Preliminary study of expression of Sfrp5/Dkk4 genes with ethylenethiourea-induced congenital anorectal malformations in rats

Abstract

Objective To detect the expression of Sfrp5/Dkk4 genes in Wistar embryos with anorectal malformations (ARM) induced by ethylene thiourea (ETU). Methods The Wistar pregnant rats received the injections of ethylene thiourea (ETU) and teratogenic ARM.At E15, E19, E17 and E21, 6 specimens were harvested in each group.Under anatomical microscopic observation of anal location, no anal opening confirmed the presence of anorectal malformations. And terminal rectum tissues of control group received normal saline (NS). The expressions of Sfrp5/Dkk4 were detected by Western blot and quantitative real-time PCR (qRT-PCR) in E15, E17, E19 and E21. And quantitative and comparative analyses were performed. Results For ETU teratogenic ARM, the protein expressions of Sfrp5 were 63.55±0.35, 24.51±0.41, 13.28±0.09 & 49.67±0.12(NE15, NE17, NE19 & NE21)in normal group, 21.43±0.18, 59.57±0.44, 61.23±0.47 & 32.73±0.51 (AE15, AE17, AE19 & AE21) in ARM group and 52.17±1.08, 23.66±0.87, 16.21±1.33 & 50.01±2.03 (NS15, NS17, NS19 & NS21) in NS group; For ETU teratogenic ARM, the protein expressions of Dkk4 were 20.07±0.09, 47.15±0.15, 39.88±0.37 & 13.75±0.47 (NE15, NE17, NE19 & NE21) in normal group, 54.27±0.61, 19.43±0.25, 21.09±0.17 & 57.53±0.49 (AE15, AE17, AE19, & AE21) in ARM group and 23.11±0.15, 45.89±0.67, 41.44±0.37 & 17.57±1.99 (NS15, NS17, NS19 & NS21) in NS group respectively. The protein expressions of Sfrp5/Dkk4 showed significant differences in E17 and E19 in normal and ARM groups(P<0.05)and significant differences existed in E17 and E19 in NS and ARM groups too (P<0.05). The mRNA levels of Sfrp5 was 1.63 folds higher in AE17 group than that in NE17 group (P=0.049), 1.77 folds higher in AE19 group than that in NE19 group (P=0.022), 2.68 folds higher in AE17 group than that in NS17 group (P=0.041) and 3.29 fold higher in AE19 group than that in NS19 group (P=0.034). The mRNA levels of Dkk4 was 1.75 folds higher in NE17 group than that in AE17 group (P=0.045), 1.87 folds higher in NE19 group than that in AE19 group (P=0.024), 2.04 folds higher in AE17 group than that in NS17 group (P=0.004) and 1.36 folds higher in AE19 group than that in NS19 group (P=0.028). Conclusions Abnormal expressions of Sfrp5/Dkk4 protein and mRNA may be two major pathogenic factors of ARM of E17 and E19. This discovery provides a new mechanistic insight of ARM. Key words: Rat; Cogenital ano-rectal malformation; Embryonic development; Gene expression