PCR-array with primer sets targeting HOX genes and their associated genes used to evaluate the changes in their expression between ectopic and eutopic tissues with control tissues and ectopic with eutopic tissues. The genes expression which were significantly (P<0.05) altered (DEGs) in ectopic, eutopic with normal groups and ectopic with eutopic groups are illustrated in Table I. Among the analyzed genes, there are 33.33% (28) genes in non-DEG. The expression levels of 30, 41 and 35 genes were significantly altered in eutopic and ectopic compared with normal samples and ectopic rather than eutopic samples , respectively. Twelve genes were common between the three groups and 10 of 41 DEGs were exclusively related to ectopic versus normal tissue, 5 of 35 DEGs were solely related to eutopic versus normal tissue and and 3 of 35 DEGs were only related to ectopic versus eutopic tissue. One of the interesting points is that at least in one of the analysed groups, the expression out of five of the seven studied HOXB genes, were significantly up-regulated. Three genes of the HOXA cluster were present. Only HOXA9 was significantly up-regulated in ectopic versus normal and eutopic samples. Seven genes of the HOXC cluster were studied and found that expression of five of them were significantly altered in three groups of analysis. Eight genes of the HOXD cluster were analyzed, and it was shown that expression of six of them underwent significant change in different studies (Fig. 1 and Table I).
DEGs were categorized into six groups based on venn diagram (Fig. 1). There are three analysis in the investigation: Comparison of expression level of the target genes in ectopic tissue with normal tissue (CtoN), comparison of expression level of the studied genes in eutopic tissue with normal tissue (UtoN) and comparison of expression level of the studied genes in ectopic tissue with eutopic tissue (CtoU). The expression of three groups of genes have changed only in one analysis such as PAX3 expression of which changes in ectopic tissue to naormal tissue. Three groups include gene expressions of which have altered in two analysis such as HLX the expression of which has changed in eutopic and ectopic tissues to normal tissue. The last group consists of the genes expression of which has siginificant ly altered in the three analysis (Fig. 1 and Table I).
For further investigation, hierarchical clustering was performed on the nine studied samples using DEGs (Fig. 3A) and non-DEGs (Fig. 3B) in the eutopic, ectopic samples and normal samples. Hierarchical clustering analysis showed a grouping according to PCA analysis based on the DEGs in ectopic, eutopic and normal samples. In the present study, three samples were properly classified into separate clusters on the basis of the DEGs. But non-DEGs could not classify the same samples together.
Seven co-expression networks were pointed out based on pairwise correlation of gene expression data and visualized using Cytoscape (Fig. 4). As pointed out above, expression about 64% (54/84) of the genes significantly changed between normal, eutopic and ectopic samples. All genes in the network B1 were significant genes in diseased tissues, and significant genes of ectopic tissues are abundant in the network A1 (71%) (Table II, Fig. 4).
The signaling pathways present in the KEGG and Reactome databases searched for genes in the networks (Table III, Fig. 4), have identified two pathways of "Signaling pathways regulating pluripotency of stem cells" and "Transcriptional misregulation in cancer" from the KEGG database and pathway of "activation of anterior HOX genes during the early embryogenesis" from the Reactome database. Fifteen of which were in these pathways and the MEIS1 was present in all three pathways.
The DEGs and the genes in seven networks (Fig. 4) were mapped to the DAVID database to investigate the GO, pathways, interference with other diseases and expression in other tissues. DAVID is the most popular tool in the field of functional annotation (Tables IV, V and S1).
Analysis of interference with other diseases: According to analysis of interference with other diseases of genes, many DEGs in ectopic tissues play a role in developmental diseases class, such as cleft lip and clubfoot and metabolic diseases class like bone mineral density.
Many DEGs in eutopic tissues are also involved in neurological diseases class like parkinson's disease and autism in addition to developmental and metabolic diseases class. Many networks A1,2,3 and 4 and B2 genes affected developmental diseases. Most genes in the network C cause neurological diseases. (Table IV).
GO annotation analysis: On the basis of the GO annotation, the most of DEGs in the eutopic and ectopic tissues, and different networks were involved in the development or morphogenesis of various systems, especially skeletal system. Only most network C genes play roles in the development of the nervous system such as dopaminergic neuron and cerebellum (Table S1).
Expression in other tissues: Many DEGs in the patient tissues were also expressed in different tissues such as craniofacial, prostate_carcinoma, superiorcervical ganglion, salivarygland and BM-CD33 + myeloid. Most genes in the networks were expressed in one or two tissues. Therefore, the most genes of A1, A3, A4, B1, B2, C and D networks appeared in embryo development, BM-CD33+Myeloid, superior cervical ganglion, spinal cord and ciliary ganglion respectively (Table. V).