| ObjectiveTo construct and screen effective RNA interference (RNAi) plasmid vector which can suppress the expression of p300, an subtype of histone acetylase (HATs), in mouse and establish the foundation for the further study of the temporal- and spatial-regulation of p300 during the development of embryonic heart and the impact of histone acetylation mediated by p300 on cardiac development.MethodsThree recombinant RNAi plasmids vectors, p300RNAi1, p300RNAi2 and p300RNAi3, which matched the conserved sequence of mouse p300 perfectly, were constructed and transfected into C3H/10T1/2 cells. The expressions of p300 in different groups were detected by RT-PCR and cellular immunofluorescence to analyze the inhibition efficiency of p300 RNAi vectors.ResultsThe results of RT-PCR and cellular immunofluorescence showed that there was no obvious changes in expression of p300 between control group and the experimental group transfected with p300RNAi2, while in experimental group transfected with p300RNAi1 or p300RNAi3 the expression of p300 was depressed obviously than control group (P<0.05). Conclusion The effective RNAi vector of mouse p300 was successfully constructed, both p300RNAi1 and p300RNAi3 can inhibit the expression of p300 effectively.ObjectiveBecause of the technical restriction, it was difficult to implement the RNAi of target genes in embryonic heart through pregnant mice. So this study chose curcumin, a small molecular specific inhibitor of p300, to replace the RNAi in embryos. The purpose of this part of experiment was to study the effect of curcumin on the HATs activity and the status of histone acetylation in mouse cardiocytes cultured in vitro.MethodsNeonatal mouse cardiocytes cultured in vitro were treated with curcumin at different concentration (10μM, 20μM, 30μM, 40μM and 50μM) for different durations (6h, 12h, 24h, 36h and 48h). ELISA was used to detect the histone acetylase (HATs) activity of cardiocytes and Western Blot was used to detect the acetylation of histone H3.ResultsThe results of ELISA and Western Blot showed thar curcumin could inhibit the HAT activity and histone acetylation of cardiocytes in a dose- and time-dependent manner. When the concentration of curcumin was lower than 30μM and the duration was shorter than 24h, there was no significant change in the HAT activity of cardiocytes between curcumin group and control group. With 30μM curcumin for 24h, the HAT activity was inhibited significantly compared with control group (0.37±0.10 vs 0.94±0.28, P<0.05) and the level of histone acetylation was decreased significantly (0.77±0.36 vs 2.99±1.10, P<0.05). If the concentration of curcumin was higher than 40μM, the incidence of dead cells was increased. If the duration was longer than 48h, the HAT activity of curcumin group would recover.ConclusionCurcumin could inhibit the HAT activity and the level of histone acetylation in cardiocytes cultured in vitro and the optimal condition was 30μM for 24h.ObjectiveTo study the effects of histone hypo-acetylation induced by curcumin on the expression of cardiac core transcription factors in cardiocytes cultured in vitro and to explore the mechanism.MethodsNeonatal mouse cardiocytes were cultured in vitro. After treatment with curcumin (30μM for 24h), real time RT-PCR was used to detect the expression of cardiac core transcription factors, GATA4, Nkx2.5 and Mef2c. Meanwhile the genome DNA of cardiocytes was precipitated by acetylated H3 antibody for chromatin immunoprecipitation (ChIP) then the quantity of precipitated DNA was detected by real time PCR with specific primers target to promoter of GATA4, Nkx2.5 and Mef2c. During the experiment,α-skeletal actin which has been proved being regulated by histone acetylation mediated by p300 was chosen as the positive control and riosomal protein L13A (RPL13A) whose expression dosen't depend on the regulation of hisotne acetylation was chosen as the negative control.Results(1) The results of real time of RT-PCR showed that after treatment with curcumin, the expression of GATA4 (0.34±0.07), Nkx2.5 (0.57±0.11) and Mef2c (0.26±0.04) was decreased significantly compared with control group (0.87±0.11, 1.03±0.23 and 0.76±0.14 respectively) (P<0.05). Besides the expression ofα-skeletal actin was reduced significantly compared with control group (1.02±0.05 vs 2.00±0.11, P<0.05). However there was no significant change in the express of RPL13A between curcumin group and control group.(2) The results of ChIP-PCR showed that the the acetylation of histone which bound with promoters of GATA4 (1.75±0.67), Nkx2.5 (1.29±0.40), Mef2c (2.13±0.99) andα-skeletal actin (2.68±0.05) in curcumin group was reduced significantly compared with control group (P<0.05). However there was no significant change in the acetylation of histone which bound with promoter of RPL13A.Conclusion(1) Hisotne hypo-acetylation induced by curcumin could inhibit the expression of cardiac core transcription factors, GATA4, Nkx2.5 and Mef2c, in cardiocytes cultured in vitro. (2) Hypo-acetylation of histone bound with promoter regions might be one of the mechanism how histone hypo-actylation inhibits the expression of GATA4, Nkx2.5 and Mef2c.ObjectiveTo study the effects of histone hypo-acetylation on the development of mouse embryonic heart and to explore the mechanism.MethodsPregnant mice were injected intraperitoneally with curcumin (50, 75, 100, 150mg/kg·d) during the development of embryonic heart (E7.5~E16.5) to establish the histone hypo-acetylation model. At different time point of the heart development (E11.5, E14.5, E17.5), HE staining was used to observe the heart structure, ELISA was used to detect the HAT activity of embryonic heart, Western Blot was used to evaluate the level of histone acetylation in embryonic heart, and real time RT-PCR was used to analyze the expression of cardiac core transcription factors. Results(1) After treatment with curcumin by intraperitoneal injection, the HAT activity and the level of histone acetylation of embryonic heart on E11.5, E14.5 and E17.5 was inhibited significantly compared with control group.(2) On E11.5, the embryonic hearts of curcumin group were smaller and the ventricular wall was thinner than control groups. Meanwhile the development of trabecular and intraventricular septum was delayed compared with control groups. On E14.5, the intraventricular septum of embryonic heart in curcumin group was complete but the ventricular wall and the intraventricular septum were thinner and there were fewer trabeculars compared with control groups. On E17.5 the structure of embryonic heart in curcumin group was normal but the ventricular wall and the intraventricular septum was a little thinner compared with the control groups.(3) On E11.5 and E14.5, the expression of GATA4, Nkx2.5 and Mef2c of embryonic heart in curcumin group was decreased significantly compared with control group (P<0.05). On E17.5 there was no significant change in the expression of Mef2c between curcumin group and control group though the expression of GATA4 and Nkx2.5 was reduced markedly (P<0.05). Conclusion(1) In curcumin group the development of embryonic heart was delayed in early stage. Up to late stage, the embryonic heart could complete the development though the ventricular wall was thinner and the trabecular was less than control groups.(2) In the early stage of embryonic heart development, histone hypo-acetylation could reduce the expression of GATA4, Nkx2.5 and Mef2c, which may be one of the mechanism how histone hypo-acetylation leads to abnormal heart development.(3) In the early stage of embryonic heart development, histone hypoacetylation could inhibit the expression of Mef2c but in the late stage, the expression of Mef2c recovered, which indicated that there were many regulation mechanisms in gene expression and these various mechanisms could be compensated each other in some case.(4) The expression of Mef2c returned to normal in the late stage of embryonic heart development, which may be one of the base that embryonic could complete the development finally.
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