Reports 21 (2020) 100895

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Gene expression profiling of 10 in PTEN-knockout (−/−) T human neural and mesenchymal stem cells: A system biology study ⁎ Hamid Fiujia, ,1, Mohammadreza Nassirib,1 a Department of Biochemistry, Faculty of Science, Payame Noor University of Mashhad (PNUM), Mashhad, Iran b Recombinant Research Group, The Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

ARTICLE INFO ABSTRACT

Keywords: The present study investigates the effects of PTEN deletion in human neuro and mesenchymal stem cells related PTEN deletion to the profile. The RNA sequencing (RNA-seq) performed on four neural and NSCs four mesenchymal stem cells. DEG analysis outcome revealed 122 for neural stem cells (57 up-regulated MSCs and 65 down-regulated genes) and 258 genes for mesenchymal stem cells (98 up-regulated and 160 down- RNA sequencing regulated genes) that were deferentially expressed in the PTEN (-/-) group compared to the normal group. Gene Hub genes ontology analysis indicated that in the NSCs upregulated DEGs were significantly enriched in transcriptional Tumor progression activator activity, phosphatidylinositol phosphate phosphatase activity, MAP activity while the down­ regulated DEGs were mainly involved in glycolytic process through -6-phosphate, canonical glycolysis, glucose catabolic process to pyruvate. Meanwhile, in MSCs, the upregulated DEGs were mainly enriched in positive regulation of cell migration, chromatin silencing complex, RNA polymerase core binding while the downregulated DEGs phosphofructokinase activity, focal adhesion, glycolytic process through glucose-6- phosphate. On the basis of the PPI network of the DEGs, the following top hub genes were detected: six hub genes (PTEN, MAPK8, VCL, PAX2, ITGB1 and RET) in neural stem cells and 9 hub genes (PTEN, CDK1, KIF11, VCL, HK1, KIF20B, SIRT1, ACTR1A and ITGB1) appeared in mesenchymal stem cells respectively in each of the top 10 gene lists. In conclusion, PTEN, VCL and ITGB are found to be the main hub genes in both neural and mesenchymal stem cells, which involve in cell cycle, tumor progression and metastasis.

1. Introduction 2013). PTEN or phosphatase and tensin homolog deleted on chromo­ some 10 (or MMAC1/TEP1) was introduced as a tumor suppressor lo­ One of the subjects of interest is identifying genes on the chromo­ cated on human chromosome 10q23 by three independent scientific some. The predicted number of genes varies due to employing different groups in 1997 (Stiles et al., 2004). approaches by the researchers. It is estimated that the gene numbers on It is reported that the numerous sporadic human cancer, such as chromosome 10 to produce protein are almost 700 to 800, playing prostate, breast, endometrial and glioblastoma are associated with de­ various roles in the body (Deloukas et al., 2004). letion and mutation in the PTEN gene (Dahia, 2000). Moreover, the Moreover, alteration in chromosome 10 in terms of number or mutation rate in human cancer is similar to that of the P53 gene structure is associated with several types of cancer. The gliomas, for (Stokoe, 2001). instance, known as a brain tumor, is resulted from a loss of all or part of The later investigation indicated that PTEN suppresses the phos­ chromosome 10. Regarding the association between the loss of chro­ phatidylinositol-3-kinase (PI3K)/AKT signaling pathway, thereby in­ mosome 10 and cancerous tumors, it is suggested that genes on this activating cell growth being influenced negatively on the survival sig­ chromosome play critical roles in either growth or cell cycle and divi­ naling pathway (Stiles et al., 2004; Downes et al., 2007). sion. Lack of these genes has led to unleashed cell division, resulting in PTEN deficiency has been reported in many investigations as reg­ cancerous status (Deloukas et al., 2004). As well as gliomas, other ef­ ulators of growth, survival, and cell proliferation by preventing the fects of chromosomal abnormalities resulted from gene deletion are PI3K-AKT-mTOR pathway in several cancers such as thyroid or breast accompanied by cellular or intracellular disabilities (Chang et al., cancers and syndrome-like Cowden syndrome (Lee et al., 2018;

⁎ Corresponding author. E-mail address: [email protected] (H. Fiuji). 1 H.F. and M.N. made equal contribution to this study. https://doi.org/10.1016/j.genrep.2020.100895 Received 3 July 2020; Received in revised form 3 September 2020; Accepted 26 September 2020 Available online 08 October 2020 2452-0144/ © 2020 Elsevier Inc. All rights reserved. H. Fiuji and M. Nassiri Gene Reports 21 (2020) 100895

Fig. 1. Differentially expressed (DE) genes between PTEN (−/−) and control groups in human neuro and mesenchymal stem cells represented in the characteristic trumpet shape of MA plots (A) Correlation analysis of gene expression changes between deleted PTEN vs. control samples in NSCs. (B) Correlation analysis of gene expression changes between deleted PTEN vs. control samples in MSCs. The log fold change is plotted on the y-axis and the mean expression of the reads counts is shown on the x-axis. Each point represents a gene at adjusted p value (adjP) ≤ 0.1.

Fig. 2. analysis classified the differentially expressed genes into 3 groups. Molecular function, biological process, and cellular component in Neural stem cells (NSCs).

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Table 1 dephosphorylating of the lipid substrate phosphatidylinositol-3, 4, 5- The top 15 enriched gene ontology terms of up-regulated DEGs and down­ triphosphate (PIP3) in the cell (Stiles et al., 2004; Downes et al., 2007). regulated DEGs. (NSCs). A, The top 15 enriched gene ontology terms of the In addition, it is illustrated that the lipid phosphatidylinositol (3,4,5)- upregulated DEGs. B, The top 15 enriched gene ontology terms of the down- triphosphate [PtdIns (3,4,5) P3], which is a direct product of PI3-kinase regulated DEGs. (PI3K), is one of the major substrates of PTEN in both in virto and in A vivo circumstances (Stambolic et al., 1998; Maehama and Dixon, 1998; Myers et al., 1998). Interestingly, PTEN deficiency in embryonic stem Category Term Count P-value (ES) cells and human cancer cell lines results in subsequent accumu­ BP Inositol phosphate dephosphorylation 2 0.0001 lation of PtdIns(3,4,5)P3 (Kimura et al., 2003). BP Phosphorylated carbohydrate dephosphorylation 2 0.0001 In the present study, a gene expression profile was downloaded from BP Neuron projection extension involved in neuron 2 0.0002 the NCBI sequence read archive (SRA) database with following ID: SRA projection guidance study: SRP047516; GEO: GSE61794 (Duan et al., 2015). We selected 8 BP Axon extension involved in axon guidance 2 0.0002 BP Inositol phosphate catabolic process 2 0.0002 cases from these data set, including neural and mesenchymal stem cells CC Meiotic cohesin complex 1 0.01 containing null and normal PTEN gene. Both Linux (Ubuntu 19.10) and CC Early 3 0.02 R packages were employed to analyze the differentially expressed genes CC Secondary lysosome 1 0.03 (DEGs); Gene Ontology (GO), Kyoto Encyclopedia of Genes and Gen­ CC COPII vesicle coat 1 0.03 omes (KEGG) and Reactome analysis were applied to analyze the CC Axon 2 0.06 MF Transcriptional activator activity, RNA polymerase 2 0.01 functional enrichment and significant pathways associated with the II core promoter proximal region sequence-specific DEGs. In addition, identification of hub genes was performed through binding integrating the DEGs into a protein-protein interaction (PPI) network MF Phosphatidylinositol phosphate phosphatase 3 0.01 for modular analysis. Eventually, this study aimed to investigate the activity MF Monoamine transmembrane transporter activity 1 0.02 knocked out PTEN effects on the different pathways and possible dis­ MF Inositol trisphosphate phosphatase activity 1 0.02 orders related to chromosome 10 genes in neural and mesenchymal MF MAP kinase activity 1 0.03 stem cells, which will give us a deeper understanding of PTEN function in the cells.

B 2. Method Category Term Count P-value 2.1. RNA-seq read alignment and transcriptome processing BP Glycolytic process through glucose-6-phosphate 3 7.16E-05 BP Canonical glycolysis 3 7.16E-05 2.1.1. Sequence retrieval BP Glucose catabolic process to pyruvate 3 7.16E-05 BP Positive regulation of metanephros development 2 0.0002 The RNA-Seq raw data files containing both neural and mesench­ BP Myoblast differentiation 2 0.001 ymal cases were downloaded from NCBI, GEO, and SRA (series ID CC Cytoplasmic vesicle lumen 4 0.0008 GSE61794) (Duan et al., 2015) These datasets included eight samples CC Mitochondrion 10 0.001 containing four human neural stem cells (NSCs) and four mesenchymal CC Focal adhesion 5 0.005 CC Membrane raft 3 0.006 stem cells (MSCs), of them, were control samples where PTEN ex­ CC Ficolin-1-rich granule lumen 3 0.007 pressed normally contrasting the remaining samples which PTEN were MF Carboxylic acid binding 2 0.005 knocked out through deletion of their first exon in PTEN gene. MF binding 4 0.009 Heterogeneity of the datasets allowed us to look at the isoform ex­ MF Hydro-lyase activity 2 0.009 pression variations across different conditions in NSCs& MSCs. These MF Kinase binding 5 0.01 MF Iron ion binding 2 0.01 data files were submitted on 26-Sep-2014. Downloading the SRA files, they then converted into Fastq files by SRA TOOLKITS assigned in DEG, differentially expressed gene; BP, biological process; CC, cellular com­ NCBI. ponent; MF, molecular function. 2.1.2. Quality control Stambolic et al., 1998). PTEN can also dephosphorylate both lipids and FastQC (Andrews, 2016) and Trimmomatic (Bolger et al., 2014) specifically. PTEN functions as a tumor suppressor that can software were used for preprocessing of raw reads. The subsequent pass through the membrane to enter the nucleus and involve genome analysis was performed by mapping the obtained clean raw reads from integrity, DNA repair, recombination, chromatin condensation, and each sample to the human reference genome individually using To­ transnational regulation (Abbas et al., 2019). pHat2 (Dobin and Gingeras, 2013) previously GRCh37 On the molecular basis, several downstream targets of activated (h19) reference downloaded from Ensembl Genome Browser and then AKT are regulated by its activities such as apoptotic was indexed by bowtie2-build software. factors (Datta et al., 1997). glycogen synthase (GSK3α and β) (Pap and Cooper, 1998) and cell cycle inhibitors p21 and p27 (Chen 2.1.3. Differential gene expression (DGE) analysis et al., 2018). FeatureCounts package (Liao et al., 2014) was implemented in Therefore, the constant activation of the PI3K/Akt pathway re­ Linux (ubunto 19.10) to produce a matrix of genewise counts provided sulting from PTEN knockout is correlated to cell cycle promotion, cell input for gene expression analysis package. Normalization and differ­ proliferation, and cell migration (Kimura et al., 2003). ential expression analysis was carried out in R software (v.3.6), using Because PTEN dephosphorylates both lipid and protein demon­ DESeq2 package (v.1.20) (Love et al., 2014). Biomart package (Durinck strated in vitro, it is also recognized as a dual function protein. et al., 2009) was executed in R to identify the official gene symbol and However, what is known as the significant biological effect of PTEN is other gene characteristics corresponding to the Ensembl gene ID. Hence

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Fig. 3. Gene Ontology analysis classified the differentially expressed genes into 3 groups. Molecular function, biological process, and cellular componentin Mesenchymal stem cells (MSCs). we made contrast between Normal sample and PTEN (−/−) in each 2.1.5. GO and pathway enrichment analyses stem cell type. Evaluation of GO terms and Pathway identifier enrich­ In this study, the EnrichR online bioinformatics database was ap­ ment was performed using Fisher's exact test. False discovery rate (FDR) plied to provide GO enrichment, KEGG and Reactome pathway analysis approach was used to control the P-values. For our research FDR and P- (Chen et al., 2013). Three approaches including the CC (cellular com­ values less than 0.05 were considered as statistically significant cor­ ponents), BP (biological processes), and MF (molecular functions) ca­ rection. tegories have opted for Gene Ontology (GO) term enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathways were analyzed to identify key genes and pathways involved in 2.1.4. Integration of PPI network analysis two types stem cells samples. Gene counts more than 2, and the p-value One of the online tools, as a resource search providing gene inter­ below 0.07 was set as the cutoff point. action analysis, either physical or psychical analyses, is STRING version 11 (https://string-db.org/cgi/input.pl) (Szklarczyk et al., 2019). In this 3. Results study PPI network of unregulated and downregulated DEGs was con­ structed using the STRING online database with a significance level 3.1. Comparison of gene expression profiling higher than 0.7. the string output was used for making nodes and edges input for Cytoscape (http://www.cytoscape.org/) software version 3.7 The obtained DEG analysis outcome revealed 122 human genes for (Shannon et al., 2003) to visualize the PPI network with a combined neural stem cells and 258 genes for mesenchymal stem cells that were score higher than 0.4 as cut off interaction score. Meanwhile, molecular deferentially expressed in the PTEN (−/−) group compared to the complex detection (MCODE) (Bader and Hogue, 2003) clustering plugin normal group. The identification of significant genes was based on the was served to construct module analysis resulting from PPI network FDR (false discovery rate) criterion (FDR ≤0.1). Approximately 57 and with the following main criterion: Node, degree and K-core score cutoff 98 genes were over-expressed with positive Log2–fold change in human was ≥2; max depth, 100. neural and mesenchymal stem cells, respectively, while 65 and 160 By applying cytohubba application in Cytoscape, three significant genes were under-expressed with negative Log2 fold change in those analyses, including Centrality criterion degree, closeness and be­ mentioned null PTEN samples respectively (Fig. 1). tweenness, were assayed. The identified subnetworks resulted from cytohubba were selected as hub genes with high centrality criterion (Chin et al., 2014). 3.2. GO functional enrichment analysis By definition, the number of nodes with the connected edges is considered as the degree and their shortest path between nodes and 3.2.1. Neural stem cells profile edges, and crossing the number of critical nodes with the shortest path To determine the functions of all DEGs, we used EnrichR online tool were identified as closeness and betweenness terms, respectively. to calculate the over-representation of GO terms divided into three

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Table 2 transcriptional activator activity, RNA polymerase II core promoter The top 15 enriched gene ontology terms of up-regulated DEGs and down­ proximal region sequence-specific binding, phosphatidylinositol phos­ regulated DEGs. (MSC). A, The top 15 enriched gene ontology terms of the phate phosphatase activity, monoamine transmembrane transporter upregulated DEGs. B, The top 15 enriched gene ontology terms of the down- activity, inositol trisphosphate phosphatase activity, MAP kinase ac­ regulated DEGs. tivity while the downregulated DEGs were mainly enriched in car­ A boxylic acid binding, actin binding, hydro-lyase activity, kinase binding, iron ion binding. Category Term Count P-value

BP Positive regulation of cell migration 8 1.31E-05 3.3. Mesenchymal stem cells profile BP Regulation of mitotic nuclear division 4 0.0007 BP Regulation of substrate adhesion-dependent cell 3 0.001 The above methods were also exerted for mesenchymal stem cells spreading with the following results: In the BP group upregulated DEGs were BP Regulation of cellular macromolecule biosynthetic 9 0.003 process mainly enriched positive regulation of cell migration, regulation of BP Positive regulation of cellular process 8 0.003 mitotic nuclear division, regulation of substrate adhesion-dependent CC Mitotic spindle 4 0.0007 cell spreading, regulation of cellular macromolecule biosynthetic pro­ CC Chromosome, centromeric region 3 0.001 cess, positive regulation of cellular process while the top five down­ CC Heterochromatin 2 0.01 regulated DEGs were mainly enriched in glycolytic process, glycolytic CC Kinesin complex 2 0.02 CC Chromatin silencing complex 1 0.03 process through glucose-6-phosphate, canonical glycolysis, glucose MF Transcription corepressor activity 5 0.003 catabolic process to pyruvate and mesenchymal to epithelial transition. MF Transcriptional repressor activity 4 0.007 In the CC group, the upregulated DEGs were mainly enriched in mitotic RNA polymerase II transcription spindle, chromosome, centromeric region, heterochromatin, kinesin Regulatory region sequence-specific binding MF Transcription regulatory region 6 0.007 complex, chromatin silencing complex while the downregulated DEGs DNA binding were mitochondrion, nucleolar part, focal adhesion, integral compo­ MF Phosphatidylinositol-3,4,5-trisphosphate binding 2 0.01 nent of mitochondrial membrane, protein kinase complex. In the MF MF RNA polymerase core enzyme binding 2 0.01 group the upregulated DEGs were mainly enriched in transcription corepressor activity, transcriptional repressor activity, phosphatidyli­ nositol-3-phosphate binding, transcription regulatory region DNA B binding, phosphatidylinositol-3,4,5-trisphosphate binding, RNA poly­ Category Term Count P-value merase core enzyme binding while the downregulated DEGs were mainly enriched in phosphofructokinase activity, pyrophosphatase ac­ BP Glycolytic process 3 0.0007 tivity, carbohydrate kinase activity, nucleotidyl-transferase activity, BP Glycolytic process through glucose-6-phosphate 6 0.001 BP Canonical glycolysis 2 0.001 cadherin binding (Fig. 3 and Table 2). BP Glucose catabolic process to pyruvate 3 0.001 BP Mesenchymal to epithelial transition 2 0.001 3.4. Signaling pathway analysis CC Mitochondrion 21 7.18E-05 CC Nucleolar part 5 7.18E-05 CC Focal adhesion 8 0.008 3.4.1. Neural stem cells KEGG and Reactome pathways CC Integral component of mitochondrial membrane 3 0.01 After the GO enrichment analysis, the following pathways mainly CC Protein kinase complex 1 0.01 enriched in KEGG and Reactome online resources corresponding mostly MF Phosphofructokinase activity 2 0.001 to the GO enrichment. In neural stem cells, the upregulated genes were MF Pyrophosphatase activity 3 0.004 mainly enriched in Inositol phosphate , MF Carbohydrate kinase activity 2 0.004 MF Nucleotidyltransferase activity 2 0.004 Phosphatidylinositol signaling system, Human T-cell leukemia virus 1 MF Cadherin binding 7 0.01 infection, Protein processing in the endoplasmic reticulum, Focal ad­ hesion, ErbB signaling pathway, Insulin resistance while in Reactome DEG, differentially expressed gene; BP, biological process; CC, cellular com­ ponent; MF, molecular function. pathways were enriched mainly in FGFR2 ligand binding and activation Homo sapiens, Negative regulation of the PI3K/AKT network Homo sa­ groups as follows: biological processes (BP), cellular component (CC), piens, Axon guidance Homo sapiens, PI-3K cascade: FGFR1 Homo sapiens, and molecular function (MF) (Fig. 2 and Table 1). In the BP group, the PI3K/AKT activation Homo sapiens, Netrin-1 signaling Homo sapiens, top five upregulated DEGs were mainly enriched in inositol phosphate Inositol phosphate metabolism Homo sapiens. The downregulated genes dephosphorylation, phosphorylated carbohydrate dephosphorylation, were mainly enriched in Central carbon metabolism in cancer, neuron projection extension involved in neuron projection guidance, Glycolysis/Gluconeogenesis, Proteoglycans in cancer, Galactose meta­ axon extension involved in axon guidance and inositol phosphate bolism, Fructose and mannose metabolism, Thyroid cancer, Prostate catabolic process while the downregulated DEGs were mainly enriched cancer while in Reactome pathway were mainly enriched Glycolysis in the glycolytic process through glucose-6-phosphate, canonical gly­ Homo sapiens and Glucose metabolism Homo sapiens, Smooth Muscle colysis, glucose catabolic process to pyruvate, positive regulation of Contraction Homo sapiens, Beta oxidation of hexanoyl-CoA to butanoyl- metanephros development, myoblast differentiation. In the CC group, CoA Homo sapiens, Beta oxidation of lauroyl-CoA to decanoyl-CoA-CoA the upregulated DEGs were mainly enriched in meiotic cohesin com­ Homo sapiens, Beta oxidation of octanoyl-CoA to hexanoyl-CoA Homo plex, early endosome, secondary lysosome, COPII vesicle coat and axon sapiens, Beta oxidation of decanoyl-CoA to octanoyl-CoA-CoA Homo while the downregulated DEGs were mainly cytoplasmic vesicle lumen, sapiens (Fig. 4 and Table 3; A, B). mitochondrion, focal adhesion, membrane raft, ficolin-1-rich granule lumen. In the MF group the upregulated DEGs were mainly enriched in 3.4.2. Mesenchymal stem cells KEGG and Reactome pathways In mesenchymal stem cells the upregulated genes were mainly

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Fig. 4. Top 7 most significantly Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway enrichment analysis for up- and down-regulated genes between normal vs null PTEN samples in NSCs. A) KEGG pathways in up-regulated genes. B) Reactome pathway in up-regulated genes C) KEGG pathways in down- regulated genes. D) Reactome pathway in down-regulated genes. ErbB, erythroblastic leukemia viral oncogene; FGFR2, fibroblast growth factor receptor 2; PI3K, phosphatidylinositol-3-kinase; AKT, protein kinase B. enriched in Axon guidance, NF-kappa B signaling pathway, Prostate PAX2, ITGB1 and RET were appeared in all the methods after calcu­ cancer, Transcriptional misregulation in cancer, MicroRNAs in cancer, lation (Table 4; A). Among these 6 hub genes, PTEN and MAPK8 pre­ Phenylalanine, tyrosine and tryptophan biosynthesis, p53 signaling sented the highest degree, 11 and 9, respectively. The Cytoscape plugin pathway while in Reactome pathways were enriched mainly in cGMP MCODE showed the top two modules with scores of 2.8, 2.667 (Fig. 7). effects Homo sapiens, Hemostasis Homo sapiens, Nitric oxide stimulates Then, the obtained genes in these three modules selected for more guanylate cyclase Homo sapiens, Resolution of Sister Chromatid analyzes by functional enrichment. Proteins Involved in Insulin Re­ Cohesion Homo sapiens, Mitotic Prometaphase Homo sapiens, Netrin-1 sistance, Proteins Involved in Insulin Resistance and Proteins Involved signaling Homo sapiens, G2/M DNA replication checkpoint Homo sa­ in Alzheimer's Disease. Module 2 was mainly associated with the Cen­ piens. The downregulated genes were mainly enriched in Fructose and tral carbon metabolism in cancer, Glycolysis/Gluconeogenesis, and mannose metabolism, Central carbon metabolism in cancer, Galactose Galactose metabolism. metabolism, Glycolysis/Gluconeogenesis, Inositol phosphate metabo­ The above methods and conditions were served for Mesenchymal lism, Phosphatidylinositol signaling system, Biosynthesis of unsaturated stem cells with the following features; 423 nodes and 423 edges fatty acids while in Reactome pathway were mainly enriched in (Fig. 8). The selected 9 hub genes for this data set were PTEN, CDK1, Glycolysis Homo sapiens, Glucose metabolism Homo sapiens and KIF11, VCL, HK1, KIF20B, SIRT1, ACTR1A and ITGB1 after calculation Metabolism Homo sapiens, Synthesis of PIPs at the early endosome (Table 4; B). Among these 10 hub genes, PTEN and CDK1 presented the membrane Homo sapiens, metabolism of carbohydrates Homo sapiens, highest degree, 22 and 20, respectively. The Cytoscape plugin MCODE MAP2K and MAPK activation Homo sapiens, metabolism of lipids and showed the top two modules with scores of 7, 6.571 (Fig. 9). Then, the lipoproteins Homo sapiens (Fig. 5 and Table 3; C, D). genes in these two modules were analyzed by functional enrichment. Proteins Involved in p53 signaling pathway, Gap junction and Cell Cycle Module 2 were mainly associated with Ribosome biogenesis in 3.4.3. Protein-protein interaction (PPI) network and modular analysis in , RNA polymerase and Cytosolic DNA-sensing pathway. neural and mesenchymal stem cells All the DEGs (|log2 fold change (FC)| ≥ 0.2) were analyzed using the STRING database. Then, we analyzed these data by using Cytoscape 4. Discussion software to draw a PPI network containing 110 nodes and 110 edges (Fig. 5). This data was applied as input for Cytoscape software and the The role of PTEN deficiency in several types of solid tumors, in­ CytoHubba application to observe possible intersections from the three cluding NSCLC, breast, colorectal, endometrial, and ovarian cancers has calculations methods (Degree, Closeness, Betweenness) (Fig. 6) and been documented (Alfieri et al., 2017). then assigned 6 hub genes in these DEGs, including PTEN, MAPK8, VCL, For example, alteration by PTEN expression in NSCLCs has been

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Table 3 The top six enriched pathways of upregulated differentially expressed genes and downregulated differentially expressed genes in both NSCs and MSCs. A, The top 6 enriched pathways of the upregulated DEGs (NSCs). B, The top 6 enriched pathways of the downregulated DEGs (NSCs). C, The top 6 enriched pathways of the upregulated DEGs (MSCs). D, The top 6 enriched pathways of the downregulated DEGs (MSCs).

A

Term Count Genes P. value Combined score

Inositol phosphate metabolism 3 INPP5A; INPP5F; PTEN 0.001 95.28 Phosphatidylinositol signaling system 3 INPP5A; INPP5F; PTEN 0.002 62.36 Insulin resistance 2 MAPK8; PTEN 0.03 21.24 Negative regulation of the PI3K/AKT network Homo sapiens R-HSA-199418 3 FGF8;NRG3; PTEN 0.002 71.76 Axon guidance Homo sapiens R-HSA-422475 6 NRP1; FGF8; DPYSL4; NRG3; SLIT1; DOCK1 0.002 23.25 Inositol phosphate metabolism Homo sapiens R-HSA-1483249 2 INPP5A;PTEN 0.006 79.15

B

Term Count Genes P. value Combined score

Central carbon metabolism in cancer 4 RET; PGAM1; PFKP; HK1 5.92E-05 184.30 Glycolysis/Gluconeogenesis 3 PGAM1; PFKP; HK1 0.001 89.08 Prostate cancer 2 TCF7L2; PLAU 0.03 20.46 Glycolysis Homo sapiens R-HSA-70171 3 PGAM1; PFKP; HK1 0.03 253.62 MAP2K and MAPK activation Homo sapiens R-HSA-5674135 1 VCL 0.05 17.41 Metabolism of lipids and lipoproteins Homo sapiens R-HSA-556833 5 PHYH; ECHS1; ALOX5; PSAP; ANKRD1 0.06 6.46

C

Term Count Genes P. value Combined score

Prostate cancer 3 ZEB1; PLAU; PTEN 0.06 27.8 Transcriptional misregulation in cancer 4 HHEX; ZEB1; PLAU; JMJD1C 0.01 18.9 MicroRNAs in cancer 5 ZEB1; PLAU; DDIT4; PTEN; SIRT1 0.01 14.13 Resolution of Sister Chromatid Cohesion Homo sapiens R-HSA-2500257 3 CDK1; SMC3; ZWINT 0.01 26.97 Mitotic Prometaphase Homo sapiens R-HSA-68877 3 CDK1; SMC3; ZWINT 0.01 23.77 G2/M DNA replication checkpoint Homo sapiens R-HSA-69478 1 CDK1 0.02 151.78

D

Term Count Genes P. value Combined score

Central carbon metabolism in cancer 4 PGAM1; HKDC1; PFKP; HK1 0.02 48.49 Glycolysis/Gluconeogenesis 4 PGAM1; HKDC1; PFKP; HK1 0.002 45.13 Biosynthesis of unsaturated fatty acids 2 HACD1; SCD 0.01 36.41 Glycolysis Homo sapiens R-HSA-70171 4 PFKFB3; PGAM1; PFKP; HK1 0.0001 141.17 Synthesis of PIPs at the early endosome membrane Homo sapiens R-HSA-1660516 2 INPP5F; PI4K2A 0.0001 93.12 MAP2K and MAPK activation Homo sapiens R-HSA-5674135 2 APBB1IP; VCL 0.005 21.68

DEG, differentially expressed gene. reported in 8.2–59% of squamous cell lung cancer (SCC, squamous mice harboring germline and Cowden syndrome (CS) (Chalhoub and hystotype) (Fumarola et al., 2014). The correlation between PTEN Baker, 2009; Suzuki et al., 2008; Zbuk and Eng, 2007). Modern mole­ knocked-out with the later TNM stage of breast cancer was positive that cular biology has highlighted the alteration in gene expression profiles investigated by meta-analyses research (Chen et al., 2016). Null PTEN of cells involved in diseases. Our understanding can be elevated by also involved in 20% of colorectal cancer at the first stage while its analyzing the genes detected by the high-throughput sequencing tech­ effects rose by 58.9% at the fourth stage represented through alter­ nology, thereby predicting gene patterns with their alteration. To date, nating in the promoter hyper-methylation, declining in DNA copy myriads of deferentially expressed genes (DEGs) have been analyzed; number and reducing of protein expression (Lin et al., 2015). Further­ however, the methods might be varied (Kimura et al., 2003; Li et al., more, a wide range of frequencies (between 16 and 41% of tumor 2018; Ohno-Urabe et al., 2018). Therefore, further analysis, hub genes samples) of PTEN loss in prostate cancer was shown; however, the and pathways can be obtained by serving high throughput screening homozygous deletion was the most frequent event (Wise et al., 2017). tools (Wang et al., 2019). PTEN inactivation in the form of extensive PTEN loss is found in 70% of glioblastomas describing the most ag­ range mutations including missense and nonsense mutations, deleted gressive grade of astrocytic tumors (Chow and Baker, 2006). PTEN form of mono or bi-allelic of genomic locus, inhibition by oncogenic mutation is also associated with endometrial carcinoma's initial stage microRNAs and hypermethylation of promoter can be found in the (Di Cristofano and Ellenson, 2007). It is predisposed that PTEN muta­ somatic cancers such as glioblastoma, breast cancer and prostate cancer tions might have an essential role in the primary endometrial carci­ (Papa and Pandolfi, 2019). nomas and progression glioblastoma regarding PTEN mutation in the In the present study, R studio was used to extract the genetic

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Fig. 5. Top 7 most significantly Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway enrichment analysis for up- and down-regulated genes between normal vs null PTEN samples in MSCs. A) KEGG pathways in up-regulated genes. B) Reactome pathway in up-regulated genes C) KEGG pathways in down- regulated genes. D) Reactome pathway in down-regulated genes. PIPs, phosphatidylinositol phosphate; MAPK, mitogen-activated protein kinase. information from SRP047516; GSE61794 (Duan et al., 2015). We Mos–dependent cell transformation (Cowley et al., 1994; Hughes et al., identified 122 mRNA DEGs in human neural stem cells, including 57 1997; Lenormand et al., 1993; Okazaki and Sagata, 1995; Whalen et al., upregulated DEGs and 65 downregulated DEGs (fold change > 0.2, P- 1997). value < 0.05). We also used two different resources, including KEGG PTEN can decrease cell proliferation and cell migration by down­ and Reactome analysis to better monitoring of various relating enriched regulation of Ras-mediate MAP kinase activation, and meanwhile, it pathways; however, P-value < 0.07 were used as criteria for choosing reduces the activity of actin cytoskeletal organization and focal adhe­ main pathways involved in two sample types. sion which eventually cell proliferation and cell migration were af­ The upregulated DEGs were mainly enriched in transcriptional ac­ fected (Gu et al., 1998). tivator activity, phosphatidylinositol phosphate phosphatase activity, The correlation between the PI3K pathway and cancer was in­ MAP kinase activity while the downregulated DEGs were mainly in­ troduced in the 1980s, when its activity linked to the src protein of Rous volved in glycolytic process through glucose-6-phosphate, canonical sarcoma vitus and the middle-T protein of polyoma virus, known as two glycolysis, glucose catabolic process to pyruvate. Moreover, the KEGG oncoproteins (Sugimoto et al., 1984; Whitman et al., 1985). In human enrichment analysis results showed that the upregulated DEGs were cancer, two specific target p110α and PTEN mutations are highly associated with the Inositol phosphate metabolism, connected to the increasing activity of PI3K pathway, which provides Phosphatidylinositol signaling system, Insulin resistance while in clear genetic evidence for an integral role of PI3K in human cancer Reactome pathway were Negative regulation of the PI3K/AKT network (Chalhoub and Baker, 2009). Cell metabolism, angiogenesis and cancer Homo sapiens, Axon guidance Homo sapiens, Inositol phosphate meta­ growth are controlled through regulation of PI3K-AKT-mTOR pathway bolism Homo sapiens whereas the downregulated DEGs were mainly by PTEN, leading to the limited glucose uptakes. This is processed by enriched in the Central carbon metabolism in cancer, Glycolysis/ controlling the plasma membrane expression of GLUT1 exerted by Gluconeogenesis, Prostate cancer while in Reactome pathway were PTEN on either direct dephosphorylation of AKT or PtdIns (Li et al., Glycolysis Homo sapiens, MAP2K and MAPK activation Homo sapiens, 1997a; Perrone et al., 2009; Wu et al., 2003) PTEN conversely decrease metabolism of lipids and lipoproteins Homo sapiens. the anti-Warburg effect through glycolytic in this process MAP kinase pathway plays an integral role in transformation and (Alfieri et al., 2017). Interestingly, it has shown that various deleted cell cycle regulation. Cell growth, cell adhesion and spreading can be AKT isoforms rescue the PTEN loss driven tumorigenesis in several suppressed through the blocking of MAP kinase, which in turn inhibit tissues, especially in prostatic gland and endometrium (Papa and the upstream signaling components such as c-Ras– and v- Pandolfi, 2019). Besides, reduced glucose uptake and increased level of

8 H. Fiuji and M. Nassiri Gene Reports 21 (2020) 100895

Fig. 6. PPI network constructed with the differentially expressed genes in NSCs. PPI network constructed with the differentially expressed genes. Red nodes represent hub GENE analysis by cytoHubba.

Table 4 oxidative phosphorylation in mitochondria were revealed in the A: Key hub genes in the protein-protein interaction network found in NSCs. B: transgenic mouse model due to PTEN overexpression (Garcia-Cao et al., Key hub genes in the protein-protein interaction network found in MSCs. 2012). So, the possibility of cancer prevention and therapy may be A increased by triggering the inhibition of the metabolic reprogramming induced by PTEN loss (Zhou et al., 2019). Gene Full name Degree Regulation In prostate cancer cases, PTEN is highly mutated or deleted pro­ moting the PI3K/Akt signaling pathway, tumorigenesis (Li et al., PTEN Phosphatase and tensin homolog 11 Up MAPK8 Mitogen-activated protein kinase 8 10 Up 1997b; Majumder et al., 2003) and the c-Jun NH2-terminal kinase VCL Vinculin 9 Down (JNK) pathway (Vivanco et al., 2007). Besides, P53 activity can be di­ PAX2 Paired box 2 9 Down rectly regulated by PTEN interaction in an Akt-independent manner ITGB1 Integrin subunit beta 1 8 Down (Chang et al., 2008; Freeman et al., 2003). The lipid metabolism in RET Ret proto-oncogene 7 Down prostate cancer is more exposed to alteration comparing other epithelial cancers highlighted by the overexpressing of fatty acid syntheses re­ B sulted from PTEN loss, which consequently leads to cholesterol and fatty acid accumulation in human tumors, particularly in prostate Gene Full name Degree Regulation cancer (Zhou et al., 2019). Meanwhile, 258 mRNA DEGs in human mesenchymal stem cells PTEN Phosphatase and tensin homolog 22 Up CDK1 Cyclin dependent kinase 1 20 Up included 98 upregulated DEGs and 160 downregulated DEGs (fold KIF20B Kinesin family member 20B 13 Up change > 0.2, P-value < 0.05). The upregulated DEGs were mainly SIRT1 Sirtuin 1 13 Up enriched in positive regulation of cell migration, chromatin silencing KIF11 Kinesin family member 11 15 Down complex, RNA polymerase core enzyme binding while the down­ VCL Vinculin 15 Down HK1 1 14 Down regulated DEGs phosphofructokinase activity, focal adhesion, glycolytic ACTR1A ARP1 actin-related protein 1 homolog A, 12 Down process through glucose-6-phosphate. Moreover, the KEGG enrichment centractin alpha analysis results showed that the upregulated DEGs were associated with ITGB1 Integrin subunit beta 1 11 Down Prostate cancer, Transcriptional misregulation in cancer, MicroRNAs in cancer, Reactome pathway was Resolution of Sister Chromatid Cohesion Homo sapiens, Mitotic Prometaphase Homo sapiens, G2/M

9 H. Fiuji and M. Nassiri Gene Reports 21 (2020) 100895

Fig. 7. Top 2 modules from the protein-protein interaction network designed for NSCs. Nodes and links represent human proteins and protein interactions. (A) The enriched pathways of module 1; (B) the enriched pathways of module 2.

DNA replication checkpoint Homo sapiens. In contrast, the down-regu­ family member (miR-200a and miR-200b), mir-25A, mir-22, mir-21 can lated DEGs were mainly enriched in the Central carbon metabolism in regulate the PTEN through attaching to the specific segment of the cancer, Glycolysis/Gluconeogenesis, Biosynthesis of unsaturated fatty 3′UTR region of PTEN mRNA to interrupt PTEN expression. This in­ acids Reactome pathway were Glycolysis Homo sapiens, Synthesis of teraction can also be directly targeted by such miR-93 reported recently PIPs at the early endosome membrane Homo sapiens, MAPK activation in non-small cell lung cancer (NSCLC) (Alfieri et al., 2017). Interest­ Homo sapiens. ingly, this direct reaction of Micro-RNA with PTEN is appeared in miR- Although the function of PTEN as a tumor suppressor is associated 21 investigated in hepatocellular carcinoma (HCC) (Meng et al., 2007) with the inhibition of the PI3K pathway in cytoplasmic localization, its however, it has not yet reported in glioma in both in vivo and vitro role in the nucleus to arrest the cell cycle progression independent of (Huse et al., 2009). Furthermore, PTEN is triggered by miR-19a and PI3K pathway has been also reported (Hou et al., 2017). miR-214 as reported in different cancers (Pezzolesi et al., 2008; Yang The nuclear PTEN contributes to DNA repairs in response to geno­ et al., 2008). toxic stress, and meanwhile maintain chromosomal stability (Bassi Based on pathways obtained in this research, cell polarization and et al., 2013; Shen et al., 2007). Moreover, PTEN modulates both cell mobility can also be affected by deleting PTEN. The cellular po­ chromosome segregation and DNA replication by engaging directly larization is induced through combining of PtdIns (4,5)P2 to CDC42 in with transmitting genetic complexes (Hou et al., 2017). Chromosomal the apical region, promoting PAR-6/aPKC complex binding. The epi­ instability (CIN) in terms of both structure and number can be led to thelial formation being abrogated as a resultant of PTEN deletion led to incorrect chromosome transition resulted from null PTEN in the cells, the transition from epithelial to mesenchymal (Alfieri et al., 2017). marked by the centromere breakage, justifying the critical role of PTEN Furthermore, Cdc42 is required to promote mitosis and G1-S phase in preserving the chromosomal stability (Shen et al., 2007). progression through constituting the Cdc42-Par6-aPKC signaling com­ According to our results, PTEN also promotes chromosome con­ plex. The mitotic spindle orientation and cell proliferation can be in­ densation and stabilizes the heterochromatin status through its func­ duced by PTEN deletion (Stengel and Zheng, 2011). PTEN possibly tional interaction with non-histone proteins, like heterochromatin interrupts the formation of the focal adhesion through focal adhesion protein 1 (HP1) or histone proteins (Chen et al., 2014; Gong et al., kinase, which plays a critical role in controlling several pathways such 2015). Chromosome compaction can be disrupted when deleted PTEN as invasion, cell growth, survival and cell spreading. It has documented or its C terminus domain promote neutralizing positive charge of his­ that the FAK expression is reduced in MKN28 cells, a preclinical model tones through inducing H4 acetylation, resulting in chromatin decon­ of gastric cancer, when over-expressing the PTEN, thereby leading to densation (Hou et al., 2017). PTEN also monitors checkpoints to inhibit preventing cell migration and invasiveness in vivo tumor growth the cell cycle progression with unpaired and twisted DNA damage. The (Alfieri et al., 2017). loss of PTEN results in improper surveillance of impaired DNA gener­ As the last procedure in our investigation, the key DEGs were ated during both the G2-M transition and DNA replication, thereby identified through building PPI network. Constructing the PPI network, triggering the critical task of segregation in the genome during mitosis. we found that six hub genes (PTEN, MAPK8, VCL, PAX2, ITGB1, and The loss of PTEN in the embryonic stem cells represented an inaccurate RET) in neural stem cells and 9 hub genes (PTEN, CDK1, KIF11, VCL, DNA damage checkpoint, failing to respond to ionizing radiation (IR) HK1, KIF20B, SIRT1, ACTR1A and ITGB1) appeared in mesenchymal being correlated to defective phosphorylation of CHK1 as well as as­ stem cells respectively in each of the top 10 gene lists in terms of de­ sociation with increased frequency of aneuploidy of aberrant PTEN gree, betweenness and closeness. PTEN, VCL and ITGB are found to be expression in breast carcinoma (Puc et al., 2005). Therefore, these the central hub genes in both neural and mesenchymal stem cells. The findings prove that PTEN plays a crucial role in the activation of the cytoplasmic and nuclear role of PTEN in several pathways was dis­ DNA damage checkpoint, and correspondingly contribute to G2 cussed above. Vinculin (VCL) involving cell-cell adhesion and cell checkpoint screening for inhibiting CIN and aneuploidy (Hou et al., matrix junction regulates E-cadherin expression that is served to fila­ 2017). ments to the membrane and, meanwhile, plays a critical role in cell Some non-coding miRNAs with 20–25 nucleotide, such as mir-200 locomotion and cell morphology (Ai et al., 2017). It has also

10 H. Fiuji and M. Nassiri Gene Reports 21 (2020) 100895

demonstrated that the lack of VCL in the majority of cancers like prostate cancer being expressed with long alteration in the normal cell (Thorsen et al., 2008). Integrin-beta 1 (ITGB1) is one of the components of the integrin family embedding alpha and beta transmembrane sub­ units. It is reported that ITGB1 is highly expressed in normal and tu­ mors. Interestingly, several development processes like angiogenesis, tumor progression and metastasis are monitored by the ITGB1. It is also documented that this gene show resistance to radio-therapies or other targeted therapies in many cancer types. Its correlation with the diag­ nosis of several types of cancer (e.g., breast cancer, prostate cancer, correctional cancer and lung cancer) in the RNA or protein level has been investigated, highlighting its role as the anti-tumor therapies. However, there is still inconsistency between the research reports (Sun et al., 2018). Homozygous PTEN deletion in embryo leads to flawed placentation and eventually to induce death by embryonic day (E) 7.512 (Gregorian et al., 2009). In the embryonic NSCs, null PTEN results in negative regulation of NSCs proliferation, self-renewal, and survival in vivo and in vitro circumstances (Groszer et al., 2006; Groszer et al., 2001). More investigation revealed that PTEN deletion in NS/PCs contributes to accelerating of self-renewal capacity, and meanwhile increasing the rate of G0-G1 cell cycle entry, thereby shortening the cell cycle period. This process is associated with enhanced self-renewal in NSCs that promote the evading of proliferation control from homeostatic me­ chanisms (Groszer et al., 2006; Groszer et al., 2001). Therefore, impermanent PTEN deletion can be considered in certain neurodegenerative diseases treatment when its effects on embryonic NSCs self-renewal, proliferation, and survival enhance the limited adult NSCs population (Gregorian et al., 2009). Whether MSCs impacts on tumor progression is still elusive despite a massive investigation in this scope. Controversially, however, results have dual views toward MSC function in tumor progression, some of which revealed that MSCs can modify the signaling pathways toward tumor progression while other studies suggested that proliferation can be suppressed by the MSCs promoting apoptosis (Lin et al., 2013; Yulyana et al., 2015). Recently, it has been revealed that MSCs are involved in the mediating Wnt and AKT signaling, leading to invasion and survival of tumor cells. How­ ever, these signals can be inhibited by the up-regulation of PTEN, for instance, in gliomas (Lin et al., 2019).

5. Conclusion and future outlook

In the end, High-throughput sequencing tools were implemented to analyze and evaluate differential expression of RNA seq reads and distinguished several key genes and pathways that were highlighted in different tumors or related diseases, particularly prostate cancer. Based on the outcome obtained in this study, we discussed each pathway se­ parately and showed a wide variety of null PTEN effects on chromo­ some 10 gene expression in both neural and mesenchymal stem cells, which is confirming the previous studies that PTEN status play critical roles in gene expression of stem cells and involve in numerous path­ ways in and out of the cell. In conclusion, although weaknesses exist, gene expression profiling of chromosome 10 where explain the PTEN status provide a promising approach to examine the molecular and cellular mechanism involved in different pathways contributing to the tumorigenesis and tumor progression.

CRediT authorship contribution statement PPI network constructed with the differentially expressed genes in MSCs. PPI network constructed with the differentially expressed genes. Red nodes represent hub GENE analysis by cytoHubba.

H. F. and M. N. designed and performed differentially experiment

Fig. 8. gene analysis. All authors discussed the results and contributed to the final manuscript.

11 H. Fiuji and M. Nassiri M. and Fiuji H. 12

Fig. 9. Top 2 modules from the protein-protein interaction network designed for MSCs. Nodes and links represent human proteins and protein interactions. (A) The enriched pathways of module 1; (B) the enriched Gene Reports21(2020)100895 pathways of module 2. H. Fiuji and M. Nassiri Gene Reports 21 (2020) 100895

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