Differential expression of microRNAs in the late stage terminal hindgut development in fetal rats / 中华实用儿科临床杂志

ABSTRACT

Objective To detect the expression of microRNA(miRNAs)in the late stage terminal hindgut de-velopment in fetal rats.Methods Twenty -four female rats were randomly divided into 2 groups.They were matched in proportion of male and female 21 .The experimental rats(n =1 2)received 1 0 g/L ethylenethiourea (1 25 mg/kg)by gavage on gestational day 1 1 ,and the control rats (n =1 2)received same amount of distilled water.The fetal rats were obtained by caesarean section on the gestational day 1 6 in each group.One centimeter rectum was taken out from 2 similar weight fetal rats for extracting total RNA by Trizol method.There were 24 fetal rats in each group.Chip hybri-dization was conducted after Poly(A)and biotin added to the RNA.Then,the chip was washed and dyed,and scanned thereafter.According to the differences in the expression profile of miRNAs and target gene analysis results,the miRNAs probably regulating the key gene of hindgut development was selected for target genes expression analysis.Results Compared with control group,expressions of 1 1 1 miRNAs in terminal hindgut of experimental group were up -regulated on the gestational day 1 6,and 1 1 7 miRNAs were down -regulated.Ten miRNAs of biggest differential expression be-tween them were selected for target genes prediction,pathway analysis,and Gene Ontology analysis.The results showed that some genes were closely related to rat fetus terminal hindgut growth and development,such as Shh,Hoxd13,and so on.According to the differential expression of miRNAs and target genes analysis,miR -1 93 might have an important role in the Hoxd13 gene for anorectal development.Real -Time PCR and Western blot showed that the expression of Hoxd13 and its protein level were significantly decreased when miR -1 93 highly expressed in rat intestinal epithelial cells,and the difference was statistically significant compared with the control group(1 .00 ±0.1 2 vs 0.71 ±0.1 0) (0.88 ±0.06 vs 0.75 ±0.08)(t =3.329,3.1 30;P =0.029,0.01 1 ).Conclusions miRNAs probably have an im-portant regulatory role in their target gene expression in terminal hindgut development of fetal rats,and miR -1 93 can significantly inhibit the expression of Hoxd13 gene in rat intestinal epithelial cells.