Long Noncoding RNA SOX2-OT Exacerbates Hypoxia-Induced Cardiomyocytes Injury by Regulating miR-27a-3p/TGFβR1 Axis

Figures

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  Figure 1. SOX2-OT is upregulated in HCMs treated by hypoxia. (a) Data from GSE66360 showed an upregulation of SOX2-OT in patients' blood. (b) CCK-8 assay was used to measure cell viability. #P < 0.01 vs. control. (c) Flow cytometry was utilized to assess cell apoptosis. #P < 0.01 vs. control. (d) RT-qPCR was performed to detect lncRNAs (SOX2-OT, RNY5, HOX11-AS, PCGEM1, SRA1, BCYRN1, CDKN2B-AS, RMST, H19, and RMRP) levels. #P < 0.01 vs. control.

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  Figure 2. SOX2-OT directly interacts with miR-27a-3p in hypoxic HCMs. (a) The binding site of miR-27a-3p on SOX2-OT was predicted by DIANA tools. (b, e) The overexpression and knockdown efficacy of miR-27a-3p or SOX2-OT was conducted in RT-qPCR. #P < 0.01 vs. miR-NC. (c) The binding capacity between SOX2-OT and miR-27a-3p was evaluated by using 4 mutations of SOX2-OT in luciferase reporter assay. #P < 0.01 vs. miR-NC. (d, f) RT-qPCR validated that SOX2-OT and miR-27a-3p negatively regulated each other. #P < 0.01 vs. miR-NC or NC. (g) RIP assay was adopted to confirm the combination between SOX2-OT and miR-27a-3p. #P < 0.01 vs. IgG. (h) The level of miR-27a-3p in HCM cells was measured by RT-qPCR. #P < 0.01 vs. control.

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  Figure 3. SOX2-OT cooperates with miR-27a-3p to modulate cellular processes of hypoxic HCMs. (a–e) Proliferation, apoptosis, caspase-3 activity, migration, and invasion of HCMs were determined by CCK-8, flow cytometry, caspase-3 activity, and trans-well assays separately. #P < 0.01 vs. NC and ΦP < 0.01 vs. sh-SOX2-OT.

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  Figure 4. SOX2-OT regulates TGFβR1 level by collaborating with miR-27a-3p in hypoxic HCMs. (a) The binding sequences between miR-27a-3p and TGFBR1 were hypothesized by Targetscan. (b, d) The luciferase activity of wild-type or mutant pGLO-TGFBR1-3′UTR was measured by luciferase reporter assay in HCMs. #P < 0.01 vs. miR-NC or NC. ΦP < 0.01 vs. sh-SOX2-OT. (c, e) The mRNA level of TGFBR1 was monitored by RT-qPCR assay. #P < 0.01 vs. miR-NC or NC. ΦP < 0.01 vs. sh-SOX2-OT. (f, g) The detection of TGFBR1 protein level was carried out in western blot assay. #P < 0.01 vs. Mock/NC and ΦP < 0.01 vs. Double KD.

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  Figure 5. MiR-27a-3p inhibits heart failure development by modulating TGFβR1 in HCM cells. (a–g) CCK-8, flow cytometry, caspase-3 activity, and trans-well assays were separately implemented to evaluate proliferation, apoptosis, migration, and invasion of HCMs in miR-NC/pcDNA3.1, miR-27a-3p/pcDNA3.1, and miR-27a-3p/TGFβR1 groups. #P < 0.01 vs. miR-NC/pcDNA3.1 and ΦP < 0.01 vs. miR-27a-3p/pcDNA3.1.

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  Figure 6. SOX2-OT facilitates HCM cell injury via TGFβR1. (a–g) Proliferation, apoptosis, migration, and invasion of HCMs transfected with NC/pcDNA3.1, sh-SOX2-OT/TGFβR1, or sh-SOX2-OT/pcDNA3.1 were assessed by CCK-8, flow cytometry, caspase-3 activity, and trans-well assays. #P < 0.01 vs. miR-NC/pcDNA3.1 and ФP<0.01 vs. sh-SOX2-OT/pcDNA3.1.