Wu Y, Zhang Y W, Yue H D, Gao S H, He Z D, Chen Y, Yu Z L, Liu X Z
Department of Stomatology, Henan Provincial People's Hospital, Zhengzhou 450003, China.
Clinical Medical Research Center, Henan Provincial People's Hospital, Zhengzhou 450003, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2022 Apr 9;57(4):397-402. doi: 10.3760/cma.j.cn112144-20220113-00014.
To explore the molecular mechanism of cleft palate in mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The pregnant mice were randomly divided into TCDD-treated group (=42) and control group (=42). TCDD-treated group was given by gavage a single dose of TCDD (64 μg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence. The localization and expression of maternally expressed gene3 (MEG3) in mouse embryonic palatal mesenchymal cells was detected by situ hybridization and real-time PCR (RT-PCR). The key protein expressions of transforming growth factor-β (TGF-β)/Smad signaling pathway in mouse embryonic palatal mesenchyme were analyzed by Western blotting. The interaction of MEG3 and TGF-β receptor Ⅰ (TGF-βRⅠ) was examined by RNA binding protein immunoprecipitation (RIP). At GD13 and GD14, compared with the control group, the ratio of BrdU-positive cells in the palatal mesenchyme of TCDD-treated fetuses decreased significantly (GD13, 6.66, =0.003; GD14, 6.56, =0.003). However, at GD15, the ratio of BrdU-positive cells was significantly increased (5.98, =0.004). MEG3 was mainly expressed in the nuclei of fetal mouse palatal mesenchymal cells, and the expression of MEG3 in TCDD group was significantly increased at GD13, GD14 and GD15(GD13, 39.28, =0.012; GD14, 18.75, =0.042; GD15, 28.36, =0.045). At GD14, TCDD decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells (p-Smad2, 9.48, =0.001;Smad4, 63.10, =0.001), whereas the expression of Smad7 was significantly increased at GD14 (30.77, <0.001). The results of the RIP experiment showed that the amount of TGF-βRⅠ-bound MEG3 in mouse embryonic palatal mesenchymal cells in the TCDD group (23.940±1.301) was higher than that in the control group (8.537±1.523)(24.55, <0.001). MEG3 is involved in the suppression of mouse embryonic palatal mesenchymal cell proliferation, functioning at least in part via interacting with the TGF-βRⅠ protein and thereby suppressing Smad signaling in the context of TCDD induced cleft palate.
探讨2,3,7,8-四氯二苯并对二恶英（TCDD）诱导小鼠腭裂的分子机制。将孕鼠随机分为TCDD处理组（n = 42）和对照组（n = 42）。TCDD处理组在妊娠第10天（GD10）上午8:00经口灌胃给予单剂量TCDD（64μg/kg），对照组经口灌胃给予等体积玉米油。在GD13 - GD15，通过HE染色观察胎鼠腭部发育情况。采用5-溴-2-脱氧尿苷（BrdU）免疫荧光法检测小鼠胚胎腭间充质细胞增殖情况。通过原位杂交和实时荧光定量PCR（RT-PCR）检测母源表达基因3（MEG3）在小鼠胚胎腭间充质细胞中的定位及表达。采用蛋白质免疫印迹法分析转化生长因子-β（TGF-β）/Smad信号通路关键蛋白在小鼠胚胎腭间充质中的表达。通过RNA结合蛋白免疫沉淀（RIP）实验检测MEG3与TGF-β受体Ⅰ（TGF-βRⅠ）的相互作用。在GD13和GD14时，与对照组相比，TCDD处理的胎鼠腭间充质中BrdU阳性细胞比例显著降低（GD13，6.66，P = 0.003；GD14，6.56，P = 0.003）。然而，在GD15时，BrdU阳性细胞比例显著升高（5.98，P = 0.004）。MEG3主要表达于胎鼠腭间充质细胞核中，在GD13、GD14和GD15时，TCDD组MEG3表达显著升高（GD13，39.28, P = 0.012；GD14，18.75, P = 0.042；GD15，28.36, P = 0.045）。在GD14时，TCDD降低了胚胎腭间充质细胞中p-Smad2和Smad4的水平（p-Smad2，9.48, P = 0.001；Smad4，63.10, P = 0.001），而在GD14时Smad7表达显著升高（30.77, P < 0.001）。RIP实验结果显示，TCDD组小鼠胚胎腭间充质细胞中与TGF-βRⅠ结合的MEG3量（2,394.0±130.1）高于对照组（853.7±152.3）（P = 24.55, P < 0.001）。MEG3参与抑制小鼠胚胎腭间充质细胞增殖，至少部分是通过与TGF-βRⅠ蛋白相互作用，从而在TCDD诱导腭裂的情况下抑制Smad信号通路。
