Differential miRNA expression analysis during late stage terminal hindgut development in fetal rats

Terminal hindgut deformity is the leading digestive tract malformation, however, the etiology and pathogenesis remained unknown. To date, gene expression abnormalities were considered the primary cause of these diseases. miRNAs have been found to play an important role in regulating the expression of genes.

A total of 24 pregnant rats were randomly divided into two groups. The experimental group (n=12) received 1% ethylenethiourea (125mg/kg) by gavage on gestational day 11, while the control group (n=12) received the same volume of distilled water. From each group, fetal rats were obtained by cesarean section on gestational day 16. For the extraction of total RNA, 1 cm rectum samples were obtained from four fetal rats that had similar weights. Chip hybridization was conducted after poly(A) and biotin were added to the RNA samples, and this was followed by washing, dyeing, and scanning of the chip. Differences identified in the miRNA expression profiles and the target gene analysis results were further analyzed to identify potential regulators of terminal hindgut development.

Compared with the control group, 111 miRNAs expressed in the terminal hindgut of the experimental group were up-regulated on gestational day 16, while 117 miRNAs were down-regulated. The ten miRNAs with the greatest differential expression profiles between the experimental and control samples were selected for target gene prediction, pathway analysis, and gene ontology analysis. A subset of these miRNAs was found to be closely related to rat fetus terminal hindgut growth and development. In addition, target gene analysis showed that miR-193 may have an important role in regulating a key gene in anorectal development, Hoxd13. This role was confirmed in a dual luciferase reporter assay when miR-193 was able to inhibit expression of a reporter gene under the control of the 3' untranslated region of the Hoxd13 gene in the human embryonic kidney cell line, 293T. Real-time PCR and Western blotting experiments further showed that the expression of Hoxd13 was significantly lower when miR-193 was highly expressed in rat intestinal epithelial cells. The differences in both sets of experiments were statistically significant compared with the negative control group (P<0.05).

These data support an important regulatory role for miRNAs in the expression of target genes during terminal hindgut development in fetal rats. In particular, miR-193-mediated inhibition of Hoxd13 was found to be significant in rat intestinal epithelial cells.