The Preliminary Research On The Role Of NKX2.1 In Neural Tube Defects Induced By ATRA

Objective:1.To verify the Nkx2.1 gene expression of embryonic brain tissue of the normal group and deformity group using the high-throughput transcriptome.2.To determine the temporal and spatial characteristic of Nkx2.1 gene expression in mouse embryos by whole embryo in situ hybridization.3.To study the mechanism of NKX2.1 in the occurrence of neural tube defects induced by ATRA.Methods:1.When C57BL/6 mice were pregnant for 7.5 day(E7.5),all-trans retinoic acid(ATRA)was injected into mice in a dose of body weight to establish the model of mouse embryo NTDs;then the embryonic brain tissue was taken out at E8.5,E9.5 and E10.5.2.The expression of Nkx2.1 mRNA in normal and malformed brain tissues at E8.5,E9.5 and E10.5 was detected by real-time PCR,and the results of RNA sequencing were verified.3.The spatial expression of Nkx2.1 at E8.5,E9.5 and E10.5 was detected by whole embryo in situ hybridization.4.The si RNA was transfected into the Neuro-2a cells by liposomes to interrupt the expression of Nkx2.1.The experiment was divided into three groups: blank control group(untreated normal control group),negative control group(control vector plasmid transfection group without Nkx2.1 sequence)and siRNA-Nkx2.1 interference group(Nkx2.1 interference vector plasmid transfection group).Then,the transfection results were detected by real-time PCR and Western blotting.5.The cell proliferation ability of the two groups was detected by Cell Counting Kit-8(CCK-8)in negative control group and siRNA-Nkx2.1 interference group.6.The changes of cell cycle were observed by flow cytometry in negative control group and siRNA-Nkx2.1 interference group.7.The changes of cell apoptosis were observed by flow cytometry in negative control group,siRNA-Nkx2.1-24 h interference group(Nkx2.1 interference vector plasmid transfection 24 h)and siRNA-Nkx2.1-48 h interference group(Nkx2.1 interference vector plasmid transfection 48 h).8.The activation of Caspase3 in negative control group,siRNA-Nkx2.1-24 h interference group and siRNA-Nkx2.1-48 h interference group was observed under fluorescence microscope by activated Caspase3 staining.9.The protein levels expression of Shh,Ptch1 and Smo were detected by Western blotting.10.Statistical analysis: datas were expressed as mean ± standard deviation(X ± S),SPSS17.0 software was used to analyze datas,P < 0.05 means that the difference was statistically significant;datas for Graph used the GraphPad Prism 5 software.Results:1.The model of mouse embryonic NTDs was successfully established by using ATRA of 21 mg/kg.The expression of Nkx2.1 in malformed brain tissues at E8.5,E9.5 and E10.5 was significantly lower than that in the control group by real-time PCR.The descending ratio was about 4 times,20 times and 36 times,this ratio was statistically significant(P < 0.01).Experimental datas were consistent with the RNA sequencing results,basically.2.The expression of Nkx2.1 was detected by whole embryo in situ hybridization.In the normal group,Nkx2.1 at E8.5 was weakly expressed in the forebrain and significantly expressed in the forebrain,midbrain and hindbrain at E9.5 and E10.5.However,almost no expression was found in the deformed group.3.The expression of Nkx2.1 mRNA and protein expression were reduced after the small interfering fragment of Nkx2.1 entered the Neuro-2a cells by liposome.At the mRNA level,the relative expression of the blank control group,negative control group and siRNA-Nkx2.1 interference group was 1.01 ± 0.09,0.93 ± 0.05 and 0.35 ± 0.03,respectively.The difference was statistically significant(P < 0.001).At the protein level,The relative expression was 0.76 ± 0.02,0.66 ± 0.12 and 0.31 ± 0.02,respectively.Compared to the control group,the differences were statistically significant(P < 0.001).4.CCK-8 results showed that there was no significant difference between the negative control group and the siRNA-Nkx2.1 interference group on the first and second day of transfection.However,OD value of the siRNA-Nkx2.1 interference group was significantly lower than the negative control group on the third day of transfection.Compared to the control group,the differences were statistically significant(P < 0.05).5.The results of the flow cytometry showed that the proportion of G1 phase cells in the cell cycle of siRNA-Nkx2.1 interference group was significantly lower than that in negative control group.The proportion of G1 phase cells was(64.23 ± 1.67)% and(49.31± 2.51)% in negative control group and siRNA-Nkx2.1 interference group,respectively.The difference was statistically significant(P < 0.01).6.The results of the flow cytometry showed that the apoptosis rate of siRNA-Nkx2.1interference group was significantly higher than that of negative control group,and the apoptotic rate increased with the prolongation of time.The apoptotic rates of the negative control group,siRNA-Nkx2.1-24 h interference group and siRNA-Nkx2.1-48 h interference group were(9.93 ± 0.56)%,(12.93 ± 0.63)% and(21.37 ± 1.51)%,respectively.Compared to the control group,the differences were statistically significant(P < 0.05).7.The fluorescent staining results of activated Caspase3 in the siRNA-Nkx2.1interference group were higher than that in the negative control group,and the number of Caspase3 positive staning cells were increased with the prolongation of the time.The number of Caspase3 positive staning cells of the negative control group,siRNA-Nkx2.1-24 h interference and the siRNA-Nkx2.1-48 h group were 5.25 ± 1.71,16.25 ± 3.53 and 27.02± 2.24,respectively.Compared to the control group,the differences were statisticallysignificant(P < 0.001).8.The expression of Shh,Ptch1 and Smo protein was detected by Western blotting,and the expression of Shh,Ptch1 and Smo protein in siRNA-Nkx2.1 interference group was significantly lower than that in negative control group(P < 0.01).Conclusions:1.The transcription factor NKX2.1 reduction may play an important role in the occurrence of neural tube defects induced by ATRA.2.The low expression of NKX2.1 may lead to the occurrence of NTDs by participating in Shh signaling pathways,by affecting decreased cell proliferation and increased apoptosis.