Studies On The Role And Mechanism Of Circular RNA And Rno_circ₀004002/miR-342-5p/Wnt3a Axis In The Development Of Anorectal Malformations

Background: Anorectal malformations（ARM）are the most common congenital anomalies with an incidence of approximately 1:5000 to 1:1500 of live births.At present,most of the diseases related to ARM can be treated by operation.Although with the perfect treatment,postoperative complications required long-term medical treatment,which had severely affected the physical and mental health of the patients,and brought a heavy burden to the family.Current studies indicated that despite an unclear and complex multifactorial etiology,genetic factors including gene mutations and the interaction of molecules in the signaling pathway might be related to the occurrence of ARM.Wnt signaling pathway played an important role in all ARM-related networks.It has been proved that the canonical Wnt/β-catenin pathway was irreplaceable in several physiological processes such as embryonic development,cell proliferation,cell differentiation and cell apoptosis,which was indispensable in the normal development of anorectum.However,whether the expression and regulation mechanism of upstream molecules related to ARM remains unclear.As a large member of the non-coding RNA family,circular RNAs（circRNA）have unique characteristics.Differ from the linear RNA,circRNA are closed circular molecules with covalently closed loop structure that lack 5′–3′ polarity or a polyadenylated tail.CircRNA are molecules of conservation,diversity and enrichment,and it is often specifically expressed in the tissue or developmental stage.It has been shown that circRNA endow numerous potential functions,such as acting as competing endogenous RNAs to interact with miRNA,regulating splicing or transcription to influence gene expression,and binding to RNA-associated proteins to form RNA-protein complexes that regulate gene transcription.CircRNA play a critical role in biological processes and participate in multiple processes involved in disease progression.Thus,this evidence suggested the existence of opportunities to target circRNA for therapeutic intervention and as novel diagnostic biomarkers.However,until now,it has not been reported whether circRNAs can act as ceRNAs to be involved in anorectal development during ARM in rat embryos.In the present study,we used the Wistar rat model induced by ethylenethiourea（ETU）to study circRNAs in ARM and normal rat embryos.We used high-throughput sequencing technology to identify differentially expressed circRNAs related to anorectal development in ARM and normal rat embryos.After qRT-PCR verification,selected rno_circ₀004002 as the target circRNA.Functionally,explored regulation role of rno_circ₀004002 by cell proliferation,cell apoptosis and transition of epithelial to mesenchymal in IEC-6 cells.Moreover,using qRT-PCR and Western-blot to detect the expression of rno_circ₀004002,miR-342-5p and Wnt3 a in ARM and normal anorectal tissues.Mechanistically,dualluciferase reporter assay and cell transfection was performed to detect the relationship between rno_circ₀004002 and miR-342-5p,miR-342-5p and Wnt3 a,rno_circ₀004002 and Wnt3 a,which demonstrated that rno_circ₀004002 acted as sponge of miR-342-5p to regulate expression of Wnt3 a and cell functions.Overall,we provided a scientific basis for the role of rno_circ₀004002 in ARM,and rno_circ₀004002 might be an effective therapeutic target for the treatment of ARM.Methods: 1.Animal model and tissue collection.Female（220²50g）and male（250³00g）Wistar rats were mated overnight（ratio 4:1）,gestational day 0（GD0）was defined as when sperm were observed in vaginal smears after overnight mating.Some gravid Wistar rats were randomly divided into ARM and normal group.In the ETU-induced group,the gravid rats were administered a single dose of 125 mg/kg of 1% ETU via oral gavage on GD10.As control,another rats were administered an equal dose of saline,without ETU,via oral gavage on GD10.Embryos were harvested via cesarean delivery on GD17,GD19 and GD21,divided into ARM embryos（short or no tail without anus）or normal embryos（normal tail with normal anus）.The anorectal tissues were dissected,removed from surrounding tissues under magnification,and immediately frozen in liquid nitrogen to prepare for high-throughput sequencing,qRT-PCR and Western-blot.2.High-throughput sequencing technology were used to identify differentially expressed circRNAs related to anorectal development in ARM and normal rat embryos on GD19.Bioinformatic tools,including Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis were undertaken to predict the functions of parental genes.After validated expression of six selected circRNAs by qRT–PCR,binding miRNAs were predicted by miRanda database.3.PCR reverse cloning and Sanger sequencing were used to confirm the full-length sequence of rno_circ₀004002.qRT-PCR was conducted to confirm the expression of rno_circ₀004002 in anorectal tissues and in IEC-6 cells.After transfected with siRNA or pLO5-ciR vectors to down-regulate or up-regulate expression of rno_circ₀004002,the behavior of cell proliferation,apoptosis and EMT were detected in IEC-6 cells.4.Dual-luciferase reporter assay and cell transfection were used to validate the regulation of rno_circ₀004002 and miR-342-5p,miR-342-5p and Wnt3 a.qRT–PCR was conducted to confirm the expression of miR-342-5p and Wnt3 a in ARM and normal anorectal tissues.After transfected with si-rno_circ₀004002,miR-342-5p inhibitor or sirno_circ₀004002+miR-342-5p inhibitor,expression of Wnt3 a and behavior of cell proliferation,apoptosis and EMT were detected in IEC-6 cells.5.Statistical analysis.SPSS 21.0 and Prism 7.0 software were used for statistical analysis.Student’s t-test and one-way ANOVA were used to analyze the differences,with at least three biological replicates.All data were presented as mean±standard deviation（SD）,and p<0.05 was considered statistically significant.Results: 1.High-throughput sequencing analysis showed that 78 circRNAs were differentially expressed in ARM and normal anorectal tissues on GD19.Compared with normal group,37 circRNAs were down-regulated and 41 circRNAs were up-regulated in ARM group.GO and KEGG analysis demonstrated that Oxygen and Tight junctions were the major and meaningful enrichment of circRNA parental genes.Validation of six selected circRNAs by qRT-PCR indicated that the expression patterns were consistent with the sequencing results,however,with the different fold changes.Therefore,our data confirmed the existence of circRNAs in anorectal tissues,with differential expression observed in ARM and normal embryos.2.Confirmed by PCR reverse cloning and Sanger sequencing,the full-length sequence of rno_circ₀004002 was obtained and the length was 410 bp.qRT-PCR results showed that,on GD17,GD19 and GD21,the expression of rno_circ₀004002 was downregulated in ARM anorectal tissues relative to the normal tissues,and it was remarkably decreased on GD19 and GD21.In IEC-6 cells,rno_circ₀004002 was mostly in cytoplasm,and silencing of rno_circ₀004002 obviously increased cell apoptosis,inhibited cell proliferation and repressed transition of epithelial to mesenchymal,while,overexpression of rno_circ₀004002 had the opposite functions.Therefore,our data indicated rno_circ₀004002 played a key role in later stage of anorectal development.Downregulation of rno_circ₀004002 might lead to ARM and repressed multiple functions of IEC-6 cells.3.Dual-luciferase reporter assay and cell transfection results showed that rno_circ₀004002 acted as sponge of miR-342-5p and Wnt3 a was a direct downstream target of miR-342-5p.Rno_circ₀004002 and miR-342-5p were co-transfected to measure the effect on Wnt3 a expression and cell functions.When transfected with sirno_circ₀004002,Wnt3 a was decreased;and miR-342-5p inhibitor could partially upregulate Wnt3 a expression level when co-transfected with si-rno_circ₀004002.Additionally,miR-342-5p inhibitor could partially rescue the suppression of sirno_circ₀004002 on cell proliferation and EMT,and miR-342-5p inhibitor could also partly reverse the promotion of si-rno_circ₀004002 on cell apoptosis.In anorectal tissues,on GD17,GD19 and GD21,compared with normal group,miR-342-5p was up-regulated in ARM group and it was significantly increased on GD19 and GD21,while,Wnt3 a was down-regulated in ARM group and it was significantly decreased on GD19 and GD21.Our data suggested that,up-regulation of miR-342-5p and down-regulation of Wnt3 a would contribute to ARM.Rno_circ₀004002 acted as sponge of miR-342-5p to regulate expression of Wnt3 a.Rno_circ₀004002 played a regulatory role in cell proliferation,apoptosis and EMT through rno_circ₀004002/miR-342-5p/Wnt3 a axis.Conclusions: 1.CircRNA exist in anorectal tissues,with differential expression observed in ARM and normal embryos.2.Rno_circ₀004002 played a key role in later stage of anorectal development.Down-regulation of rno_circ₀004002 might lead to ARM,and increased cell apoptosis,inhibited cell proliferation and repressed transition of epithelial to mesenchymal.3.Up-regulation of miR-342-5p and down-regulation of Wnt3 a would contribute to ARM.Rno_circ₀004002 acted as sponge of miR-342-5p to regulate expression of Wnt3 a.Rno_circ₀004002 played a regulatory role in cell proliferation,apoptosis and EMT through rno_circ₀004002/miR-342-5p/Wnt3 a axis.