组蛋白乙酰化/去乙酰化失衡对平面细胞极性途径关键基因影响的研究

Effect of histone acetylation/deacetylation imbalances on key gene of planar cell polarity pathway

摘要:

目的 观察组蛋白乙酰化/去乙酰化失衡对胎鼠心脏的致畸作用及对H9C2心肌细胞平面细胞极性（PCP）途径关键基因Vangl2、Scrib、Rac1表达的影响。方法 将40只C57/B6孕小鼠随机分为空白对照组（n=10）、溶剂对照组（n=10）和丙戊酸（VPA）组（n=20）；应用组蛋白去乙酰化酶（HDAC）抑制剂VPA单次剂量700 mg/kg腹腔注射妊娠第10.5天（E10.5 d）VPA组孕鼠，溶剂对照组孕鼠腹腔注射等量生理盐水，空白对照组不做任何处理。于E15.5 d处死孕鼠，统计死胎率；取活胎鼠心脏行苏木精-伊红（HE）染色，观察VPA对胎鼠心脏的致畸作用。培养H9C2心肌细胞并将其分为空白对照组、溶剂对照组和VPA组，VPA组以不同浓度（2.0、4.0、8.0 mmol/L）作用于H9C2心肌细胞，溶剂对照组加入等量生理盐水，空白对照组不进行任何处理。实时荧光定量PCR和Western blot检测HDAC1~3及Vangl2、Scrib、Rac1基因在VPA干预后24、48、72 h mRNA及其蛋白表达水平；比色法测定总HDAC活性变化。结果 VPA组胎鼠死亡率为31.7%，心脏畸形发生率显著高于两对照组（P < 0.05）。与两对照组相比，不同浓度VPA干预后各时间点，HDAC1 mRNA表达水平均显著升高（P < 0.05），而蛋白表达水平于48 h及72 h显著降低（P < 0.05）；不同浓度VPA干预后，HDAC2 mRNA仅在24 h表达显著下降（P < 0.05），而蛋白表达水平在各时间点均显著下调（P < 0.05）；HDAC3 mRNA在VPA（4.0 mmol/L、8.0 mmol/L）干预的各时间点均表达增高（P < 0.05），而蛋白表达水平在不同浓度VPA干预后各时间点均下调（P < 0.05）。与两对照组相比，不同浓度VPA干预后，Vangl2、Scrib mRNA及其蛋白表达水平在48 h、72 h均显著下降（P < 0.05），Vangl2蛋白表达仅在72 h降低（P < 0.05）。与两对照组相比，VPA（4.0及8.0 mmol/L）干预后24 h，总HDAC活性显著降低（P < 0.05）；干预后48 h、72 h，不同浓度VPA组总HDAC活性均明显降低（P < 0.05）。结论 VPA可能通过直接抑制HDAC1~3蛋白表达水平及总HDAC活性致乙酰化/去乙酰化失衡，从而导致PCP途径关键分子Vangl2、Scrib mRNA及蛋白表达水平下调，这可能是先天性心脏病发生的机制之一。

Abstract:

Objective To investigate the effect of histone acetylation/deacetylation imbalances on embryonic hearts of mice and its effect on key genes of planar cell polarity (PCP) pathway-Vangl2, Scrib and Rac1 in H9C2 cells. Methods Forty pregnant C57/B6 mice were randomly assigned into three groups:blank group (n=10), vehicle group (n=10), and valproic acid (VPA)-treated group (n=20). In the VPA-treated group, VPA, a histone deacetylase (HDAC) inhibitor, was administered to each individual dam intraperitoneally at a single dose of 700 mg/kg on embryonic day 10.5 (E10.5). The vehicle and blank groups received equivalent saline or no interventions, respectively. Dams were sacrificed on E15.5, and death rates of embryos were evaluated. Subsequently, embryonic hearts of survival fetus were removed to observe cardiac abnormalities by hematoxylin-eosin (HE) staining. H9C2 cells were cultured and allotted to the blank, vehicle, and VPA-treated groups:the VPA treated group received VPA exposure at concentrations of 2.0, 4.0 and 8.0 mmol/L; the vehicle and blank groups received equivalent saline or no interventions, respectively. HDAC1-3 as well as Vangl2, Scrib and Rac1 mRNA and protein expression levels were determined by quantitative real-time PCR and Western blot, respectively. The total HDAC activity was analyzed by colorimetric assay. Results The fetus mortality rate after VPA treatment was 31.7%, with a significantly higher rate of cardiac abnormalities in comparison with the controls (P < 0.05). In comparison with the blank and vehicle groups, HDAC1 mRNA was significantly increased at various concentrations of VPA treatment at all time points of exposure (P < 0.05), together with a reduction of protein level after 48 and 72 hours of exposure (P < 0.05). The inhibition of HDAC2 mRNA after various concentrations of VPA incubation was pronounced at 24 hours of exposure (P < 0.05), while the protein levels were reduced at all time points (P < 0.05). HDAC3 mRNA was prominently induced by VPA (4.0 and 8.0 mmol/L) at all time points of treatment (P < 0.05). In contrast, the protein level was inhibited after VPA treatment (P < 0.05). In comparison with the blank and vehicle groups, Vangl2 mRNA as well as Scrib mRNA/protein expression levels were markedly reduced after 48 and 72 hours of VPA treatment (P < 0.05), together with a reduction of protein level in Vangl2 at 72 hours (P < 0.05). Compared with the blank and vehicle groups, a significant repression in the total HDAC activity was observed in the VPA-treated group at concentrations of 4.0 and 8.0 mmol/L after 24 hours of treatment (P < 0.05), and the effect persisted up to 48 and 72 hours, exhibiting pronounced inhibition at all concentrations (P < 0.05). Conclusions VPA might result in acetylation/deacetylation imbalances by inhibiting HDAC1-3 protein expression and total HDAC activity, leading to the downregulation of mRNA and protein expression of Vangl2 and Scrib. This could be one of the mechanisms contributing to congenital heart disease.