Research / 2022 / Article / Fig 6

Research Article

Downregulation of circ-ZNF609 Promotes Heart Repair by Modulating RNA N6-Methyladenosine-Modified Yap Expression

Figure 6

circ-ZNF609 regulates YAP expression via YTHDF3 in cardiomyocytes. (a) The expression of circ-ZNF609 was validated by qRT-PCR ( wells/group) and western blot ( wells/group). (b) Validation of m6A modification in Yap mRNA using meRIP-qPCR (/group). (c) qRT-PCR demonstrated the expression level of Ythdf1, Ythdf2, and Ythdf3 treated with sh-circ-ZNF609 (; ns: nonstatistically significant. wells/group). (d) Western blot analysis demonstrated the YTHDF1, YTHDF2, and YTHDF3 expression levels treated with sh-circ-ZNF609 (; ns: nonstatistically significant. wells/group). (e) The amount of Ythdf3 mRNA in AC16 cardiomyocytes treated with actinomycin D with or without circ-ZNF609 knockdown at indicated time points ( at each time point, /group). (f) qRT-PCR demonstrated the expression level of Yap with or without YTHDF3 knockdown ( wells/group). (g) Western blot analysis demonstrated the expression level of YAP with or without YTHDF3 knockdown ( wells/group). (h) qRT-PCR demonstrated the expression levels of Yap at indicated groups ( wells/group). (i) Western blot demonstrated the expression of levels of YAP at indicated groups ( wells/group). (j) Quantification of the relative TUNEL-positive NRCMs showed that YTHDF3 overexpression could block the protective effect of circ-ZNF609 knockdown ( wells/group). (k) RNA immunoprecipitation assay demonstrated YTHDF3 binding to circ-ZNF609 and Yap (/group). (l) Left: silver staining of the circ-ZNF609-protein complex pulled down by circ-ZNF609 junction antisense probe or sense probe with cell lysates from AC16 cardiomyocytes. Right: western blot and qRT-PCR analysis suggested that YTHDF3 protein and Yap mRNA were specifically pulled down by circ-ZNF609 antisense probe (/group). (m) RNA immunoprecipitation assay demonstrated the binding activity of YTHDF1 to Yap mRNA with or without circ-ZNF609 knockdown (/group). (n) RNA immunoprecipitation assay demonstrated the binding activity of YTHDF2 to Yap mRNA with or without circ-ZNF609 knockdown (/group). Scr: shRNA scrambled control; sh-circ-ZNF609: shRNA targeted to circ-ZNF609; Fugw: empty vector without YTHDF3 overexpression; sh-YTHDF3: shRNA targeted to YTHDF3; YTHDF3: YTHDF3 overexpression construct; NC: siRNA negative control; si-circ-ZNF609: siRNA targeted to circ-ZNF609; OGD/R: oxygen-glucose deprivation/reperfusion. (a–g, k, l) Independent-sample -test; (h–j, m, n) two-way ANOVA test.