|Ahead of print publication
Bioinformatics analysis of regulated MicroRNAs by placental growth factor signaling in cancer stem cells
Zohreh Salehi, Hassan Akrami, Homeyra Seydi
Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran
Department of Biology, Faculty of Science, Razi University, Kermanshah
Source of Support: None, Conflict of Interest: None
Aim of Study: Since the effect of placental growth factor (PlGF) on MicroRNAs (miRNAs) at molecular level was remained unknown, the aim was to predict the transcription factors (TFs) and their regulated miRNAs that activated by PlGF and analysis the function, biological processes, and cancer stem cells (CSCs)-related signaling pathways of miRNAs that regulated in PlGF signaling pathway.
Subjects and Methods: The aim of this study is to find the TFs that activated by PlGF, we used three online software programs PCViz, PubAngioGen, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Then, the regulatory miRNAs downstream of the TFs were identified by four software TMHD, chipbase, circuits, and transmir databases. Target genes of miRNAs were predicted by three online software program TargetScan, Pictar, and miRanda algorithms. Moreover, Mirwalk database was used to find the validated miRNAs in angiogenesis process. Furthermore, Gene ontology (GO) biological process, GO molecular function, KEGG pathway, BIOCARTA pathway, Panther pathway, and Reactome pathway in Database for Annotation and Visualization and Integrated Discovery tools were used to find the functions and signaling pathways of target genes.
Results: Many target genes of miRNAs in PlGF pathway were involved in CSCs-related signaling pathways such as Hedgehog, Wnt/b-catenin, Notch, mTOR, epidermal growth factor EGF, and transforming growth factor-beta signaling pathways. Regulatory miRNAs in PlGF signaling pathway probably promote cell proliferation, migration, tubulogenesis, and metastasize in CSCs.
Conclusions: Bioinformatic analysis revealed that regulatory miRNAs and their target genes in PlGF pathway played important roles in the progression of CSCs-related signaling pathways.
Keywords: Cancer stem cell, Database for Annotation, Visualization and Integrated Discovery database, Kyoto Encyclopedia of Genes and Genomes database, miRNA, placental growth factor
| > Introduction|| |
Placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, is a prognostic tumor marker. PlGF promotes survival, migration, the proliferation of tumor, endothelial cells, and collateral vessel growth. PlGF stimulates angiogenesis by upregulating angiogenic factors including VEGF-A, platelet-derived growth factor (PDGF), and matrix metalloproteinase 9. PlGF expression level was increased in many cancers such as gastric, non-small cell lung cancer, breast, and renal cell carcinoma.,,
Tumor initiating cells, also known as cancer stem cells (CSCs) can lead to tumor formation, progression, angiogenesis, and resistance to therapy. A significant higher level expression of PlGF gene was reported in CSCs. CSCs secreted multiple factors that promote angiogenesis and long-term growth of CSCs. Many signaling pathways such as Sonic hedgehog homolog, Wnt/b-catenin, Notch, mTOR, and transforming growth factor-beta (TGF-β) participate in the regulation of CSCs functions.,,,, Regulatory transcription factors (TFs) in PlGF signaling pathways can regulate expression of oncomiRs and tumor-suppressor MicroRNAs (miRNAs). miRNAs are noncoding, endogenous, and small RNAs that play important regulatory roles on targeting specific mRNAs. Overexpression of oncomirs and down-expression of tumor suppressive miRNAs were reported in many cancers such as gastric, breast, and lung cancer. Oncomirs promote cancer development and tumor suppressive miRNAs decrease the oncogenes in different cancers. In addition, some miRNAs suppress oncogenes and tumor suppressor genes and have dual roles in cancer progression., PlGF signaling pathways influence on TFs that involved in regulation of gene and miRNA expression profiles. The aim of this study was to predict the miRNAs in PlGF signaling pathways of CSCs and analysis of their functions, biological processes, and CSCs-related signaling pathways such as Wnt/b-catenin, Notch, and mTOR signaling pathways.
| > Subjects and Methods|| |
Prediction of activated-transcription factors in placental growth factor signaling pathway
Activated-TFs by PlGF were taken from three online software program including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (www.genome.jp/kegg/pathway), PubAngioGen (http://www.megabionet.org/aspd/), and PCViz (http://www.pathwaycommons.org/pcviz/) databases.
Identification of MicroRNAs that regulated by activated-transcription factors in placental growth factor signaling pathway
Four software programs included TMHD (http://184.108.40.206:8080/TMREC/), chipbase (http://deepbase.sysu.edu.cn/chipbase/), circuits (http://biocluster.di.unito.it/circuits/tn/), and transmir (http://www.cuilab.cn/transmir) database were used for the identification of regulated miRNAs by TFs that were activated in PlGF signaling pathways.
MicroRNA targets prediction
Three online software programs, TargetScan (http://www.targetscan.org), Pictar (http://www.pictar.org/cgi-bin/PicTar_vertebrate.cgi), and miRanda algorithm (http://www.microrna.org/), were used for miRNA targets prediction with regarding P value and the statistically significant threshold adjusted at the value of P ≤ 0.05. The validated miRNA-target interactions were aggregated by mirwalk database (www.umm.uni-heidelberg.de/apps/zmf/mirwalk/).
Analysis function and signaling pathways of MicroRNAs-target genes
We used Database for Annotation, Visualization, and Integrated Discovery (DAVID) tools (http://www.david.abcc.ncifcrf.gov/) to the analysis of regulated miRNAs by PlGF signaling pathways. Gene ontology (GO) biological process (BP), GO molecular function (MF), KEGG pathway, BIOCARTA pathway, Panther pathway, and Reactome pathway in DAVID tools were used for analysis the functions, and signaling pathways of miRNAs target genes involved in angiogenesis signaling pathway. Target genes of miRNAs were submitted to DAVID tools. Homo sapiens species was selected in background tab module in the DAVID database and the statistically significant threshold at a value of P ≤ 0.05. Results of GO BP, GO MF, KEGG, BIOCARTA, Reactome, and Panther pathway were gotten to analysis of miRNAs target genes and focused on target genes that involved in progression of CSCs-related signaling pathways such as Hedgehog, Wnt/b-catenin, Notch, mTOR, EGF, TGF-β signaling pathway and cell proliferation, migration, metastasis, and tubulogenesis.
| > Results|| |
Activated-TFs in PlGF signaling pathway were gotten from PubAngioGen, KEGG, and PCViz pathway databases. Then, the regulatory miRNAs in PlGF signaling pathway were taken from four software TMHD, chipbase, circuits, and TransmiR databases. Target genes of miRNAs were identified by three online software programs, TargetScan, Pictar, and miRanda algorithms. For instance, the miRNA-target genes interactions for nuclear factor kappa B (NFk-B) are shown in Supplementary 1. miRWalk database was used to found validated miRNAs and their target genes involved in angiogenesis signaling pathway [Supplementary 2]. The miRNA-target genes in PlGF signaling pathway were submitted in DAVID knowledgebase database and selected H. sapiens species. We got results of KEGG, BIOCARTA, Reactome, and Panther pathway for analyzing of miRNA target genes at the value of P ≤ 0.05. Most of these targets were related to regulation of actin cytoskeleton, FAS signaling pathway, apoptosis, MAPK signaling pathway, NGF, fibroblast growth factor (FGF) signaling pathways, cell cycle, angiogenesis, VEGF signaling pathway, TGF-beta signaling pathway, p53 pathway, Jak-STAT, Wnt signaling pathway, Hedgehog signaling pathway, PDGF signaling pathway, PI3 kinase pathway, signaling by epidermal growth factor receptor, pathways in cancer, FGF signaling pathway, etc. Bioinformatics results were shown that many miRNAs involved in CSCs-related signaling pathways such as Hedgehog, Wnt/b-catenin, Notch, mTOR, EGF, and TGF-β signaling pathways. miRNAs which regulated JUN TFs includes hsa-mir-4448, hsa-mir-4252, hsa-mir-4257, hsa-mir-3679, hsa-mir-4290, hsa-mir-4787, and hsa-mir-3681 took part in Wnt signaling pathway. Moreover, hsa-mir-4666, hsa-mir-3197, hsa-mir-125, hsa-mir-3681, hsa-mir-451b, and hsa-mir-449c play important roles in Hedgehog signaling pathway. Our data showed hsa-mir-301a, hsa-mir-3199, hsa-mir-4257, hsa-mir-3117, and hsa-mir-3681 involved in mTOR signaling pathway. Hsa-mir-3197 targeted Indian Hedgehog and BMP8B and involved in the Hedgehog signaling pathway. We arranged the JUN TF-miRNA interactions and their signaling pathways and a network using cytoscape [Figure 1]. For example, among all TFs, miRNAs, and target genes of miRNAs and their signaling pathways for ETS1 that extracted by KEGG, BIOCARTA, Reactome, and Panther pathway are shown in Supplementary 3. Most of miRNA-target genes involved in CSCs signaling pathways. For instance, signaling pathway analysis revealed ESR1, a TF that activated by PlGF, targeted 78, 498, 49, 58, 165, and 107 miRNAs involved in the Hedgehog, Wnt, mTOR, EGF, TGF-β, and NOTCH signaling pathways, respectively. The part of these results is shown in [Figure 2].
|Figure 1: The pathway related to JUN transcription factor. Placental growth factor-activated JUN transcription factors regulate MicroRNA expression. Target genes of MicroRNAs take part in cancer stem cells-related signaling pathways such as Hedgehog, Wnt/b-catenin, Notch, mTOR, epidermal growth factor, and transforming growth factor-beta signaling pathway|
Click here to view
|Figure 2: Summary of cancer stem cells-related signaling pathways regulated by activated-transcription factors in placental growth factor signaling pathway|
Click here to view
GO BP and GO MF module in the DAVID database were used for analyzing the miRNA-target genes functions. GO BP results revealed that miRNAs-target genes implicated in regulation of cyclin-dependent protein kinase activity, G1/S transition of mitotic cell cycle, mitotic cell cycle, regulation of cell growth, induction of apoptosis, cell cycle, negative regulation of cell proliferation, regulation of cell size, glial cell differentiation, negative regulation of phosphorus metabolic process, regulation of cell death, induction of programmed cell death, regulation of phosphate metabolic process, cell cycle process, angiogenesis, blood vessel development, patterning of blood vessels, response to hypoxia, in utero embryonic development, morphogenesis of a branching structure, cell activation, etc., For instance, hsa-mir-4794, hsa-mir-4779, hsa-mir-4632, hsa-mir-4265, hsa-mir-630, and hsa-mir-533 were regulated by NFk-B, and their target genes might be take part in positive regulation of angiogenesis, blood vessel morphogenesis, artery morphogenesis, and vasculature development. A part of the miRNAs and TFs network in PlGF signaling pathway is shown in [Figure 3].
|Figure 3: The regulatory pathway between placental growth factor, MicroRNAs, their target genes and their biological roles in cell proliferation, apoptosis, differentiation, and cell migration|
Click here to view
Moreover, GO MF results indicated that target genes of miRNAs might participate in enzyme inhibitor activity, phospholipase inhibitor activity, integrin binding, growth factor activity, protein complex binding, cell surface binding, lipase inhibitor activity, actin binding, cytoskeletal protein binding, and tubulin binding. The complete results of GO MF and GO BP for each regulated miRNA target of ETS1 by TFs are shown in Supplementary 3.
| > Discussion|| |
PlGF blockage inhibits angiogenesis, lymphangiogenesis, proliferation, growth, and metastasis of various tumors. In this study, three online software KEGG pathway, PubAngioGen, and PCViz were used to find the activated-TFs in PlGF signaling pathway.
MiRNAs play important roles in proliferation, differentiation, angiogenesis, and cellular physiology in development, and disease such as different types of cancer by negative regulating of gene expression., The regulatory miRNAs of TFs were identified by TMHD, chipbase, circuits, and transmiR databases. The miRNAs signaling pathways analysis which performed by KEGG pathway, BIOCARTA pathway, Panther pathway, and Reactome pathway module in the DAVID database were indicated that miRNAs in PlGF signaling pathways take part in CSCs process. The study results showed LEF1 regulated several miRANs such as hsa-mir-630, hsa-mir-371, hsa-mir-448, hsa-mir-519a, hsa-mir-222, hsa-mir-199a, and hsa-mir-802 that their target genes were taken part in Wnt signaling pathway and the validated results concurred with our results that obtained by bioinformatics analysis. For example, hsa-mir-371, hsa-mir-372, and hsa-mir-373 which activated by LEF1, take part in wnt/β-catenin-signaling pathway. NF-κB regulates hsa-mir-21 expression and plays an important role in angiogenesis signaling pathway. We showed the target genes of regulated miRNAs by NF-κB such as hsa-mir-21, hsa-mir-448, hsa-mir-760, and hsa-mir-4420 take part in angiogenesis signaling pathway. We showed that hsa-mir-221, hsa-mir-331, hsa-mir-410, hsa-mir-429, and hsa-mir-21 involved in cancer development and various studies were shown that hsa-mir-21 was regulated by STAT and played in cancer cell growth, migration, invasion, and self-renewal of mouse embryonic stem cells. Hsa-mir-125 controls of the Hedgehog signaling in cerebellar neuronal progenitor and tumor cells and signaling pathway tools results were shown that the miRNAs target genes by JUN, such as hsa-mir-3197, hsa-mir-125, and hsa-mir-3681, participated in the Hedgehog signaling pathway. Results from various studies have indicated that hsa-mir-144, hsa-mir-141, and hsa-mir-200a involved in Wnt signaling pathway and development. Our results also indicated that many regulated miRNAs by PlGF pathway such as hsa-mir-141, hsa-mir-200a, and hsa-mir-4290 play important roles in Wnt signaling pathway.
Our results from GO BP and GO MF were shown that the most target genes of regulatory miRNAs in PlGF pathways play regulatory roles in cell cycle, blood vessel morphogenesis, cell migration, etc. Recent studies on angiogenesis indicated that the let-7 family involved in cardiovascular diseases and cardiovascular differentiation of stem cells. CLIP methods were indicated that hsa-mir-378 played important regulatory roles in angiogenesis process. My results obtained by bioinformatics studies were shown that hsa-mir-378 was regulated by HNF4A, MYOD1, and LEF1 TFs and played important roles in the regulation of cell proliferation and angiogenesis.
| > Conclusions|| |
This study result indicated that activated-TFs in PlGF signaling pathway might regulate the expression of oncomiRs and tumor-suppressor miRNAs in CSCs signaling pathways. Therefore, PlGF promotes stemness properties in CSCs by the regulation of miRNAs expression.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
De Falco S. The discovery of placenta growth factor and its biological activity. Exp Mol Med 2012;44:1-9.
Li B, Sharpe EE, Maupin AB, Teleron AA, Pyle AL, Carmeliet P, et al.
VEGF and plGF promote adult vasculogenesis by enhancing EPC recruitment and vessel formation at the site of tumor neovascularization. FASEB J 2006;20:1495-7.
Fischer C, Mazzone M, Jonckx B, Carmeliet P. FLT1 and its ligands VEGFB and plGF: Drug targets for anti-angiogenic therapy? Nat Rev Cancer 2008;8:942-56.
Wei SC, Tsao PN, Yu SC, Shun CT, Tsai-Wu JJ, Wu CH, et al.
Placenta growth factor expression is correlated with survival of patients with colorectal cancer. Gut 2005;54:666-72.
Parr C, Watkins G, Boulton M, Cai J, Jiang WG. Placenta growth factor is over-expressed and has prognostic value in human breast cancer. Eur J Cancer 2005;41:2819-27.
Chen CN, Hsieh FJ, Cheng YM, Cheng WF, Su YN, Chang KJ, et al.
The significance of placenta growth factor in angiogenesis and clinical outcome of human gastric cancer. Cancer Lett 2004;213:73-82.
Bose B, Shenoy S. Stem cell versus cancer and cancer stem cell: Intricate balance decides their respective usefulness or harmfulness in the biological system. J Stem Cell Res Ther 2014;4:173. Doi: 10.4172/2157-7633.1000173.
Chen Y, Jiang T, Mao A, Xu J. Esophageal cancer stem cells express PLGF to increase cancer invasion through MMP9 activation. Tumour Biol 2014;35:12749-55.
Zhao Y, Bao Q, Renner A, Camaj P, Eichhorn M, Ischenko I, et al.
Cancer stem cells and angiogenesis. Int J Dev Biol 2011;55:477-82.
Martin J, Donnelly JM, Houghton J, Zavros Y. The role of sonic hedgehog reemergence during gastric cancer. Dig Dis Sci 2010;55:1516-24.
Cai C, Zhu X. The Wnt/β-catenin pathway regulates self-renewal of cancer stem-like cells in human gastric cancer. Mol Med Rep 2012;5:1191-6.
Yeh TS, Wu CW, Hsu KW, Liao WJ, Yang MC, Li AF, et al.
The activated notch1 signal pathway is associated with gastric cancer progression through cyclooxygenase-2. Cancer Res 2009;69:5039-48.
Howard BM, Gursel DB, Bleau AM, Beyene RT, Holland EC, Boockvar JA, et al.
EGFR signaling is differentially activated in patient-derived glioblastoma stem cells. J Exp Ther Oncol 2010;8:247-60.
Miyazono K, Suzuki H, Imamura T. Regulation of TGF-beta signaling and its roles in progression of tumors. Cancer Sci 2003;94:230-4.
Martinez NJ, Walhout AJ. The interplay between transcription factors and microRNAs in genome-scale regulatory networks. Bioessays 2009;31:435-45.
Bartel DP. MicroRNAs: Target recognition and regulatory functions. Cell 2009;136:215-33.
Liu C, Tang DG. MicroRNA regulation of cancer stem cells. Cancer Res 2011;71:5950-4.
Hime GR, Somers WG. Micro-RNA mediated regulation of proliferation, self-renewal and differentiation of mammalian stem cells. Cell Adh Migr 2009;3:425-32.
Wang X, Yu H, Lu X, Zhang P, Wang M, Hu Y, et al.
MiR-22 suppresses the proliferation and invasion of gastric cancer cells by inhibiting CD151. Biochem Biophys Res Commun 2014;445:175-9.
Franchina T, Amodeo V, Bronte G, Savio G, Ricciardi GR, Picciotto M, et al.
Circulating miR-22, miR-24 and miR-34a as novel predictive biomarkers to pemetrexed-based chemotherapy in advanced non-small cell lung cancer. J Cell Physiol 2014;229:97-9.
Fischer C, Jonckx B, Mazzone M, Zacchigna S, Loges S, Pattarini L, et al.
Anti-plGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell 2007;131:463-75.
Zhou AD, Diao LT, Xu H, Xiao ZD, Li JH, Zhou H, et al.
β-catenin/LEF1 transactivates the microRNA-371-373 cluster that modulates the Wnt/β-catenin-signaling pathway. Oncogene 2012;31:2968-78.
Li X, Gao L, Cui Q, Gary BD, Dyess DL, Taylor W, et al.
Sulindac inhibits tumor cell invasion by suppressing NF-κB-mediated transcription of microRNAs. Oncogene 2012;31:4979-86.
Löffler D, Brocke-Heidrich K, Pfeifer G, Stocsits C, Hackermüller J, Kretzschmar AK, et al.
Interleukin-6 dependent survival of multiple myeloma cells involves the stat3-mediated induction of microRNA-21 through a highly conserved enhancer. Blood 2007;110:1330-3.
Ferretti E, De Smaele E, Miele E, Laneve P, Po A, Pelloni M, et al.
Concerted microRNA control of hedgehog signalling in cerebellar neuronal progenitor and tumour cells. EMBO J 2008;27:2616-27.
Song JL, Nigam P, Tektas SS, Selva E. MicroRNA regulation of Wnt signaling pathways in development and disease. Cell Signal 2015;27:1380-91.
Bao MH, Feng X, Zhang YW, Lou XY, Cheng Y, Zhou HH, et al.
Let-7 in cardiovascular diseases, heart development and cardiovascular differentiation from stem cells. Int J Mol Sci 2013;14:23086-102.
Kishore S, Jaskiewicz L, Burger L, Hausser J, Khorshid M, Zavolan M, et al.
A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins. Nat Methods 2011;8:559-64.
[Figure 1], [Figure 2], [Figure 3]