A Preliminary Study On The Mechanism Of MiR-222-3p Targeting Ddit4 To Regulate Neural Tube Closure In Mice

Objective:1.Verify the expression of miR-222-3p and Ddit4 genes in miRNA-Seq of normal embryonic brain tissue and ATRA deformed groups;verify that the target gene of miR-222-3p is Ddit4.2.Observe the effect of low expression of miR-222-3p on the function of HT-22 cells,and initially determine the effect of miR-222-3p on cell function.3.The effects of miR-222-3p on Wnt signaling pathway and HT-22 cell function were observed by interfering with the expression of Ddit4,and the mechanism of miR-222-3p in the development of neural tube defects in mouse embryo induced by ATRA was explored.Methods:1.Establish mouse embryo NTDs model:By intragastric administration,on the 7.5th day of pregnancy?E7.5?of C57BL/6 mice,all-trans retinoic acid?ATRA?was injected into pregnant mice at a dose of 28 mg/Kg to establish embryonic neural tube defects.?NTDs?model,and then continued to extract embryonic brain tissue at E8.5,E9.5 and E10.5,respectivel.2.Verify the abnormal expression of microRNA and genes and the relationship between the two:Real-time PCR was used to detect the expression of miR-222-3p and Ddit4 mRNA levels in normal and abnormal brain tissues of E8.5,E9.5 and E10.5.miR-seq results for verification;The target gene of miR-222-3p was detected by the double luciferase reporter gene technology;the chemically synthesized miR-222-3p mimic?mim-222?and miR-222-3p inhibitor?Anti-222?transfected into HT-22 cells;and the expression of the downstream target gene Ddit4 was detected by Real-time PCR and Western blotting;3.Detection of cell phenotype changes after interfering with miR-222-3p expression:Transfection of chemically synthesized Anti-222 into HT-22 cells via liposomes,and then cell proliferation?CCK-8 method,Western blotting?,apoptosis?Flow cytometry,AO-EB,Western blotting?,cell cycle?flow cytometry?and migration?cell scratch test?levels.The research was divided into three groups:Con group?untreated HT-22 cells?,Anti-NC group?transfected with empty vector HT-22 cells?,Anti-222 group?miR-222-3p inhibitor transfected HT-22 cells?.4.To detect the effect of miR-222-3p downstream genes on cell phenotype:Chemically synthesized Ddit4 small interfering RNA?siR-Ddit4?was transfected into HT-22 cells via liposomes to interfere with Ddit4 expression,and cell proliferation,apoptosis,cycle and scratch experiments were performed.The experiment was divided into three groups:Anti-NC group?transfected with empty vector HT-22 cells?,Anti-222 group?miR-222-3p inhibitor transfected HT-22 cells?,and Anti-222+siR-Ddit4 group?MiR-222-3p inhibitor and siRNA-Ddit4 co-transfect HT-22 cells?.5.Detect the expression of Wnt signaling pathway related indicators:Real-time PCR was used to detect changes in the expression of Wnt signaling pathway indicators?-catenin and TCF4;Western blotting was used to detect changes in the expression of Wnt signaling pathway marker proteins?-catenin and TCF4 and downstream indicators C-myc CycinD1protein expression.6.Statistical analysis methods:Means±standard deviation?X±S?are used to represent the data.SPSS17.0 software is used for statistical analysis.GraphPad Prism 8software is used for data mapping.Image-J software is used for gray value analysis.Differences between sample numbers.*Indicates P<0.05,**indicates P<0.01,***indicates P<0.001,and the difference is statistically significant when P<0.05.Results:1.28 mg/kg ATRA gavage was used to successfully establish C57BL/6 mouse embryo NTDs model.The levels of miR-222-3p mRNA in E8.5,E9.5,and E10.5 brain tissues were detected by Real-time PCR.The expression of miR-222-3p in RA group was significantly lower than Con group?P<0.001?.;Ddit4 expression in RA group was significantly higher than Con group?P<0.01?;experimental data are consistent with miR-seq results.2.Earlier analysis by Targetscan,PicTar,miRanda bioinformatics software found that the target gene of miR-222-3p was Ddit4;dual luciferase reporter gene technology found that dual-luciferase after the wild-type Ddit4 vector was combined with miR-222-3p Activity decreased?P<0.001?,and the activity of dual luciferase did not change after the mutant Ddit4 vector was combined with miR-222-3p,indicating that the downstream target gene of miR-222-3p was Ddit4;by fluorescence microscopy and flow cytometry The transfection efficiency was detected to be more than 80%,and the expression level of Ddit4 was detected after transfection.The expression of Ddit4 in Anti-222 group was significantly higher than that in Con group and Anti-NC group?P<0.001?,and in mim-222 group Compared with Con group and mim-NC group,the expression was significantly decreased?P<0.01?.3.The results of cell proliferation show:The results of CCK-8 method showed that the absorbance value?OD?at 0 h of Anti-NC group and Anti-222 group was not significantly different from that of Con group.The OD value of Anti-222 group was significantly lower than Con in the 2-4 days after transfection.Group and Anti-NC group?P<0.01?;the OD value of Anti-222+siR-Ddit4 group was significantly higher than that of Anti-222 group?P<0.01?;Western blotting results showed that the expression level of PCNA protein in Anti-222 group was decreased?P<0.001?;the expression level of PCNA protein in Anti-222+siR-Ddit4 group was higher than that of Anti-222?P<0.05?.4.The results of apoptosis show:Flow cytometry results showed that the early and late withering rates of cells in Anti-222 group were significantly higher than those in Con and Anti-NC groups?P<0.001?;the early withering rate of cells in Anti-222+siR-Ddit4 group And the rate of late withering was significantly lower than that in the Anti-222 group?P<0.001?;The AO-EB results showed that there were significantly more early apoptotic cells and late apoptotic cells in Anti-222 than in the Con and Anti-NC groups,with green and orange-red,which were contracted or bead-shaped?P<0.001?;Western blotting results showed that the expression level of Cleaved Caspase-3 protein in Anti-222 group was significantly increased?P<0.001?;the expression level of PCNA protein in Anti-222+siR-Ddit4 group was significantly lower than that in Anti-222 group?P<0.001?.5.The cell cycle results show:Flow cytometry results showed that the number of cells in the G1 phase of the Anti-222 group was significantly higher than that of the Con and Anti-NC groups,while the number of cells in the S and G2 phase was significantly lower than that of the Con and Anti-NC groups(P_G1<0.001,P_S<0.001);The number of cells in the Anti-222+siR-Ddit4 group in the G1 phase was significantly lower than that in the Anti-222 group,and the number of cells in the S and G2 phase was significantly higher than in the Anti-222 group(P_G1<0.001,P_S<0.001).6.The cell migration results showed that the cell scratching results showed that the Anti-222 group had significantly reduced cell migration ability?P<0.001?compared with the Con and Anti-NC groups;the Anti-222+siR-Ddit4 group migration ability was significantly enhanced compared to Anti-222?P<0.001?.7.Real-time PCR results showed that?-catenin and TCF4 mRNA expression was significantly reduced in the Anti-222 group compared with the Con and Anti-NC groups?P<0.001?;the Anti-222+siR-Ddit4 group was significantly lower than the Anti-The expression was significantly increased in 222 groups?P<0.05?.8.Western blotting results showed that compared with the Con and Anti-NC groups,the expression levels of?-catenin,TCF4,C-myc,and CycinD1 in the Anti-222 group were significantly reduced?P<0.01?;Anti-222+siR-The expression of Ddit4 group was significantly higher than that of Anti-222 group?P<0.001?.Conclusions:1.Reduced miR-222-3p expression may play an important role in the development of neural tube defects induced by ATRA.2.The target gene of miR-222-3p is Ddit4.3.miR-222-3p may cause the occurrence of NTDs by affecting Ddit4 expression and participating in the Wnt signaling pathway,causing abnormal cell proliferation,apoptosis,and migration.