Bioinformatic Analysis For Pathogenesis Of Pulmonary Hypoplasia In Congenital Diaphragmatic Hernia And Its Candidate Therapeutic Compounds

BackgroundCongenital diaphragmatic hernia (CDH) is a birth defect of the diaphragm. Malformation of the diaphragm allows the abdominal organs to push into the chest cavity, hindering proper lung formation. Newborns with CDH often have severe respiratory distress. CDH is a life-threatening pathology in neonates and a major cause of death due to the complications:pulmonary hypoplasia. The incidence of CDH ranges from 1 in 2,500 to 5,000 births in recent population-based studies. Although there was great improvement in the treatment of CDH in past decades, the mortality remains high, ranging from 40% to 60%. The pathogenesis of pulmonary hypoplasia of CDH is still unclear and the absence of effective treatment persists. Therefore, a great mount of studies focus on pathogenesis and intervention of lung hypoplasia.Lung development is a complex process under precise control, involved with cell growth, proliferation, differentiation and apoptosis, as well as the interactions between cells, mesenchymal-epithelial. This process involves a great number of cytokine, including growth fators, transcription factors, extracellular matrix, which construct a powerful network related to the regulation of lung development during various developmental stages. Recent studies have revealed that a new class of small non-coding RNA (microRNA, miRNA) is involved in the process of lung development process. MicroRNAs is a small non-coding RNA molecule (containing about 22 nucleotides) that play key roles in the regulation of gene expression. Acting at the post-transcriptional level, these fascinating molecules may fine-tune the expression of as much as 60% of all mammalian protein-encoding genes. MicroRNAs usually induce gene silencing by binding to target sites found within the 3’UTR of the targeted mRNA. This interaction prevents protein production by suppressing protein synthesis and/or by initiating mRNA degradation. Since most target sites on the mRNA have only partial base complementarity with their corresponding microRNA, individual microRNAs may target as many as 100 different mRNAs. Moreover, individual mRNAs may contain multiple binding sites for different microRNAs, resulting in a complex regulatory network. Their discovery added a new dimension to the understanding of complex gene regulatory networks in humans and animals alike. It was inappropriate that explanation for pathogenesis of pulmonary hypoplasia depended on mono-biological factor decades ago owing to the technique at that time. With the rapid development of modern science and technology, bio-chip was designed for revealing the essence of biology. It is an efficient way for revealing the mechanism of lung hypoplasia in CDH.Bio-chips are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions. Bio-chips enable researchers to quickly screen large numbers of biological analytes for a variety of purposes, such as discovery of causative gene, disease diagnosis and prognosis evaluation. Bioinformatics is an interdisciplinary field that develops methods and software tools for understanding biological data. As an interdisciplinary field of science, bioinformatics combines computer science, statistics, mathematics, and engineering to analyze and interpret biological data. Bioinformatics has been used for in silico analyses of biological queries using mathematical and statistical techniques.In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which new candidate medications are discovered. Historically, drugs were discovered through identifying the active ingredient from traditional remedies or by serendipitous discovery. Later chemical libraries of synthetic small molecules, natural products or extracts were screened in intact cells or whole organisms to identify substances that have a desirable therapeutic effect in a process known as classical pharmacology. Since sequencing of the human genome which allowed rapid analysis, it has become common practice to use high throughput screening of large compounds libraries against isolated biological targets which are hypothesized to be disease modifying in a process known as reverse pharmacology. With the advent of microarray and bioinformatics, the gene-based pattern is the major change in drug discovery and development. Drugs from these screens are efficient for those rare diseases.The purpose of present study is to analyze the miRNA and mRNA expression profiles in hypoplastic lung from Nitrofen-induced CDH rat model by bio-chip and the essential connection between them through bioinformatic analysis. The data from above could help for revealing the pathogenesis of pulmonary hypoplasia, developing new ways in prenatal interventions.Objectives1) To detail the mRNA expression profiles in hypoplastic lung with Nitrofen-induced rat model by microarray technology2) To reveal the pathogenesis of pulmonary hypoplasia in CDH through the data of miRNA microarray and bioinformatic analysis of miRNA-mRNA.3) To predicte efficient drug for reversion of pulmonary hypoplasia prenatally by compounds screening in Connectivity Map.Methods1) Nitrofen-induced CDH rat modelObservation of sperm-positive vaginal smears was considered a proof of pregnancy and represented day 0.5 of gestation. Pregnant rats at day 9.5 of gestation randomly received by gavage either a single-dose of 100 mg of 2,4-dichorophenyl-p-nitrophenylether (nitrofen) dissolved in 1 mL of olive oil for CDH group or an equal volume of olive oil for control grounp. From the control and CDH group, fetuses were both removed by caesarian section at E21.5. Left lung from control group and lungs from the side shown hernia in nitrofen-exposed fetuses were excised and stored at -80℃ for array and real time QPCR studies or fixed in formalin for further use of hematoxylin and eosin (HE) stain studies and immunohistochemistry.2) Analysis of mRNA expression profilesA microarray (Agilent Whole Rat Genome Oligo Microarray 4 X 44K) study was performed to analyse mRNA expression levels in hypoplastic lung of Nitrofen-induced CDH. The results were verified with the data in FACTA and Pubmed.3) Examination of miRNA expression profiles and its integration with mRNA expression profilesThe combination was underwent between data from miRNA microarray and predited target genes from TargetScan. Then the bioinformatic analysis was performed for gene ontology and signaling pathway.4) Verification for selected miRNAs and mRNAsThe expression levels of miRNA and their target genes were tested by real-time quantitative PCR. The tissue location of target genes were evaluated by immunohistochemistrical stain. The 3’-untranslated region (3’-UTR) of target genes were detectd by luciferase assay.5) Analysis of potential compounds for prenatal interventionPotential compounds for prenatal intervention of pulmonary hypoplasia were screened via Connectivity Map base on the database of mRNA expression.Results1) Hypoplastic lungs in nitrofen-induced CDH rat modelSeventy-one fetuses were collected from control group and 59 from nitrofen-exposed group. The fetuses from control group all had normal diaphragms, however 64.4%(38/59) fetuses from exposed group had CDH. All the defects in diaphragms occurred in left side. Generally, livers, stomachs and bowl could come into thoracic cavity through the defects in diaphragms. Hypoplastic lungs in CDH group were observed.2) mRNA expression profilesA cDNA microarray analysis found 10,121 transcripts being up-regulated and 7,558 transcripts down-regulated in the CDH group. From the database of Pubmed and FACTA, it was found that 63 genes have been tested and 50 of them shared the same pattern in Nitrofen-induced CDH lung.3) Intergrated analysis of miRNA and mRNAThe miRNAs microarray and bioinformatic analysis indicated that 13 miRNA specie being up-regulated, involved 3,126 target genes and 716 signaling pathways in which TGFβ signaling pathway and GnRHR signaling pathway are the common pathway; 8 miRNA specie down-regulated, involved 2,729 target genes and 564 signaling pathways in which WNT signaling pathway, TGFβ signaling pathway, FGF signaling pathway and GnRHR signaling pathway are the same pathway. Mir-141-3p, mir-375-3p, TGF-β2 and FZD8 were selected for further study.4) The Expression of mir-141-3p and TGF-β2 in Fetal Lungs in Rat Model of Nitrofen-induced CDHThe relative quantities of mir-141-3p and TGF-β2 mRNA expressed in CDH group and control group were 1.57±0.28 vs 1.00±0.18,0.58±0.17 vs 1.00±0.15, respectively. Immunohistochemistry studies showed that TGF-β2 is expressed predominantly in the airway epithelium and the immunological activity (shown by the average optical density) in CDH group was lower than in the control group (20.32±3.54 vs 45.14±4.56). All the differences reach statistical significance (p< 0.05). The luciferase activity of the reporter construct containing the 3’-UTR of TGF-β2 was significantly decreased by mir-141-3p.5) The Expression of mir-375-3p and FZD8 in lungs of Nitrofen-induced CDH Rat ModelThe relative quantities of mir-375-3p and FZD8 mRNA expressed in CDH group and control group were 0.41±0.15 vs 1.00±0.17,1.62±0.20 vs 1.00±0.16, respectively. Their differences reach statistical significance (p< 0.05). Immunohistochemistrical stain indicated that FZD8 locate in the airway epithelium and the immunological activity (shown by the average optical density) in CDH group was significant higher than in the control group (38.32±8.54 vs 20.14±5.76,p<0.05). The luciferase activity of the reporter construct containing the 3’-UTR of FZD8 was significantly induced by mir-375-3p.5) Study of potential compounds for prenatal therapyThree compounds with top negative-enrichment were trapidil, tretinoin and orciprenaline, which related to genes in TGFFβ signaling pathway. In addition, iloprost, corticosterone and sulmazole were compounds with top negative-enrichment, which involved genes in WNT signaling pathway.Conclusion1) The bioinformatic analysis of miRNA-mRAN expression profiles indicates nitrofen-induced hypoplastic lungs have an abnormal transcriptome that may lead to impaired development. Potential pathways relevant to nitrofen-induced lung hypoplasia include WNT, TGFβ, FGF and GnRHR signaling pathway.2) TGF-β2 was identified as the target of mir-141-3p and they might be involved in the pathogenesis of pulmonary hypoplasia in nitrofen-induced CDH.3) FZD8 was verified as the target of mir-375-3p and they might be relevant to the mechanism of abnormal lung morphogenesis in CDH induced by nitrofen.4) Based on the analysis from Connectivity Map, it is suggested that trapidil, tretinoin and orciprenaline could normalize hypoplastic lung of nitrofen-induced CDH owing to the inversion of genes in TGFβ signaling pathway. In addition, iloprost, corticosterone and sulmazole might rescue the abnormal lung because of the reversion of genes in WNT signaling pathway.