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Differential miRNA expression analysis during late stage terminal hindgut development in fetal rats
Journal of Pediatric Surgery 52 (2017) 1516–1519
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Basic Science
Differential miRNA expression analysis during late stage terminal hindgut development in fetal rats☆ Shuguang Jin ⁎, Junxiang Wang, Hong Chen, Bo Xiang Department of Pediatric Surgery, West China Hospital of Sichuan University, Chengdu 610041, China
a r t i c l e
i n f o
Article history: Received 4 October 2016 Received in revised form 20 January 2017 Accepted 20 February 2017 Key words: MiRNA Hindgut Gene expression Development Rat
a b s t r a c t Background: 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. Methods: A total of 24 pregnant rats were randomly divided into two groups. The experimental group (n = 12) received 1% ethylenethiourea (125 mg/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. Results: 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, 293 T. 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 b 0.05). Conclusion: 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. © 2017 Elsevier Inc. All rights reserved.
Terminal hindgut deformity, or congenital anorectal malformation, is the leading digestive tract malformation diagnosed, and has an incidence of 1/5000 to 1/1500 [1]. It has been reported that approximately one-third of these patients exhibit some degree of functional disorders of defecation, with 86.4% experiencing high anorectal malformation, 47.9% experiencing median anorectal malformation, and 27% experiencing low anorectal malformation [1]. Moreover, the higher the position of an imperforate anus, the higher the incidence of defecation dysfunction and the greater the impact on quality of life. To date, the etiology and
pathogenesis of anorectal malformation have remained unknown because of a wide disease spectrum, phenotypic diversities, and contributions by multiple genes, among other factors. More recently, miRNAs have been found to play an important role in regulating the expression of genes related to biological growth and development and incidence of disease. Therefore, the aim of this study was to conduct a preliminary analysis of miRNA expression in the late stage of rat fetus terminal hindgut development. 1. Materials and methods
☆ Supportive foundations: The study was supported by fund programs of Doctoral Program of Higher Education of China (20130181120042) and Basic Research Project of Sichuan Province (2013JY0174) ⁎ Corresponding author. E-mail address: [email protected] (S. Jin). http://dx.doi.org/10.1016/j.jpedsurg.2017.02.015 0022-3468/© 2017 Elsevier Inc. All rights reserved.
1.1. Experimental animals A total of 36 healthy and mature Sprague Dawley (SD) rats of similar weight (24 females, 12 males) were purchased from the
S. Jin et al. / Journal of Pediatric Surgery 52 (2017) 1516–1519
1517
Fig. 1. Cluster analysis of miRNAs of differential expression between two groups.
animal experiment center of West China Medical Center of Sichuan University. The rats were divided into two equal groups. Two female rats were mated to one male rat. Gestational day 0 was defined as the presence of sperm in a vaginal smear after overnight mating. The experimental group received an intragastric administration of 1% ethylenethiourea (ETU, 125 mg/kg) on gestational day 11. In parallel, the control group was administered an equal amount of distilled water. On gestational day 16, four rat fetuses of similar weight were acquired by cesarean section from each of pregnant rats, thereby resulting in the use of approximately 48 fetal rats in each of groups. One centimeter resections of terminal hindgut tissue were collected for the extraction of RNA with Trizol methods.
1.2. Chip detection of miRNA expression RNA quality was inspected with a NanoDrop 2000 and Agilent Bioanalyzer 2100. The RNA samples then underwent addition of a poly(A) tail and biotin labeling. Following hybridization of these prepared RNA samples to a GeneChip miRNA 4.0, the chip was washed and dyed with a GeneChip Hybridization Wash and Stain Kit (Affymetrix). Then it was scanned to obtain images for data analysis. 1.3. Differential gene expression profiles of miRNAs and target gene analysis MiRNAs expressed in the terminal hindgut samples that exhibited differential expression between the experimental group and control
Fig. 2. Target gene enrichment analysis of miRNAs of differential expression between two groups.
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S. Jin et al. / Journal of Pediatric Surgery 52 (2017) 1516–1519
1
2
3
Hoxd-13 GAPDH Fig. 5. Western blot assays performed in IEC-6 rat intestinal epithelial cells (1 for oligonucleotide negative control; 2 for small interfering RNA positive control; 3 for miR193 experimental group).
1.5. Regulatory action of miRNAs on the expression of their corresponding target genes
Fig. 3. Dual luciferase experiments performed in human embryonic 293 T kidney cells (1 for 3′UTR-empty vector + miR-193; 2 for 3′UTR vector + miR-193; 3 for 3′UTR-mutant vector + miR-193; 4 for 3′UTR-positive control + miR-193-empty vector; 5 for 3′UTRpositive comtrol + miRNA-positive control).
group samples on gestational day 16 were subjected to cluster analysis. The screening criterion to identify a significant difference was a |foldchange | N 2. The ten miRNAs with the largest differential expression levels were selected for target predicting using Targetscan, microRNA. org and miRDBase database to identify potential target genes of miRNAs. Target gene enrichment analysis was subsequently conducted with the gene pathways listed in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta databases. Target gene functional classifications were analyzed by Gene Ontology Enrichment Analysis.
1.4. MiRNA regulation via the 3′untranslated region (UTR) of target genes Dual luciferase experiments were performed in human embryonic 293 T kidney cells to determine whether the miRNAs of interest maintained a regulatory role via the 3′UTR of their target genes. For this, primers were designed and synthesized for PCR amplification of the 3′ UTR sequences of the predicted target genes expressed in the hindgut of fetal rats. Restriction enzyme recognition sites were introduced in the upstream and downstream primers for subsequent cloning into reporter plasmids. After PCR amplification, digestion and purification of the PCR products, the 3′UTR cassettes were individually ligated into reporter plasmids and transformed into competent cells for amplification. Extracted plasmids were sequenced. A mutant luciferase reporter plasmid was constructed using the same method with primers that introduced mutated regions of interest. Both the plasmids containing the miRNAs and the reference plasmids were transfected into 293 T cells and luciferase activity was assayed.
IEC-6 rat intestinal epithelial cells were transfected with miRNAs, a negative control oligonucleotide, and a positive control small interfering RNA with Lipofectamine 2000 reagent (Invitrogen). Briefly, total RNAs were extracted and two-step Real-time PCR was conducted with primers designed for the miRNAs and target genes. Solubility curves of the amplified products were subsequently generated and relative quantitations were analyzed using F = 2 − ΔΔCt. In addition, total protein samples were extracted and separated by SDS-PAGE electrophoresis. Following transfer of these separated proteins onto PVDF membranes, expression levels of Hoxd13 protein were detected following the incubation of these membranes with primary and secondary antibodies, membrane washing, and X-ray radiography.
1.6. Statistical analysis Statistical analysis was performed using SPSS statistical software, version 19.0. Between-group differences were tested with independentsample student t test, analysis of variance, or nonparametric test as appropriate. A probability value of P b 0.05 was taken to represent a significant difference.
2. Results 2.1. MiRNAs differential expression profile and target gene analysis Compared with the control group, 111 miRNAs were up-regulated in the experimental rat fetus terminal hindgut group, while 117 miRNAs were down-regulated. The results of a cluster analysis are shown in Fig. 1. The target genes for the ten miRNAs that exhibited maximum differential expression were predicted, and both enrichment analysis (Fig. 2) and gene ontology functional classification analyses were performed. Genes previously shown to regulate fetal rat terminal hindgut development were identified, including Shh, Gli2/Gli3, Hoxd13, Hoxa13, Hoxa11, and the downstream targets EphB2 and EphB3. In combination, these data support an important regulatory role for miR-193 in the expression of Hoxd-13 in the rat fetus terminal hindgut.
2.2. Regulatory role of miR-193 in Hoxd13 gene expression
Fig. 4. Real-time PCR experiments performed in IEC-6 rat intestinal epithelial cells.
To further characterize the regulatory role of miR-193, the 3′UTR of Hoxd13 was assayed in human embryonic 293 T kidney cells. Expression of miR-193 significantly inhibited the expression of Hoxd13 via the 3′ UTR of the Hoxd13 gene (P b 0.05, Fig. 3). Real-time PCR also showed that mRNA levels of Hoxd13 were significantly decreased compared with the control group when miR-193 was highly expressed in IEC-6 rat intestinal epithelial cells (P b 0.05, Fig. 4). The upstream and downstream primers used for Hoxd13 were: 5′-GTGGAGAAGTACATGGACGT3′ and 5′-TAGCCCTGGTAGAAGGACACT-3′, respectively. Similarly, Western blot assays showed that expression of Hoxd13 significantly decreased compared with the negative control group (P b 0.05, Fig. 5).
S. Jin et al. / Journal of Pediatric Surgery 52 (2017) 1516–1519
3. Discussion MiRNAs are a category of noncoding single-stranded RNA molecules with a length of approximately 22 nucleotides. The main function of miRNAs is to act on the 3′-terminal noncoding region of target gene mRNAs to inhibit translation or mRNA degradation, thereby providing post-transcriptional regulation of gene expression. In recent years, it has been demonstrated that miRNAs play an important role in the development of many systems, such as nerve, heart, blood, and gastrointestinal tract [2–5]. In the present study, gene expression abnormalities were the primary cause of the hindgut or anorectal malformations that occurred. In a study conducted by Mandhan et al. [6], the expression levels of Shh, BMP-4, Hoxa13, and Hoxd13 were lower in the rectums of rats with anorectal malformations than in rats with normal rectums by RT-PCR and real-time PCR. In a study by Dan et al. [7], expression of Hoxd-13 also appeared to decrease in the urorectal septum, intestinal epithelium, cloacal membrane, and anal canal epithelium when anorectal malformations were present in rat fetuses as detected by immunohistochemical examination and RT-PCR. Furthermore, in the distal rectum of children, decreased expression of HOXD13 has been detected in association with anorectal malformations [8]. The above mentioned data confirm that the Hox gene plays a key role in the development of the anorectum. In the present study, the teratogenic effect of ETU that was applied during a critical embryonic period between days 11 and 13 manifested as differences in miRNA expression during the late stage of fetal rat terminal hindgut development [9]. These differences were subsequently analyzed in combination with target gene analyses. Both miRNAs and their target genes are highly conserved heredity genes. The sequences of miRNA genes usually derive from inverted repeats of the 3′-terminal UTR of their target genes, and miRNA binding sites and their target genes are often located near protein coding genes [10]. The expression patterns of both miRNA genes and their target genes have been found to be similar, and the expression profiles of the miRNAs resembled those of developmental switches which control the range and intensity of target gene expression [11]. Shh has been found to be an important signaling pathway in the regulation of anorectum development [6,12–14]. Shh is a highly conserved heredity gene, its protein product is a secreted protein, and it is an important regulator of primary function for individual morphology development. Shh is mainly expressed in the endoderm of the epithelial layer and it acts on its receptor Ptc. When Shh combines with Ptc, the inhibition on protein Smo is eliminated. The zinc finger protein transcription factor Gli of Shh is activated and downstream intracellular signaling is triggered [15]. As a result, the proliferation of epithelial stem and neural stem cells is promoted [16,17]. In the present study, target gene predictions for the differentially expressed miRNAs detected included important genes of the Shh signaling pathway, i.e., Shh, Gli2/Gli3, and others [18,19]. It has been demonstrated that Shh regulates intestinal development via control of the Hox gene. In chicken embryos, abnormal expression of Shh leads to ectopic expression of Hoxd13 [20]. Hox genes are members of a highly conserved family of homologous box genes, and these genes control many aspects of development, especially in the human hindgut [21,22]. The human Hox gene family contains 39 genes which have been
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divided into four clusters, HOXA-D [23]. It has been predicted that most Hox genes are targets of miRNAs, and 30/39 human Hox genes contain one or more miRNA binding sites [24,25]. In the present study, a subset of Hox genes involved in the control of anorectum development were identified, including Hoxd13, Hoxa13, Hoxa11, and their downstream targets. Lower levels of Hoxd13 expression were also found to be related to higher expression levels of miR-193 in fetal rat terminal hindgut tissue. Thus, the present results provide a new perspective and direction for future studies of the etiology and pathogenesis of anorectal malformations. References [1] Shi CR, Jin XQ, Li ZZ. Pediatric surgery. 4th ed. Beijing: People's Medical Publishing House; 2009 324–33. [2] Chen W, Qin C. General hallmarks of microRNAs in brain evolution and development. RNA Biol 2015;12:701–8. [3] Fuller AM, Qian L. MiRiad roles for microRNAs in cardiac development and regeneration. Cell 2014;3:724–50. [4] Raghuwanshi S, Karnati HK, Sarvothaman S, et al. microRNAs: key players in hematopoiesis. Adv Exp Med Biol 2015;887:171–211. [5] Liang G, Malmuthuge N, McFadden TB, et al. Potential regulatory role of microRNAs in the development of bovine gastrointestinal tract during early life. PLoS One 2014; 9:e92592. [6] Mandhan P, Quan QB, Beasley S, et al. Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in ethylenethiourea-exposed fetal rats. J Pediatr Surg 2006;41:2041–5. [7] Dan Z, Bo ZZ, Tao Z, et al. Hoxd-13 expression in the development of hindgut in ethylenethiourea-exposed fetal rats. J Pediatr Surg 2010;45:755–61. [8] Yuzuo BAI, Hong GAO, Weilin WANG. Expression of Hoxd 13 gene in children with congenital anorectal malformation. Zhongguo Dang Dai Er Ke Za Zhi 2003;5:201–4. [9] Hirai Y, Kuwabara N. Transplacentally induced anorectal malformations in rats. Pediatr Surg 1990;25:812–6. [10] Allen E, Xie Z, Gustafson AM, et al. Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat Genet 2004;36:1282–90. [11] Stefani G, Slack FJ. Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol 2008;9:219–30. [12] Liu G, Moro A, Zhang JJ, et al. The role of Shh transcription activator Gli2 in chick cloacal development. Dev Biol 2007;303:448–60. [13] Mandhan P, Beasley S, Hale T, et al. Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats. Pediatr Surg Int 2006;22:31–6. [14] Huang Y, Zhang P, Zheng S, et al. Hypermethylation of SHH in the pathogenesis of congenital anorectal malformations. J Pediatr Surg 2014;49:1400–4. [15] Rimkus TK, Carpenter RL, Qasem S, et al. Targeting the sonic hedgehog signaling pathway: review of smoothened and GLI inhibitors. Cancers (Basel) 2016;8. http:// dx.doi.org/10.3390/cancers8020022 [pii: E22]. [16] Shyer AE, Huycke TR, Lee C, et al. Bending gradients: how the intestinal stem cell gets its home. Cell 2015;161:569–80. [17] Yu YH, Narayanan G, Sankaran S, et al. Purification, visualization, and molecular signature of neural stem cells. Stem Cells Dev 2016;25:189–201. [18] Zhang ZB, Gao H. Expression of Gli2 gene in congenital anorectal malformation. Chin J Pediatr Surg 2001;22:325–7. [19] Jin SG, Zhao YY. Research progress of sonic hedgehog gene and intestinal malformations. Chin J Pediatr Surg 2005;26:436–8. [20] Roberts DJ, Johnson RL, Burke AC, et al. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 1995;121:3163–74. [21] Illig R, Fritsch H, Schwarzer C. Spatio-temporal expression of HOX genes in human hindgut development. Dev Dyn 2013;242:53–66. [22] Wang C, Li L, Cheng W. Anorectal malformation: the etiological factors. Pediatr Surg Int 2015;31:795–804. [23] Bhatlekar S, Fields JZ, Boman BM. HOX genes and their role in the development of human cancers. J Mol Med (Berl) 2014;92:811–23. [24] Pearson JC, Lemons D, McGinnis W. Modulating Hox gene functions during animal body patterning. Nat Rev Genet 2005;6:893–904. [25] Lewis B, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell 2003;115:787–98.
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Basic Science
Differential miRNA expression analysis during late stage terminal hindgut development in fetal rats☆ Shuguang Jin ⁎, Junxiang Wang, Hong Chen, Bo Xiang Department of Pediatric Surgery, West China Hospital of Sichuan University, Chengdu 610041, China
a r t i c l e
i n f o
Article history: Received 4 October 2016 Received in revised form 20 January 2017 Accepted 20 February 2017 Key words: MiRNA Hindgut Gene expression Development Rat
a b s t r a c t Background: 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. Methods: A total of 24 pregnant rats were randomly divided into two groups. The experimental group (n = 12) received 1% ethylenethiourea (125 mg/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. Results: 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, 293 T. 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 b 0.05). Conclusion: 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. © 2017 Elsevier Inc. All rights reserved.
Terminal hindgut deformity, or congenital anorectal malformation, is the leading digestive tract malformation diagnosed, and has an incidence of 1/5000 to 1/1500 [1]. It has been reported that approximately one-third of these patients exhibit some degree of functional disorders of defecation, with 86.4% experiencing high anorectal malformation, 47.9% experiencing median anorectal malformation, and 27% experiencing low anorectal malformation [1]. Moreover, the higher the position of an imperforate anus, the higher the incidence of defecation dysfunction and the greater the impact on quality of life. To date, the etiology and
pathogenesis of anorectal malformation have remained unknown because of a wide disease spectrum, phenotypic diversities, and contributions by multiple genes, among other factors. More recently, miRNAs have been found to play an important role in regulating the expression of genes related to biological growth and development and incidence of disease. Therefore, the aim of this study was to conduct a preliminary analysis of miRNA expression in the late stage of rat fetus terminal hindgut development. 1. Materials and methods
☆ Supportive foundations: The study was supported by fund programs of Doctoral Program of Higher Education of China (20130181120042) and Basic Research Project of Sichuan Province (2013JY0174) ⁎ Corresponding author. E-mail address: [email protected] (S. Jin). http://dx.doi.org/10.1016/j.jpedsurg.2017.02.015 0022-3468/© 2017 Elsevier Inc. All rights reserved.
1.1. Experimental animals A total of 36 healthy and mature Sprague Dawley (SD) rats of similar weight (24 females, 12 males) were purchased from the
S. Jin et al. / Journal of Pediatric Surgery 52 (2017) 1516–1519
1517
Fig. 1. Cluster analysis of miRNAs of differential expression between two groups.
animal experiment center of West China Medical Center of Sichuan University. The rats were divided into two equal groups. Two female rats were mated to one male rat. Gestational day 0 was defined as the presence of sperm in a vaginal smear after overnight mating. The experimental group received an intragastric administration of 1% ethylenethiourea (ETU, 125 mg/kg) on gestational day 11. In parallel, the control group was administered an equal amount of distilled water. On gestational day 16, four rat fetuses of similar weight were acquired by cesarean section from each of pregnant rats, thereby resulting in the use of approximately 48 fetal rats in each of groups. One centimeter resections of terminal hindgut tissue were collected for the extraction of RNA with Trizol methods.
1.2. Chip detection of miRNA expression RNA quality was inspected with a NanoDrop 2000 and Agilent Bioanalyzer 2100. The RNA samples then underwent addition of a poly(A) tail and biotin labeling. Following hybridization of these prepared RNA samples to a GeneChip miRNA 4.0, the chip was washed and dyed with a GeneChip Hybridization Wash and Stain Kit (Affymetrix). Then it was scanned to obtain images for data analysis. 1.3. Differential gene expression profiles of miRNAs and target gene analysis MiRNAs expressed in the terminal hindgut samples that exhibited differential expression between the experimental group and control
Fig. 2. Target gene enrichment analysis of miRNAs of differential expression between two groups.
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S. Jin et al. / Journal of Pediatric Surgery 52 (2017) 1516–1519
1
2
3
Hoxd-13 GAPDH Fig. 5. Western blot assays performed in IEC-6 rat intestinal epithelial cells (1 for oligonucleotide negative control; 2 for small interfering RNA positive control; 3 for miR193 experimental group).
1.5. Regulatory action of miRNAs on the expression of their corresponding target genes
Fig. 3. Dual luciferase experiments performed in human embryonic 293 T kidney cells (1 for 3′UTR-empty vector + miR-193; 2 for 3′UTR vector + miR-193; 3 for 3′UTR-mutant vector + miR-193; 4 for 3′UTR-positive control + miR-193-empty vector; 5 for 3′UTRpositive comtrol + miRNA-positive control).
group samples on gestational day 16 were subjected to cluster analysis. The screening criterion to identify a significant difference was a |foldchange | N 2. The ten miRNAs with the largest differential expression levels were selected for target predicting using Targetscan, microRNA. org and miRDBase database to identify potential target genes of miRNAs. Target gene enrichment analysis was subsequently conducted with the gene pathways listed in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta databases. Target gene functional classifications were analyzed by Gene Ontology Enrichment Analysis.
1.4. MiRNA regulation via the 3′untranslated region (UTR) of target genes Dual luciferase experiments were performed in human embryonic 293 T kidney cells to determine whether the miRNAs of interest maintained a regulatory role via the 3′UTR of their target genes. For this, primers were designed and synthesized for PCR amplification of the 3′ UTR sequences of the predicted target genes expressed in the hindgut of fetal rats. Restriction enzyme recognition sites were introduced in the upstream and downstream primers for subsequent cloning into reporter plasmids. After PCR amplification, digestion and purification of the PCR products, the 3′UTR cassettes were individually ligated into reporter plasmids and transformed into competent cells for amplification. Extracted plasmids were sequenced. A mutant luciferase reporter plasmid was constructed using the same method with primers that introduced mutated regions of interest. Both the plasmids containing the miRNAs and the reference plasmids were transfected into 293 T cells and luciferase activity was assayed.
IEC-6 rat intestinal epithelial cells were transfected with miRNAs, a negative control oligonucleotide, and a positive control small interfering RNA with Lipofectamine 2000 reagent (Invitrogen). Briefly, total RNAs were extracted and two-step Real-time PCR was conducted with primers designed for the miRNAs and target genes. Solubility curves of the amplified products were subsequently generated and relative quantitations were analyzed using F = 2 − ΔΔCt. In addition, total protein samples were extracted and separated by SDS-PAGE electrophoresis. Following transfer of these separated proteins onto PVDF membranes, expression levels of Hoxd13 protein were detected following the incubation of these membranes with primary and secondary antibodies, membrane washing, and X-ray radiography.
1.6. Statistical analysis Statistical analysis was performed using SPSS statistical software, version 19.0. Between-group differences were tested with independentsample student t test, analysis of variance, or nonparametric test as appropriate. A probability value of P b 0.05 was taken to represent a significant difference.
2. Results 2.1. MiRNAs differential expression profile and target gene analysis Compared with the control group, 111 miRNAs were up-regulated in the experimental rat fetus terminal hindgut group, while 117 miRNAs were down-regulated. The results of a cluster analysis are shown in Fig. 1. The target genes for the ten miRNAs that exhibited maximum differential expression were predicted, and both enrichment analysis (Fig. 2) and gene ontology functional classification analyses were performed. Genes previously shown to regulate fetal rat terminal hindgut development were identified, including Shh, Gli2/Gli3, Hoxd13, Hoxa13, Hoxa11, and the downstream targets EphB2 and EphB3. In combination, these data support an important regulatory role for miR-193 in the expression of Hoxd-13 in the rat fetus terminal hindgut.
2.2. Regulatory role of miR-193 in Hoxd13 gene expression
Fig. 4. Real-time PCR experiments performed in IEC-6 rat intestinal epithelial cells.
To further characterize the regulatory role of miR-193, the 3′UTR of Hoxd13 was assayed in human embryonic 293 T kidney cells. Expression of miR-193 significantly inhibited the expression of Hoxd13 via the 3′ UTR of the Hoxd13 gene (P b 0.05, Fig. 3). Real-time PCR also showed that mRNA levels of Hoxd13 were significantly decreased compared with the control group when miR-193 was highly expressed in IEC-6 rat intestinal epithelial cells (P b 0.05, Fig. 4). The upstream and downstream primers used for Hoxd13 were: 5′-GTGGAGAAGTACATGGACGT3′ and 5′-TAGCCCTGGTAGAAGGACACT-3′, respectively. Similarly, Western blot assays showed that expression of Hoxd13 significantly decreased compared with the negative control group (P b 0.05, Fig. 5).
S. Jin et al. / Journal of Pediatric Surgery 52 (2017) 1516–1519
3. Discussion MiRNAs are a category of noncoding single-stranded RNA molecules with a length of approximately 22 nucleotides. The main function of miRNAs is to act on the 3′-terminal noncoding region of target gene mRNAs to inhibit translation or mRNA degradation, thereby providing post-transcriptional regulation of gene expression. In recent years, it has been demonstrated that miRNAs play an important role in the development of many systems, such as nerve, heart, blood, and gastrointestinal tract [2–5]. In the present study, gene expression abnormalities were the primary cause of the hindgut or anorectal malformations that occurred. In a study conducted by Mandhan et al. [6], the expression levels of Shh, BMP-4, Hoxa13, and Hoxd13 were lower in the rectums of rats with anorectal malformations than in rats with normal rectums by RT-PCR and real-time PCR. In a study by Dan et al. [7], expression of Hoxd-13 also appeared to decrease in the urorectal septum, intestinal epithelium, cloacal membrane, and anal canal epithelium when anorectal malformations were present in rat fetuses as detected by immunohistochemical examination and RT-PCR. Furthermore, in the distal rectum of children, decreased expression of HOXD13 has been detected in association with anorectal malformations [8]. The above mentioned data confirm that the Hox gene plays a key role in the development of the anorectum. In the present study, the teratogenic effect of ETU that was applied during a critical embryonic period between days 11 and 13 manifested as differences in miRNA expression during the late stage of fetal rat terminal hindgut development [9]. These differences were subsequently analyzed in combination with target gene analyses. Both miRNAs and their target genes are highly conserved heredity genes. The sequences of miRNA genes usually derive from inverted repeats of the 3′-terminal UTR of their target genes, and miRNA binding sites and their target genes are often located near protein coding genes [10]. The expression patterns of both miRNA genes and their target genes have been found to be similar, and the expression profiles of the miRNAs resembled those of developmental switches which control the range and intensity of target gene expression [11]. Shh has been found to be an important signaling pathway in the regulation of anorectum development [6,12–14]. Shh is a highly conserved heredity gene, its protein product is a secreted protein, and it is an important regulator of primary function for individual morphology development. Shh is mainly expressed in the endoderm of the epithelial layer and it acts on its receptor Ptc. When Shh combines with Ptc, the inhibition on protein Smo is eliminated. The zinc finger protein transcription factor Gli of Shh is activated and downstream intracellular signaling is triggered [15]. As a result, the proliferation of epithelial stem and neural stem cells is promoted [16,17]. In the present study, target gene predictions for the differentially expressed miRNAs detected included important genes of the Shh signaling pathway, i.e., Shh, Gli2/Gli3, and others [18,19]. It has been demonstrated that Shh regulates intestinal development via control of the Hox gene. In chicken embryos, abnormal expression of Shh leads to ectopic expression of Hoxd13 [20]. Hox genes are members of a highly conserved family of homologous box genes, and these genes control many aspects of development, especially in the human hindgut [21,22]. The human Hox gene family contains 39 genes which have been
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divided into four clusters, HOXA-D [23]. It has been predicted that most Hox genes are targets of miRNAs, and 30/39 human Hox genes contain one or more miRNA binding sites [24,25]. In the present study, a subset of Hox genes involved in the control of anorectum development were identified, including Hoxd13, Hoxa13, Hoxa11, and their downstream targets. Lower levels of Hoxd13 expression were also found to be related to higher expression levels of miR-193 in fetal rat terminal hindgut tissue. Thus, the present results provide a new perspective and direction for future studies of the etiology and pathogenesis of anorectal malformations. References [1] Shi CR, Jin XQ, Li ZZ. Pediatric surgery. 4th ed. Beijing: People's Medical Publishing House; 2009 324–33. [2] Chen W, Qin C. General hallmarks of microRNAs in brain evolution and development. RNA Biol 2015;12:701–8. [3] Fuller AM, Qian L. MiRiad roles for microRNAs in cardiac development and regeneration. Cell 2014;3:724–50. [4] Raghuwanshi S, Karnati HK, Sarvothaman S, et al. microRNAs: key players in hematopoiesis. Adv Exp Med Biol 2015;887:171–211. [5] Liang G, Malmuthuge N, McFadden TB, et al. Potential regulatory role of microRNAs in the development of bovine gastrointestinal tract during early life. PLoS One 2014; 9:e92592. [6] Mandhan P, Quan QB, Beasley S, et al. Sonic hedgehog, BMP4, and Hox genes in the development of anorectal malformations in ethylenethiourea-exposed fetal rats. J Pediatr Surg 2006;41:2041–5. [7] Dan Z, Bo ZZ, Tao Z, et al. Hoxd-13 expression in the development of hindgut in ethylenethiourea-exposed fetal rats. J Pediatr Surg 2010;45:755–61. [8] Yuzuo BAI, Hong GAO, Weilin WANG. Expression of Hoxd 13 gene in children with congenital anorectal malformation. Zhongguo Dang Dai Er Ke Za Zhi 2003;5:201–4. [9] Hirai Y, Kuwabara N. Transplacentally induced anorectal malformations in rats. Pediatr Surg 1990;25:812–6. [10] Allen E, Xie Z, Gustafson AM, et al. Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana. Nat Genet 2004;36:1282–90. [11] Stefani G, Slack FJ. Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol 2008;9:219–30. [12] Liu G, Moro A, Zhang JJ, et al. The role of Shh transcription activator Gli2 in chick cloacal development. Dev Biol 2007;303:448–60. [13] Mandhan P, Beasley S, Hale T, et al. Sonic hedgehog expression in the development of hindgut in ETU-exposed fetal rats. Pediatr Surg Int 2006;22:31–6. [14] Huang Y, Zhang P, Zheng S, et al. Hypermethylation of SHH in the pathogenesis of congenital anorectal malformations. J Pediatr Surg 2014;49:1400–4. [15] Rimkus TK, Carpenter RL, Qasem S, et al. Targeting the sonic hedgehog signaling pathway: review of smoothened and GLI inhibitors. Cancers (Basel) 2016;8. http:// dx.doi.org/10.3390/cancers8020022 [pii: E22]. [16] Shyer AE, Huycke TR, Lee C, et al. Bending gradients: how the intestinal stem cell gets its home. Cell 2015;161:569–80. [17] Yu YH, Narayanan G, Sankaran S, et al. Purification, visualization, and molecular signature of neural stem cells. Stem Cells Dev 2016;25:189–201. [18] Zhang ZB, Gao H. Expression of Gli2 gene in congenital anorectal malformation. Chin J Pediatr Surg 2001;22:325–7. [19] Jin SG, Zhao YY. Research progress of sonic hedgehog gene and intestinal malformations. Chin J Pediatr Surg 2005;26:436–8. [20] Roberts DJ, Johnson RL, Burke AC, et al. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 1995;121:3163–74. [21] Illig R, Fritsch H, Schwarzer C. Spatio-temporal expression of HOX genes in human hindgut development. Dev Dyn 2013;242:53–66. [22] Wang C, Li L, Cheng W. Anorectal malformation: the etiological factors. Pediatr Surg Int 2015;31:795–804. [23] Bhatlekar S, Fields JZ, Boman BM. HOX genes and their role in the development of human cancers. J Mol Med (Berl) 2014;92:811–23. [24] Pearson JC, Lemons D, McGinnis W. Modulating Hox gene functions during animal body patterning. Nat Rev Genet 2005;6:893–904. [25] Lewis B, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell 2003;115:787–98.