,000; Cell Signaling Technology) for 1 hour at room temperature. Signals were visualized using SuperSignal Pico PLUS chemiluminescent substrate (Pierce, Rockford, IL, USA). GAPDH served as a loading control. Data analysis was conducted using ImageJ software (NIH, Bethesda, MD, USA).

Dual luciferase reporter assay

The wild-type PART1 (PART1-WT) or mutant PART1 (PART1-MUT), which contained miR-302a-3p binding site, were inserted in a dual-luciferase reporter vector (pRL-TK) by Promega (Madison, WI, USA). Besides, the sequences of wild (TFAP2C-WT) and mutant type (TFAP2C-MUT) of TFAP2C 3’untranslated region (UTR) containing miR-302a-3p binding sites were synthesized and cloned into pRL-TK vector (Promega). The above constructs were co-transfected into cells with miR-302a-3p mimics or mimics NC by Lipofectamine 2000 (Invitrogen).

To verify the interaction between DUSP5 and TFAP2C, sequences of DUSP5, including wild-type or mutant binding sites of TFAP2C, were cloned into pGL3 vector (Promega). Cells were co-transfected with vector or OE-TFAP2C and DUSP5-WT or DUSP5-MUT using Lipofectamine 2000 (Invitrogen). After 48 hours, cells were subjected to luciferase assays (Promega).

Lactate dehydrogenase (LDH) assay

A enzyme-linked immunosorbent assay (ELISA) kit (Abcam) was utilized to evaluate LDH levels. Briefly, cell media was collected and cleared to remove cell debris. Samples were enzymically reacted with substrates in 96-well plates. Reacted plates were determined with a microplate reader at 450 nm (Bio-Rad).

A mouse model of myocardial ischemia-reperfusion injury

The left anterior descending coronary artery (LAD) was used to establish a MIRI mouse model.12) Male C57BL/6 mice (Shanghai Animal Center, Shanghai, China; 20–25 g, n=40, 8–10 weeks) were subjected to 12-hour light and dark cycles and the temperature was maintained at 20–25°C. Mice were randomly divided into 4 groups (n=5/group, we conducted two batches of model construction, one for pathological detection and the other for RNA and protein isolation and identification): control group, I/R group, I/R+vector group, and I/R+oe-PART1 group. Briefly, mice were anesthetized by intraperitoneal injection of 3.5% chloral hydrate (10 mL/kg). After a left thoracotomy between the fourth and fifth ribs, the LAD was seen and ligated with a 6-0 prolene suture. Ischemia is confirmed by visual examination of the discoloration of the occlusive distal myocardium. After 30 minutes of ischemia, the ligation was removed. The sham group used the same method of thoracotomy without ligation. In the process of I/R modeling, an injection of lentivirus particles (1×10⁸ PFU/mL, 100 μL) carrying oe-PART1 or vector was administered into the left ventricle, followed by clamping of the aorta for a duration of 10 seconds. The viral transfection into the myocardium was achieved by delivering the virus through the coronary artery, taking advantage of cardiac contraction.13)

2,3,5-Triphenyltetrazolium chloride (TTC) staining

Measurement of infarct volume was conducted as previously described.14) Briefly, myocardial tissues from mice were sliced into sections (1-mm-thick coronal sections), and stained with a 2% solution of TTC (Sigma) for 20 minutes at 37°C, followed by fixation with 4% paraformaldehyde. Afterwards, TTC stained sections were imaged using a digital camera and analyzed by Image J (NIH).

Histopathological examination (hematoxylin and eosin [H&E] and transferase-mediated dUTP nick-end labeling [TUNEL] staining)

Paraformaldehyde (4%) was adopted to fix the hearts from mice. Myocardial tissues were dehydrated and embedded in paraffin. The 4-um sections were stained with H&E staining and then analyzed utilizing light microscopy. Terminal deoxynucleotidyl TUNEL method with Onestep TUNEL Apoptosis Assay Kit (Beyotime) was used to identify apoptotic cells in myocardial sections according to the manufacturer’s instructions. TUNEL-positive nuclei were identified using a microscopy, and apoptosis rate of cardiomyocytes was calculated.

Immunohistochemistry (IHC)

Mouse myocardial tissue sections were initially subjected to a 2-hour baking process at 55°C in an oven. Subsequently, deparaffinization and rehydration steps were performed, followed by antigen retrieval using sodium citrate. Immunostaining was then conducted on the samples using anti-cleaved caspase3 (#9661, 1:1,000; Cell Signaling Technology) antibody at 4°C overnight. Afterward, sections were incubated with a secondary antibody labeled with HRP for 1 hour. Diaminobenzidine (DAB; MXB Biotechnologies, Fuzhou, China) was utilized for section development. Finally, sections were counterstained with hematoxylin and examined utilizing a microscope (Olympus).

ELISA assay

The serum of mice was collected after centrifugation, and serum concentration of creatine kinase MB (CK-MB) was detected utilizing an ELISA kit (Biosciences) according to manufacturer’s instructions.

Statistical analysis

Data were analyzed using GraphPad Prism 8 and expressed as mean ± standard deviation (SD) of 3 independent biological replicates and technical replicates in vitro and 5 mice/per group in vivo. The Kolmogorov-Smirnov test was used to determine the normality of the distribution of the data in each group, and the in vitro data conforms to a normal distribution. Therefore, all in vitro data are analyzed using parametric analysis, differences between two groups were calculated utilizing Student’s t-test. One-way analysis of variance was conducted for multiple comparisons. While in vivo data were analyzed using non-parametric analysis, differences between groups using Mann-Whitney test. The p value <0.05 was taken as statistically significant.

Overexpression of PART1 inhibited H/R-induced cardiomyocyte autophagy and apoptosis

Firstly, we used MTT assay to detect the cell viability at different hypoxia time points. As shown in Figure 1A, cell viability was about 50% at the time point of 4 hours. At this time, cells had both certain damage and certain activity. Therefore, this hypoxia time was used for subsequent experiments. Afterwards, AC16 cells were transfected with oe-PART1 and overexpressed PART1 significantly promoted PART1 expression compared with NC group (Figure 1B). Next, the transfected AC16 cells were cultured under H/R condition to establish a MIRI model in vitro. Compared to control cells, H/R treatment inhibited PART1 expression, but PART1 upregulation reversed the effect of H/R on PART1 expression (Figure 1C). Additionally, H/R treatment greatly repressed cell viability compared with control group, whereas overexpressed PART1 induced cell viability (Figure 1D). Moreover, the secretion of LDH was increased by H/R treatment, while overexpression of PART1 reversed the induction of H/R on LDH (Figure 1E). Flow cytometry and TUNEL staining showed that H/R stimulation enhanced cell apoptosis, but oe-PART1 inhibited cell apoptosis (Figure 1F and G). Meanwhile, H/R treatment increased Bax expression, while decreased Bcl-2 level. However, PART1 upregulation overturned these effects (Figure 1H). Immunofluorescence analysis revealed that fluorescence of LC3 in cells with H/R treatment was elevated, but fluorescence intensity of LC3 was reduced after overexpression of PART1 (Figure 1I). Furthermore, H/R treatment enhanced beclin-1 expression and the ratio of LC3II/I, but decreased p62 expression. Nevertheless, overexpressed PART1 showed the opposite effects (Figure 1J). Therefore, PART1 upregulation alleviated H/R-stimulated cardiomyocyte autophagy and apoptosis.

Figure 1
Overexpression of PART1 inhibited autophagy and apoptosis in H/R-challenged cardiomyocytes. (A) MTT assay detected cell viability at different hypoxia time points. (B) PART1 transfection efficiency was verified by RT-qPCR. (C) RT-qPCR detection of PART1 level after overexpression of PART1 in H/R-treated AC16 cells. (D) MTT tested cell viability. (E) ELISA tested LDH concentration. (F) Cells apoptosis was detected utilizing Flow cytometry. (G) TUNEL staining detected apoptosis. (H) Bcl-2 and Bax expressions were measured using Western blot. (I) Immunofluorescence detection of LC3 expression in AC16 cells. (J) Western blot detected p62, LC3II/I, Beclin-1 levels. Data were shown as mean ± SD based on three independent experiments (n=3).
ELISA = enzyme-linked immunosorbent assay; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; oe- = overexpression plasmids; PART1 = prostate androgen regulated transcript 1; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TUNEL = transferase-mediated dUTP nick-end labeling.

^*p<0.05, ^**p<0.01, ^***p<0.001.

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PART1 targeted miR-302a-3p to upregulate TFAP2C expression

Next, bioinformatics tool StarBase predicted binding site of miR-302a-3p with PART1 or TFAP2C (Figure 2A). Dual luciferase reporter assay demonstrated that luciferase activities were reduced by co-transfection with PART1-WT or TFAP2C-WT with miR-302a-3p mimics in AC16 cells, whereas luciferase activities were not changed in PART1-MUT and TFAP2C-MUT groups (Figure 2B). Furthermore, overexpression of PART1 inhibited miR-302a-3p expression, but enhanced TFAP2C expression (Figure 2C-E). Additionally, compared to NC group, miR-302a-3p upregulation promoted its expression but inhibited TFAP2C expression, while miR-302a-3p downregulation had the opposite effects (Figure 2F-H). Therefore, PART1 interacted with miR-302a-3p to increase TFAP2C expression in AC16 cells.

Figure 2
PART1 targeted miR-302a-3p and upregulated TFAP2C level. (A) StarBase predicted binding site of miR-302a-3p with PART1 or TFAP2C. (B) Luciferase activity of PART1-WT/MUT (left panel) or TFAP2C-WT/MUT (right panel) in AC16 cells treated with miR-302a-3p mimics or mimics NC. (C, D) RT-qPCR assessed miR-302a-3p (C) and TFAP2C (D) levels after overexpression of PART1 in AC16 cells. (E) Western blot analyzed TFAP2C protein level. (F, G) RT-qPCR detected miR-302a-3p (F) and TFAP2C (G) after miR-302a-3p overexpression or inhibition in AC16 cells. (H) Western blot tested the protein level of TFAP2C. Data were shown as mean ± SD based on three independent experiments (n=3).
MUT = mutant type; NC = negative control; PART1 = prostate androgen regulated transcript 1; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TFAP2C = transcription factor activating enhancer-binding protein 2C; WT = wild type.

^*p<0.05, ^**p<0.01, ^***p<0.001.

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PART1 participated in H/R-induced cardiomyocyte autophagy and apoptosis by regulating miR-302a-3p

To explore the mechanism of PART1 in H/R-triggered cardiomyocyte autophagy and apoptosis, AC16 cells were transfected with oe-PART1, miR-302a-3p mimics and NCs, and then cultured under H/R condition. Compared to control group, miR-302a-3p was elevated by H/R treatment. Although PART1 overexpression suppressed miR-302a-3p expression, this effect was abolished by miR-302a-3p upregulation (Figure 3A). H/R treatment inhibited cell viability, while overexpression of PART1 increased cell viability, but co-transfection of miR-302a-3p mimics inhibited cell viability (Figure 3B). Moreover, LDH release and cell apoptosis were suppressed by PART1 upregulation in H/R-challenged cells, and they were recovered by co-transfection with miR-302a-3p mimics (Figure 3C-E). Additionally, after H/R treatment, miR-302a-3p upregulation ameliorated the inhibition of oe-PART1 on Bax expression, decreased the promotion of oe-PART1 on Bcl-2 expression (Figure 3F). H/R treatment enhanced LC3 fluorescence intensity, Beclin-1 expression and LC3II/I ratio, and decreased p62 expression, while overexpression of PART1 decreased LC3 fluorescence intensity, Beclin-1 expression and LC3 II/I ratio, increased p62 level. However, miR-302a-3p upregulation reversed the regulation of PART1 on autophagy-related proteins (Figure 3G and H). In short, PART1 regulated H/R-triggered cell autophagy and apoptosis through inhibiting miR-302a-3p.

Figure 3
PART1 participated in H/R-mediated cardiomyocyte function injury via regulating miR-302a-3p. (A) RT-qPCR determined miR-302a-3p level. (B) MTT tested cell viability. (C) ELISA tested LDH concentration. (D) Flow cytometry analyzed cell apoptosis. (E) TUNEL staining detected apoptosis. (F) Western blot tested Bcl-2 and Bax levels. (G) Immunofluorescence measured LC3 expression in AC16 cells. (H) Western blot detected the protein levels of p62, LC3II/I, Beclin-1. Data were shown as mean ± SD based on three independent experiments (n=3).
ELISA = enzyme-linked immunosorbent assay; LDH = lactate dehydrogenase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PART1 = prostate androgen regulated transcript 1; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TUNEL = transferase-mediated dUTP nick-end labeling.

^*p<0.05, ^**p<0.01, ^***p<0.001.

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Knockdown of miR-302a-3p alleviated H/R-triggered cardiomyocyte autophagy and apoptosis through TFAP2C

We next depleted TFAP2C in AC16 cells and found that sh-TFAP2C significantly reduced TFAP2C expression compared with NC group (Figure 4A). Next, AC16 cells were transfected with miR-302a-3p inhibitor, sh-TFAP2C and NCs, and then cultured under H/R condition. MiR-302a-3p inhibition eliminated the inhibitory effect of H/R treatment on cell viability, whereas co-treatment of sh-TFAP2C and miR-302a-3p inhibitor could reverse the effect of miR-302a-3p inhibition on cell viability (Figure 4B). Additionally, miR-302a-3p inhibition reduced LDH release and apoptosis in H/R-exposed cells, whereas co-transfection of sh-TFAP2C and miR-302a-3p inhibitor had the opposite effect (Figure 4C-E). Furthermore, in H/R-challenged cells, the level of Bax was reduced and Bcl-2 was increased by miR-302a-3p depletion, while co-treatment of sh-TFAP2C and miR-302a-3p inhibitor reversed the effect of miR-302a-3p knockdown (Figure 4F). Moreover, miR-302a-3p knockdown mediated-decrease of LC3 fluorescence, Beclin-1 expression and LC3II/I ratio, and increased p62 level were abrogated by sh-TFAP2C (Figure 4G and H). All in all, miR-302a-3p knockdown attenuated H/R-mediated cell autophagy and apoptosis via regulating TFAP2C in AC16 cells.

Figure 4
MiR-302a-3p inhibitor attenuated H/R-triggered cardiomyocyte autophagy and apoptosis through TFAP2C. (A) TFAP2C transfection efficiency was verified by RT-qPCR and Western blot. (B) MTT tested cell viability. (C) ELISA tested LDH concentration. (D) Flow cytometry determined apoptosis. (E) TUNEL staining detected apoptosis. (F) Western blot analyzed Bcl-2 and Bax expression levels. (G) Immunofluorescence detected LC3 expression in AC16 cells. (H) Western blot measured p62, LC3II/I, Beclin-1 levels. Data were shown as mean ± SD based on three independent experiments (n=3).
ELISA = enzyme-linked immunosorbent assay; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TFAP2C = transcription factor activating enhancer-binding protein 2C; TUNEL = transferase-mediated dUTP nick-end labeling.

^*p<0.05, ^**p<0.01, ^***p<0.001.

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TFAP2C bound to DUSP5 promoter and upregulated DUSP5 expression

Furthermore, we found the binding site between TFAP2C and DUSP5 by bioinformatics tool JASPAR (Figure 5A). Dual luciferase reporter assay revealed that the fluorescence value of DUSP5 wild-type group increased in AC16 cells with oe-TFAP2C, while that of DUSP5 mutant group did not change significantly (Figure 5B). Additionally, compared to control group, overexpression of TFAP2C significantly promoted the expression of TFAP2C and DUSP5 (Figure 5C and D). Therefore, TFAP2C induced DUSP5 expression by binding with DUSP5 promoter.

Figure 5
TFAP2C bound to DUSP5 promoter and upregulated DUSP5. (A) Binding site between TFAP2C and DUSP5 by JASPAR. (B) Dual luciferase reporter assay verified the binding of TFAP2C to DUSP5 promoter. (C, D) RT-qPCR and Western blot detected the expression of TFAP2C and DUSP5 after overexpression of TFAP2C. Data were shown as mean ± SD based on three independent experiments (n=3).
DUSP5 = dual-specificity phosphatase 5; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TFAP2C = transcription factor activating enhancer-binding protein 2C.

^*p<0.05, ^**p<0.01, ^***p<0.001.

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TFAP2C upregulation suppressed H/R-induced cardiomyocyte autophagy and apoptosis through upregulating DUSP5

To explore the molecular mechanism of the protective role of TFAP2C in MIRI. We overexpressed TFAP2C and downregulated DUSP5 in AC16 cells, and then cultured under H/R condition. H/R treatment inhibited DUSP5 expression compared with control group. When TFAP2C was overexpressed, DUSP5 level was significantly increased, which could be rescued by co-transfecting sh-DUSP5 (Figure 6A). Meanwhile, cell viability was elevated in response to TFAP2C overexpression in H/R-treated cells, while co-transfection of oe-TFAP2C and sh-DUSP5 ameliorated this effect (Figure 6B). Moreover, TFAP2C upregulation induced a reduction in LDH release and apoptosis in H/R-induced cells that was completely abolished by sh-DUSP5 (Figure 6C-E). Additionally, in H/R-exposed cells, overexpression of TFAP2C downregulated Bax and upregulated Bcl-2, these effects were reversed after co-transfection with sh-DUSP5 (Figure 6F). In addition, oe-TFAP2C transfection reduced LC3 fluorescence, Beclin-1 expression and LC3 II/I ratio, and induced p62 level in H/R-treated cells. However, these effects were overturned by sh-DUSP5 (Figure 6G and H). Taken together, TFAP2C inhibited H/R-treated cell autophagy and apoptosis via DUSP5.

Figure 6
TFAP2C upregulation suppressed H/R-challenged cardiomyocyte autophagy and apoptosis through regulating DUSP5. (A) RT-qPCR and Western blot analysis of DUSP5 expression. (B) MTT tested cell viability. (C) ELISA tested LDH concentration. (D) Flow cytometry detected apoptosis. (E) TUNEL staining detected apoptosis. (F) Western blot analyzed Bcl-2 and Bax expression levels. (G) Immunofluorescence detected LC3 in AC16 cells. (H) Western blot detected p62, LC3II/I, Beclin-1 levels. Data were shown as mean ± SD based on three independent experiments (n=3).
DUSP5 = dual-specificity phosphatase 5; ELISA = enzyme-linked immunosorbent assay; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TFAP2C = transcription factor activating enhancer-binding protein 2C; TUNEL = transferase-mediated dUTP nick-end labeling.

^*p<0.05, ^**p<0.01, ^***p<0.001.

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Enforced expression of PART1 relieved I/R-induced MIRI

To explore the effect of PART1 on MIRI in vivo, a mouse model of MIRI was established by LAD surgery. oe-PART1 vector was used to treat these mice. As shown in Figure 7A, it presents the in vitro model timeline of MIRI. TTC staining showed that myocardial infarct volume was increased in response to I/R treatment, while this effect was attenuated by PART1 upregulation (Figure 7B). H&E staining showed that I/R led to obvious damage to myocardial function, manifested as inflammatory infiltration, as well as swelling and necrosis of cardiomyocytes. However, oe-PART1 reduced severe myocardial damage (Figure 7C). ELISA assay showed that level of CK-MB in mouse serum was increased after I/R. PART1 upregulation reduced the secretion of CK-MB (Figure 7D). Furthermore, mRNA expression of PART1 in mouse myocardial tissues was reduced in response to I/R treatment; oe-PART1 ameliorated this effect (Figure 7E). TUNEL staining discovered that cardiomyocyte apoptosis increased after I/R in mouse myocardial tissues, but oe-PART1 inhibited the apoptosis (Figure 7F). IHC analysis further demonstrated that cleaved caspase3 was elevated in myocardial tissue of I/R mice, and which was inhibited by PART1 upregulation (Figure 7G). Moreover, I/R dramatically increased LC3 II/I ratio and Beclin-1 expression while decreased p62 level, however, forced PART1 expression abolished the dysregulation of these molecules (Figure 7H). Taken together, these findings revealed that oe-PART1 attenuated MIRI in mice.

Figure 7
Enforced expression of PART1 relieved I/R-induced MIRI. A mice model of MIRI was prepared and treated with a PART1 overexpression vector. (A) Mouse MIRI model construction timeline. (B) TTC staining detected myocardial infarct volume. (C) H&E staining detected myocardial histopathological changes in mice. (D) Blood level of myocardial marker CK-MB was measured by ELISA. (E) RT-qPCR detection of PART1 expression in mouse myocardial tissue. (F) TUNEL staining detected cardiomyocyte apoptosis. (G) Cleaved caspase3 expression in mouse myocardial tissue was assessed with IHC. (H) Western blot detected p62, LC3II/I, Beclin-1 levels. Data are the means ± SD for three independent experiments (n=5 mice/group).
CK-MB = creatine kinase MB; ELISA = enzyme-linked immunosorbent assay; H&E = hematoxylin and eosin; IHC = immunohistochemistry; I/R = ischemia/reperfusion; MIRI = myocardial ischemia-reperfusion injury; PART1 = prostate androgen regulated transcript 1; RT-qPCR = quantitative real-time polymerase chain reaction; SD = standard deviation; TTC = 2,3,5-Triphenyltetrazolium chloride; TUNEL = transferase-mediated dUTP nick-end labeling.

^*p<0.05, ^**p<0.01, ^***p<0.001.

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