The miR‐200 family targets Tbx5, Gata4, and Pitx2during cardiac development and regeneration

Abstract

Cardiac development requires a highly complex gene regulatory network (GRN) of transcription factors. These GRN factors are known to be modulated by microRNAs (miRs), small non‐coding RNAs that target mRNAs to alter expression levels. MiR‐200 family members, miR‐200aand ‐200c­, are highly expressed in the developing heart. These miRs are known to target cardiac transcriptions factors Tbx5, Gata4, and Pitx2. However, no in vivoinvestigation of the relationship between the miR‐200family members and these transcription factors has been done to date. We hypothesize miR‐200 family members modulate Tbx5, Gata4, and Pitx2expression to allow for proper cardiac development to occur. To study the role of miR‐200 family members, we developed the Plasmid‐based miR Inhibitor System (PMIS). PMIS works by repressing miRs through seed sequence complementation, allowing for the study of individual miR family members within a cluster. Histological analysis of PMIS‐miR‐200a, PMIS‐miR‐200c, and PMIS‐miR‐200ac hearts revealed distant abnormal phenotypes, with PMIS‐miR‐200achearts presenting with a ventricle‐septal defect. Additionally, we observe embryonic stage and chamber specific changes in Tbx5, Gata4, and Pitx2 expression when miR‐200aand ‐200c are inhibited. These aberrant expression changes result in altered expression of Tbx5, Gata4, and Pitx2 downstream transcriptional targets Nppa, Gja1, and Ace2. Tbx5, Gata4, and Pitx2 drive transcription of miR‐200a and ‐200c, resulting in a negative feedback loop on to these transcription factors. Post‐natal day 1 PMIS‐miR‐200a and PMIS‐miR‐200cpups have functional and structural phenotypes which are fully recovered by postnatal day 7. We discovered a role for miR‐200aand ‐200c in cardiac development through targeting Tbx5, Gata4, and Pitx2, with potential for using PMIS in therapeutic applications.