Gene Reports 21 (2020) 100895 Contents lists available at ScienceDirect Gene Reports journal homepage: www.elsevier.com/locate/genrep Gene expression profiling of chromosome 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 Protein 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 chromosome 10 gene expression profile. The RNA sequencing (RNA-seq) performed on four neural and NSCs four mesenchymal stem cells. DEG analysis outcome revealed 122 genes 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 kinase activity while the down- regulated DEGs were mainly involved in glycolytic process through glucose-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 enzyme 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. Gene Ontology analysis classified the differentially expressed genes into 3 groups. Molecular function, biological process, and cellular component in Neural stem cells (NSCs). 2 H. Fiuji and M. Nassiri Gene Reports 21 (2020) 100895 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 endosome 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 Actin 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) proteins specifically.