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. 2017 Oct 20;49(10):e386. doi: 10.1038/emm.2017.150

Figure 3.

Figure 3

RhoBTB1 is a target gene of miR-31a-5p. (a) Targetscan and luciferase assays showed RhoBTB1 as a direct target of miR-31a-5p. n=6 per group. (b, c) Western blot showed that miR-31a-5p endogenously negatively regulated RhoBTB1 in neonatal rat ventricular cardiomyocytes (NRVMs). n=3 per group. (d) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis demonstrated that miR-31a-3p did not regulate expression of RhoBTB2 and RhoBTB3 in NRVMs, at least at the mRNA level. n=6 per group. (e) qRT-PCR analysis of RhoBTB1 in cells transfected with its siRNA. n=3 per group. (f) Immunohistochemical stainings for sarcomeric α-actinin and 5-ethynyl-2-deoxyuridine (EdU) staining showed that RhoBTB1 knockdown induced an increase in EdU incorporation, whereas co-transfection with the RhoBTB1 siRNA and miR-31a-5p mimic did not exert an additive effect. At least 2000 cells were quantified in each group. Scale bar: 100 μm. (g) Immunohistochemical stainings for sarcomeric α-actinin and EdU staining showed that RhoBTB1 knockdown induced an increase in EdU incorporation, whereas co-transfection with the RhoBTB1 siRNA and miR-31a-5p inhibitor completely reversed the suppressive effect of the miR-31a-5p inhibitor on the EdU incorporation rate of NRVMs. At least 2000 cells were quantified in each group. Scale bar: 100 μm. *P<0.05, **P<0.01, ***P<0.001 versus respective control.