LncRNA ENSRNOT00000093006.1 Regulates Cell Proliferation,Apoptosis And Its Mechanism During The Development Of Anorectal Malformation Embryos In Rats

Objective:Anorectal malformations(ARM)is a common congenital malformation in pediatric surgery,the incidence rate is about 1/5000-1/1500.Surgeries can treat most of the diseases related to ARM.However,long-term postoperative complications have affected the growth and development of children and the quality of life.Therefore,it is very important to explore the pathogenesis of ARM.At present,the researches on ARM mainly focus on the gene-expression levels,and the specific regulation mechanism of gene remains to be further discovered.In the previous study,we screened the differentially expressed lncRNA by high-throughput sequencing of terminal hindgut tissue of rat embryo model with congenital anorectal malformation induced by ethylene thiourea(ETU).Explore the expression level of lncRNA ENSRNOT00000093006.1 in anorectal malformation rats,and explore the function of this lncRNA in IEC-6 cells after interference and overexpression,so as to speculate its role in the occurrence and development of diseases.Methods:①Animal models were made and materials were obtained.A total of 30 Wistar rats were randomly divided into two groups:male and female rats.The ratio of male and female rats was 4:1 at night.Vaginal smears were performed in the morning of the next day.When sperm or vaginal suppository were seen,it was defined as gestational day 0(GD0).Pregnant rats were randomly divided into arm group and normal group.At GD10,rats in ARM group were given ETU(dissolved in normal saline,125 mg/kg),while rats in normal group were given the same amount of normal saline.The fetal rats were removed at GD16,GD17 and GD19.The fetal rats in arm group had anal atresia,short tail or no tail deformity.The tail and anal opening of normal group were normal.GD16 and GD17 were observed by microscope.GD19 could observe the sacrococcygeal morphology of fetal rats with naked eyes.The hindgut tissue of fetal rats was aseptically removed,and the teratogenic rate was counted.②The difference of expression level of lncRNA ENSRNOT00000093006.1 in hindgut tissue of fetal rats between ARM group and normal group was detected by qRT-PCR.The difference of expression level of lncRNA ENSRNOT00000093006.1 in nucleus and nucleus was verified by nuclear cytoplasmic separation and qRT-PCR.③LncRNA ENSRNOT00000093006.1 siRNA and over-expression plasmid were used to down regulate or up regulate the expression of lncRNA ENSRNOT00000093006.1 in IEC-6 cells.Cell proliferation was measured by CCK-8(cell counting kit-8),and apoptosis was detected by flow cytometry.④Western blot was used to detect the expression of Wnt5a protein in the total protein extracted from IEC-6 cells after lncRNA ENSRNOT00000093006.1 was interfered with and over-expressed.⑤The results of qRT-PCR were analyzed by 2-△△CT showed the relative expression level,Western blot bands were analyzed semi quantitatively by ImageJ software,and the experimental data were analyzed by prism 8.0.Three biological replicates were set in each experiment.Independent sample t test was used to compare the two samples,and the difference was considered to be statistically significant when p<0.05.Results:①Compared with the normal group,the expression of lncRNA ENSRNOT00000093006.1 was down regulated in GD16,GD17 and GD19 in ARM group.The expression of lncRNA ENSRNOT00000093006.1 was down regulated significantly in GD16 and GD19,and the difference was statistically significant(P<0.01);②The expression of lncRNA ENSRNOT00000093006.1 in the cytoplasm and nucleus of IEC-6 cells was consistent,and the difference was not statistically significant(P>0.05);(3)LncRNA ENSRNOT00000093006.1 could inhibit cell proliferation and promote cell apoptosis(P<0.01).Overexpression of lncRNA ENSRNOT00000093006.1 could promote cell proliferation and inhibit cell apoptosis(P<0.01);④The expression of Wnt5a protein decreased after lncRNA ENSRNOT00000093006.1 was interfered(P<0.01),and increased after lncRNA ENSRNOT00000093006.1 was overexpressed(P<0.01).Conclusion:In conclusion,this study verified that the expression of lncRNA ENSRNOT00000093006.1 in rat anorectal tissue was down regulated compared with the normal group.Cell experiment verified that reducing the expression of lncRNA ENSRNOT00000093006.1 in IEC-6 can inhibit cell proliferation,promote cell apoptosis,and inhibit the expression of Wnt5a to participate in the occurrence and development of ARM.