Experimental Study On The Effect Of MiR-381-3p On Palatal Osteogenic Differentiation And Expression Change Of Cell Cycle-related Protein In Cleft Palate Of Fetal Mice Induced By TCDD

Part ? Down-reg?lation of miR-381-3p inhibits osteogenic differentiation of mouse embryonic palatal mesenchymal cells in TCDD-induced cleft palate of fetal miceObjective: To investigate whether down-regulation of miR-381-3p is associated with inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal(MEPM)cells in 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD)induced cleft palate of fetal mice.Methods: Thirty-two pregnant mice were randomly divided into control group and TCDD group on the embryonic day 10.5(E10.5)using random number table.As the time,TCDD treatment group was given TCDD(28?g/kg),the pregnant mice in the control group were gavaged according to the same amount of corn oil.Each group of pregnant mice was sacrificed on E13.5 and E14.5,and the fetal palate was collected for analysis.MEPM cells were extracted from miRNAs and proteins after treatment with TCDD 10 nmol/L for 0,0.5,1,2,and 3 days;MEPM cells were transfected with miR-381-3p inhibitor and mimics for 48 h to extract miRNAs and protein,respectively.The relative expression of miR-381-3p was detected by real-time quantitative PCR.The relative expression levels of osteogenic marker gene(RUNX2)and osteopontin(OPN)were detected by Western blotting.Results: on E13.4 and E14.5,The relative expression levels of miR-381-3p in the TCDD group were lower than those in the control group(P<0.05,P<0.05);on E14.5,The relative expression of RUNX2 and OPN protein in TCDD group were lower than that in the control group(P<0.05,P<0.01).The expression of miR-381-3p on the 0.5th and 1st day after TCDD treatment of MEPM cells was significantly lower than that of the control group(P<0.001,P<0.01),The expression level of miR-381-3p on the second day increased significantly compared with that on the 1st day(P<0.01).The expression of RUNX2 on day 1,2 and 3 was significantly lower than that of the control group(P<0.01,P<0.001,P<0.001).The relative expression of OPN protein decreased on the first,second and third days,and the difference was statistically significant(P<0.05,P<0.001,P<0.01).The RUNX2 protein in MEPM cells transfected with miR-381-3p inhibitor was significantly lower than that in the control group(P<0.01),and the relative expression of OPN protein was significantly decreased(P<0.01).RUNX2 protein was significantly increased in MEPM cells transfected with miR-381-3p mimics compared with the control group(P<0.05),and the relative expression of OPN protein was also increased,The difference was statistically significant(P<0.05.<0.05).Conclusion: Down-reg?lation of miR-381-3p expression may be associated with inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal cells in cleft palate of fetal mice induced by TCDD.Part ? Change of cell cycle-related molec?lar expression in cleft palate of fetal mice induced by TCDDObjective: To investigate the change of cell cycle-related molec?les in the palatal tissue of TCDD-induced cleft palate of fetal mice induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD),and to explore the mechanism of cell cycle-related molec?les in cleft palate.Methods : Forty-eight C57BL/6J pregnant mice were randomly divided into TCDD treatment group and control group by random number table method(n=24),on the embryonic day 10.5(E10.5),in TCDD treatment group pregnant mice were intragastrically administered with 28 ?g/kg(corn oil containing 5 ?g/ml TCDD),and the control group was intragastrically administered with the same volume of corn oil.The fetal palate were taken out on E13.5,E14.5 and E15.5,total ribonucleic acid and total protein were extracted and Messenger ribonucleic acid(m RNA)or protein expression levels of cell cycle-related molec?lar was detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting.HEK293 t cells were treated with different concentrations of TCDD(0.01,0.1,0.5 and 1 nmol/L),MTT assay was used to detect changes in cell proliferation activity after different concentrations of TCDD.Results : The expression level of Irf6 protein were higher in the control group(1.26 � 0.13?1.67 � 0.14 and 1.42 � 0.15)than in the TCDD group(0.81 � 0.08?1.04 � 0.02 and 0.86 � 0.12)on each time point(P = 0.0471,0.0146 and 0.0241).The expression level of P21 protein on E13.5 and 14.5 in the control group(2.26 � 0.21?1.99 � 0.21)were higher than that in the TCDD group on each time point(1.43 � 0.12?0.93 � 0.22)(P = 0.8726 and 0.0273).The expression level of cyclin D1 in the control group(1.00�0.02?0.94�0.03 and 1.11�0.09)were higher than those in the TCDD group(0.28�0.01?0.33�0.06 and 0.88�0.01)on each time point(P<0.001).The expression of cyclin E1,cyclin A2,cyclin B1,CDK6,CDK2 and CDK1 in TCDD groups were higher than that in the control groups(P <0.05),E13.5 cyclin B1 and E15.5 Cdk2 m RNA expression did not change significantly between the two groups(P>0.05).After treatment with TCDD(0.1 nmol/L)on 1,2,3 days(0.70 � 0.05?1.05 � 0.03 and 1.39� 0.04 respectively),the proliferation of HEK293 t cells increased compared with the control group(0.49 � 0.04?0.98 � 0.03 and 1.55 � 0.02 respectively)(P value wree 0.0198?0.1320 and 0.0247 respectively).Conclusion: TCDD negatively reg?lates Irf6 and P21 and interferes with the normal expression of cell cycle-associated molec?les,which in turn interferes with medial edge epithelia(MEE)cells cycle arrest and proliferation,these indicate that the disorder of spatiotemporal expression of cell cycle-related molec?les during palatal development may be involved in the mechanism of TCDD-induced cleft palate.Part ? Preliminary study on the mechanism of 14-3-3 protein sigma in TCDD-induced cleft palate of fetal miceObjective: To investigate 14-3-3? protein whether participation in mechanism of cleft palate by maternal exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD)in mice.Methods: 48 pregnant mice were randomly divided into TCDD group and control group.on the embryonic day 10.5(E10.5),in TCDD treatment group pregnant mice were intragastrically administered with 28 ?g/kg(corn oil containing 5 ?g/ml TCDD),and the control group was intragastrically administered with the same volume of corn oil.The fetal palate were taken out on E13.5,E14.5 and E15.5,fetal palates and heads were obtained for analysis.The heads were sliced for HE staining and 14-3-3? protein was detected by immunohistochemical staining.14-3-3? m RNA in palate were evaluated by RT-QPCR.Western blot was used to detect the protein expression of 14-3-3?,P53 and phosphorylated P53.Results: The 14-3-3? protein was positively expressed on E14.5 and E15.5.The control group was mainly expressed in medial edge epithelial cells and residual epithelial island cells,and was mainly expressed in the medial epithelial cells of the palate in the TCDD group.On E13.5,E14.5 and E15.5,The m RNA expressions of 14-3-3? in TCDD group were(1.92�0.18,2.68�0.20 and 5.84�0.11)higher than those in control group(0.82�0.32,0.98�0.25 and 3.84�0.36)(P<0.05,P<0.01,P<0.05).On E13.5,E14.5 and E15.5,the protein expressions of 14-3-3? in TCDD group were(2.29�0.07,3.15�0.07 and 2.02�0.19)significantly higher than those in control group(1.00�0.05,1.06�0.04,0.76�0.05)(P < 0.001,P < 0.001,P < 0.01);On E13.5,The expression of P53 protein in TCDD group was(1.18�0.04)higher than that in control group(1.00�0.03)(P<0.05);On E13.5 and E14.5,the expressions of phosphorylated P53 protein in TCDD group were significantly higher(2.26�0.08,2.11�0.02)than those in control group(0.96�0.03,1.59�0.03)(P<0.001,P<0.001).Conclusion: he expressions of 14-3-3? protein were increased on E13.5,E14.5 and E15.5 in TCDD group,probably because P53 pathway was activated during the early development of palate by TCDD,causing cell cycle arrest.Besides,the elevated expression of 14-3-3? protein in the TCDD group may inhibit epithelial mesenchymal transition which causes medial edge epithelium persistent existence and interferes with the normal development process of the palate,?ltimately resulting in cleft palate formation.