Downregulation of miR-130a, antagonized doxorubicin-induced cardiotoxicity via increasing the PPARγ expression in mESCs-derived cardiac cells

Fig. 1. Assessment of PPARγ and miR-130a expression upon Dox treatment. A Relative expression level of PPARγ in Dox-treated cardiac cells (5 µM) compared with the control group (untreated group) as explained in the Materials and Methods section. B The seeding region of miR-130a which was deduced from Target Scan. As indicated, miR-130a targets transcripts of PPARγ via complementary nucleotides at 3′ UTR. C Relative expression level of miR-130a in Dox-treated cardiac cells compared with the control group as explained in the Materials and Methods section. U6 was chosen as reference gene as indicated. As evident, miR-130a serves as a negative regulator of PPARγ. Data are represented as mean ± SEM of three independent replicates of experiment. Star indicates significant difference with control at p < 0.05

Fig. 2. Antagomir-mediated silencing of miR-130a to upregulate expression of PPARγ. A Schematic representation of protocol used for antagomir transfection. The protocol is similar with the protocol in supplementary Fig. 2A, unless 25 nM of specific antagomir against miR-130a was used. Of note, Dox was not applied to induce stress toxicity. RT-qPCR was performed to assess the relative expression level of miR-130a and PPARγ. B A decrease in expression level of miR-130a upon antagomir transfection was observed compared with the control samples (Scr: Scramble and control). C An increase in PPARγ expression was observed post-antagomir transfection compared with the control samples. Data are represented as mean ± SEM of three independent replicates of experiment. Star indicates significant difference with both of scramble and control at p < 0.05

Fig. 3. miR-130a-specific antagomir reversed Dox-induced modifications of miR-130a and PPARγ transcripts. A As obvious, miR-130a-specific antagomir was capable to decrease the expression of miR-130a even after Dox treatment comparable to the control cells. B Inversely, transcript level of PPARγ was induced by antagomir despite Dox treatment. Star indicates significant difference of antagomir-treated sample with antagomir-untreated counterpart at p < 0.05. Reference genes were GAPDH and U6

Fig. 4. miR-130a-specific antagomir attenuated Dox-induced toxicity. A Antagomir reversed Dox toxicity effect as verified by Annexin V-FITC by flow cytometry. B In similar condition, caspase-3 activity returned to the baseline level by antagomir pretransfection of cardiac cell prior to Dox induction. C RT-qPCR was also performed to assess the relative expression of BCL-2 and BAX, and NFκB-P65 which clearly represented that miR-130a-specific antagomir had a protective role against Dox. The values are from experiments done in triplicate ± SEM. Reference gene was GAPDH. D Average relative amount of phosphorylated level of P65, a subunit of NFκB transcription complex to GAPDH in lysate of cardiac cells was reduced after antagomir application. The intensity of each band was quantified by ImageJ software. Remarkably, Dox treatment increased phosphorylated level of P65 thereby, while antagomir prevented this modification. Data are represented as mean ± SEM of three independent replicates of experiment. Star indicates significant difference of antagomir-treated sample with antagomir-untreated counterpart at p < 0.05

Fig. 5. Similar to miR-130a-specific antagomir, Pio pretreatment exerted a protective role against Dox-induced toxicity. Pio was added to the cell culture as described in the Materials and Methods section. A Inductive effect of Pio on PPARγ expression against attenuating effect of Dox. B Pio reversed Dox-induced apoptotic effect as verified by Annexin V-FITC by flow cytometry. C Similarly, caspase-3 activity reduced with Pio treatment of cardiac cell prior to Dox induction. D Compared with panle (B), similar outcomes were deduced on BCL2 and BAX and NF-κB-P65 expression when cardiac cells were treated with Pio. E Relative amount of phosphorylated level of P65 showed a decrease when Pio was used. The intensity of each band was quantified by ImageJ software. The reference gene and protein was GAPDH. Data are represented as mean ± SEM of three independent replicates of experiment. Star indicates significant difference of Pio-treated sample with untreated counterpart at p < 0.05

Fig. 6. PPARγ may directly attenuate miR-130a through interacting with the respective promoter. A Pio treatment exhibited the same effect of miR-130a-specific antagomir on reduction of miR-130a. Incorporation of Pio vehicle (DMSO) on downregulation of miR-130a was excluded as DMSO treatment did not affect the content of miR-130a. B Of note, Pio was able to suppress Dox-inductive level of miR-130a. C PPARγ may directly affect the expression of miR-130a through interacting with its specific response element located at the promoter of miR-130a. The data are deduced from Diana tools, miRGen database (Version 3) (carolina.imis.athena-innovation.gr/diana_tools/web/index)

Fig. 7. Schematic representation of molecular mechanism of PPARγ-induced attenuation of miR-130a which could serve as potential approach against Dox-induced toxicity of cardiac cells. As depicted Dox-inductive effect on miR-130a increases inflammation leading to apoptosis of cardiac cells, via suppression of cellular PPARγ level. Whereas Pio acts against Dox by increasing amount of activated PPARγ which suppresses miR-130a in cardiac cells