Tanshinone IIA alleviates hypoxia/reoxygenation induced cardiomyocyte injury via lncRNA AK003290/miR-124-5p signaling

Fig. 1. TSA alleviates apoptosis, oxidative stress, and MMP loss induced by H/R. a Apoptosis of cardiomyocytes was evaluated using Annexin/VI-PI staining under different dose of TSA treatment. b, c MDA and LDH amount in cardiomyocytes were analysed using commercial kit. d The ROS of cardiomyocytes was detected using commercial kit and flow cytometry. e JC-1 staining was used to evaluate the MMP. f Western blot was carried out to detect the apoptotic protein expression such as bax, cytoplasma cyt-c, cleaved caspase3, and bcl-2. *p < 0.05

Fig. 2. TSA promoted the expression level of AK003290. a qPCR was used to evaluate the expression of several lncRNAs implicated in heart disease. b qPCR was used to evaluate the expression of AK003290 in cardiomyocytes under different dose of TSA treatment. c qPCR was used to evaluate the expression of AK003290 in myocardium subjected to H/R injury. d FISH experiment was used to evaluate the expression of AK003290 under different dose of TSA treatment. *p < 0.05

Fig. 3. Knock down of AK003290 reversed the effect of TSA. a, b qPCR was performed to evaluate the expression level of AK003290 and miR-124. c. d Apoptosis of cardiomyocytes was evaluated using Annexin/VI-PI staining under TSA treatment and si-AK003290 transfection. e, f MDA and LDH amount in cardiomyocytes were analysed using commercial kit. g The ROS of cardiomyocytes was detected using commercial kit and flow cytometry. h JC-1 staining was used to evaluate the MMP. i Western blot was carried out to detect the apoptotic protein expression such as bax, cytoplasma cyt-c, cleaved caspase3, and bcl-2. *p < 0.05

Fig. 4. MiR-124-5p directly targets AK003290 in cardiomyocytes. a The targeting region between miR-124-5p and AK003290. b Luciferase activity assay was carried out to verify whether miR-124-5p targets AK003290 in cardiomyocytes. c qPCR was performed to detect the expression of miR-124-5p. d RNA pull down using the probe of miR-124-5p was performed to detect the interaction between miR-124-5p and AK003290. e qPCR was performed to detect the expression of AK003290. f Pearson analysis was performed to investigate the correlation between circCadm1 and Pawr as well as miR-124-5p targets AK003290. *p < 0.05

Fig. 5. MiR-124-5p overexpression reversed the effect of TSA. a Apoptosis of cardiomyocytes was evaluated using Annexin/VI-PI staining under TSA treatment and miR-124-5p mimic transfection. b, c MDA and LDH amounts in cardiomyocytes were analysed using commercial kit. d The ROS of cardiomyocytes was detected using commercial kit and flow cytometry. e JC-1 staining was used to evaluate the MMP. f Western blot was carried out to detect the apoptotic protein expression such as bax, cytoplasma cyt-c, cleaved caspase3, and bcl-2. *p < 0.05

Fig. 6. MiR-124-5p knock down reversed the effect of si-AK003290. a Apoptosis of cardiomyocytes was evaluated using Annexin/VI-PI staining under TSA treatment and miR-124-5p mimic transfection. b, c MDA and LDH amounts in cardiomyocytes were analysed using commercial kit. d The ROS of cardiomyocytes was detected using commercial kit and flow cytometry. e JC-1 staining was used to evaluate the MMP. f Targetscan, DIANA-CDS and miRDB were used to predicted the targets of miR-124. Venn analysis was performed to take intersection of the three set. g David analysis was carried out to analyze and enrich those target genes. We displaced the genes related to survival or apoptosis. h The distribution of AK003290 in different organs was showed. *p < 0.05