MiR-495-3p depletion contributes to myocardial ischemia/reperfusion injury in cardiomyocytes by targeting TNC

Fig. 1. The effect of H/R induction on viability, apoptosis and TNC expression in H9C2 cells. (A) CCK-8 assay was used to measure OD value in H9C2 cells after H/R treatment. (B) Then, flow cytometry was applied to analyze apoptotic cells. (C) ELISA was employed to determine the level of inflammatory cytokines (TNF-α, IL-1β). (E) Western blot was performed to detect TNC protein level in H/R-induced H9C2 cells. GAPDH served as an internal reference. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. CTL. CCK-8, cell counting kit-8; OD, optical density; TNC, tenascin-C; ELISA, enzyme linked immunosorbent assay; TNF-α, tumour necrosis factor alpha; IL-1β, interleukin 1 beta; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H/R, hypoxia/reoxygenation.

Fig. 2. The effect of TNC silencing on viability, apoptosis and MMP of H9C2 cells after H/R treatment. (A) After siTNC transfection, qRT-PCR was performed to detect TNC expression in H/R-induced cells. GAPDH served as an internal reference. (B) Next, the protein level of TNC was determined by Western blot. GAPDH served as an internal reference. (C) CCK-8 assay was used to measure the viability of H9C2 cells with TNC silencing after H/R induction. (D) The cell apoptosis was then detected by flow cytometry. (E) The content of Ca2+ in H9C2 cells was determined by colorimetry after H/R induction or/and TNC inhibition. (F) MMP level in H9C2 cells after TNC inhibition or/and H/R stimulation was measured by JC-1 MMP assay kit. (G-H) ELISA was employed to determine the level of inflammatory cytokines (TNF-α, IL-1β) in H9C2 cells undergone H/R induction or/and TNC inhibition. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. H/R + siNC. CCK-8, cell counting kit-8; qRT-PCR, quantitative real-time polymerase chain reaction; TNC, tenascin-C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme linked immunosorbent assay; TNF-α, tumour necrosis factor alpha; IL-1β, interleukin 1 beta; MMP, mitochondrial…

Fig. 3. MiR-495-3p targeted TNC in H9C2 cells. (A) Candidate miRNAs targeting TNC were screened out by miRDB (http://mirdb.org/miRDB/index.html), Targetscan (http://www.targetscan.org/vert_71/) and microT (http://www.microrna.gr/microT). (B) The binding sites between miR-495-3p and TNC were predicted by Starbase. (C) Dual luciferase reporter assay was employed to investigate the interaction between miR-495-3p and TNC. (D) The expression of miR-495-3p in H9C2 cells after H/R induction was determined by qRT-PCR. U6 served as an internal reference. ∗∗p < 0.01 vs. CTL; ˆˆˆp < 0.001 vs. MC. qRT-PCR, quantitative real-time polymerase chain reaction; TNC, tenascin-C; H/R, hypoxia/reoxygenation; CTL, control; MC, mimic control; M, miR-495-3p mimic; WT, wild-type; MUT, mutant-type.

Fig. 4. MiR-495-3p played a regulatory effect on viability, apoptosis and MMP of H/R-induced H9C2 cells by targeting TNC. (A-B) The expressions of miR-495-3p and TNC in H/R-induced cells transfected with miR-495-3p inhibitor or siTNC or both of them were determined by qRT-PCR. U6 or GAPDH served as an internal reference. (C) Subsequently, CCK-8 assay was conducted to detect OD value for evaluating cell viability. (D) Also, the apoptosis of specified cells with H/R was assessed by flow cytometry. (E) The content of Ca2+ in H/R-induced H9C2 cells was determined by colorimetry after interference with TNC or/and miR-495-3p. (F) JC-1 MMP assay kit was used to measure MMP of H/R-induced cells interfered with TNC or/and miR-495-3p. (G-H) ELISA was employed to determine the level of inflammatory cytokines (TNF-α, IL-1β) in H/R-induced H9C2 cells after interference with TNC or/and miR-495-3p. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. H/R + IC + siNC; ˆˆp < 0.01, ˆˆˆp < 0.001 vs. H/R + IC + siTNC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. H/R + I + siNC. CCK-8, cell counting kit-8; OD, optical density; qRT-PCR, quantitative real-time polymerase chain reaction; TNC, tenascin-C; GAPDH,…