| Objective:Congenital anorectal abnormality is a complex gastrointestinal malformation common in pediatric surgery with complicated types and pathological changes,and it’s difficulte to treat,which can severely affect the life and prognosis of patients.At present,the research on this disease is mostly focused on the gene level,and the specific regulatory mechanism of the gene has yet to be further discovered.In this study,ethylenethiourea(ETU)was used to induce congenital anorectal malformation in rat models.High-throughput sequencing of hindgut tissue was performed to explore the expression level of differentially expressed lncRNA AARB07048878.1 in anorectal malformed rats.Its function in IEC-6 cells was explored by interfering and over-expressing it for further speculate its role in disease development.Methods:① Forty-two Wistar pregnant rats were divided into ARM group and normal group.Pregnant rats were given ETU at the amount of 125 mg/kg and saline at GD10, pregnant rats were given cesarean section at GD16,GD17,and GD19.Fetus at GD16 and GD17 were observed under a microscope,GD19 was observed macroscopically,calculate the fetal rat malformation rate,and collect the terminal hindgut tissue from fetal rats with anal atresia,short tail or no tail deformity.② The expression of lncRNA AARB07048878.1 in the anorectal tissue in the ARM group and the normal group was detected by real-time PCR.③ Design the specific interference sequence Ribo lncRNA smart silencer and overexpression plasmid of lncRNA AARB07048878.1,use riboFECT CP Reagen to transfect the specific interference sequence into IEC-6 cells,and use Lipofectamine 3000 to transfect the specific overexpression plasmid.CCK-8 reagent was used to detect the change of cell proliferation ability,and flow cytometry was used to detect the change of apoptosis rate.④The total protein was gained from IEC-6 cells after lncRNA AARB07048878.1 was interfered and over-expressed,and the expression level of Wnt5a protein in the cells was detected by Western blot.⑤ Statistical analysis All experiments were performed in three sets of duplicate samples.The relative expression of qRT-PCR results was expressed by 2-ΔΔCT.The western blot strips were analyzed semi-quantitatively by ImageJ software.The experimental data were analyzed by SPSS 24.0 software and Prism 8.0.,Pairwise comparison was performed using independent sample t test,P<0.05 was considered with statistical significant.Results:①In Comparison with the normal group,the expression of lncRNA AARB07048878.1 was down-regulated at 16d,17d and 19d of pregnancy in the ARM group.and the expression was significantly down regulated at 17d and there were statistical differences(P<0.05);② Interfering with the expression of lncRNA AARB07048878.1 inhibited cell proliferation and promoted apoptosis(P<0.05);③Overexpression of lncRNA AARB07048878.1 increased cell proliferation and inhibited apoptosis(P<0.05);④Interfered with lncRNA AARB07048878.1,the expression of Wnt5a protein decreased(P<0.05);after overexpression of lncRNA AARB07048878.1,Wnt5a protein expression increased(P<0.05)Conclusion:In summary,this study observes the changes and effects of lncRNA in the development of ARMs at the tissue and molecular levels.It is found that lncRNA AARB07048878.1 participates in the development of ARMs by inhibiting cell proliferation and promoting apoptosis,and its effect may be done by inhibiting Wnt5a We will also explore the molecular mechanism of lncRNA AARB07048878.1 on Wnt5a regulation through further experiments.The results of this study provide an experimental basis for further exploring the molecular mechanism of lncRNA on the development of ARMs,and provide new targets and ideas for the early detection and treatment of ARMs. |
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