Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling

  • Authors:
    • Akiko Takao
    • Kazuhiro Yoshikawa
    • Sivasundaram Karnan
    • Akinobu Ota
    • Hirotsugu Uemura
    • Marco A. De Velasco
    • Yurie Kura
    • Susumu Suzuki
    • Ryuzo Ueda
    • Tokiko Nishino
    • Yoshitaka Hosokawa
  • View Affiliations

  • Published online on: September 5, 2018     https://doi.org/10.3892/or.2018.6683
  • Pages: 2455-2466
  • Copyright: © Takao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Phosphatase and tensin homolog (PTEN) deficiency is associated with development, progression, and metastasis of various cancers. However, changes in gene expression associated with PTEN deficiency have not been fully characterized. To explore genes with altered expression in PTEN‑deficient cells, the present study generated a PTEN‑knockout cell line (ΔPTEN) from a mouse prostate cancer‑derived cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9 (CRISPR/Cas9) gene editing system. Following transfection of the CRISPR/Cas9 construct, DNA sequencing was performed to identify deletion of the Pten locus and PTEN inactivation was verified by western blotting. The ΔPTEN cell line exhibited enhanced RAC‑alpha serine/threonine‑protein kinase phosphorylation and cyclin D1 expression. In addition, an increase in cell proliferation and colony formation was observed in the ΔPTEN cell line. Gene expression profiling experiments were analyzed with microarray and microRNA (miRNA) arrays. In the microarray analysis, 111 genes exhibited ≥10‑fold increased expression compared with the parent strain and mock cell line and 23 genes were downregulated. The only miRNA with increased expression of 10‑fold or more was mmu‑miR‑210‑3p. Genes with enhanced expression included genes involved in the development, progression, and metastasis of cancer such as Tet methylcytosine dioxygenase 1, twist family BHLH transcription factor 2, C‑fos‑induced growth factor and Wingless‑Type MMTV Integration Site Family, Member 3, and genes involved in immunosuppression such as Arginase 1. The results of the present study suggest that PTEN deficiency mobilizes a variety of genes critical for cancer cell survival and host immune evasion.

Introduction

Phosphatase and tensin homolog (PTEN) is a tumor suppressor and a lipid phosphatase that catalyzes dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate. PTEN has a high frequency of mutations in various cancers including prostate cancer and glioblastoma. PTEN alterations are observed in approximately half of all malignant tumors and correlate with increased RAC-alpha serine/threonine-protein (AKT) phosphorylation and initiation of downstream targets that modulate a wide range of cellular processes associated with the progression of tumor growth and survival (18).

To investigate the function of PTEN, various knockout mice have been prepared and the effect of PTEN deficiency examined (9). The authors also established a conditional PTEN-deficient mouse model of prostate cancer driven by the PSA-Cre promoter and demonstrated its utility in various experimental settings (1013). To develop better treatment strategies requires a deeper understanding the cellular and molecular mechanisms of PTEN deficiency. However, autochthonous tumors are highly heterogeneous and are composed of a complex tumor microenvironment that consists of various cell types including epithelial cancer cells, stromal fibroblasts, immune endothelial and blood cells, in addition to other contaminants, all of which contribute to the molecular characterization of PTEN deficiency. Although small interfering RNA methodology enables us to induce PTEN-deficiency at the protein level (14), researchers often take into consideration the fact that gene expression still remains at a certain level even under conditions of gene knockdown.

Next-generation genome editing strategies have been developed and are currently considered some of the best technological tools to most efficiently characterize gene function (15). The present study generated an isogenic PTEN-deficient cell clone from a parental mouse prostate cancer cell line using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system and performed comprehensive gene expression profiling analyses to identify its impact on unique genes associated with crucial roles in cellular processes, including cancer development and immunity.

Materials and methods

Cell lines

The mouse prostate cancer cell line, 2924V, which expresses wild-type PTEN was used in the present study. This cell line was established from a prostate tumor originating from a 57 week-old PSACre;PtenloxP/loxP conditional knockout mouse (10). Cells were cultured at 37°C and 5% CO2 in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% inactivated fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 IU/ml penicillin, and 100 µg/ml streptomycin.

PTEN-knockout using the CRISPR/Cas9 system

The CRISPR/Cas9 system was used to disrupt the expression of the PTEN gene as described previously (16). pSpCas9(BB)-2A-Puro (PX459) was a gift from Feng Zhang (Broad Institute of MIT, Harvard, MA, USA; Addgene plasmid #48139; 15). Briefly, a single guide RNA (sgRNA) sequence was selected using Optimized CRISPR Design (http://crispr.mit.edu/). The sgRNA sequence targeting PTEN was 5′-GCTAACGATCTCTTTGATGA-3′. The plasmid expressing human Cas9 and the PTEN sgRNA was prepared by ligating oligonucleotides into the BbsI site of PX459 (Pten/PX459). To establish a PTEN knockout clone with a one-nucleotide deletion, 2924V cells (1×106 cells/dish) were seeded in a 10-cm dish. Cells were then transfected with 10 µg of PTEN/PX459 using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Antibiotic selection (puromycin; 2 µg/ml) was begun 72 h after transfection and continued for at least 3 days. A single clone was selected, expanded, and then used for biological assays. For sequence analysis of the PTEN gene, the following primer set was used: 5′-CGCTAATCCAGTGTACAGTA-3′ and 5′-CTGCGAGGATTATCCGTCTT-3′.

Morphology

The cells were plated in a 12-well plate. After 24 h, the cell morphology was digitally photographed at ×100 magnification using an inverted microscope system (IX73: Olympus Corporation, Tokyo, Japan).

MTT assay

Briefly, parent cells, mock cells, and ΔPTEN were seeded (5×103 cells/well) into a 96-well plate. Subsequently, 10-µl MTT solution (5 µg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well and cells were incubated at 37°C in 5% CO2 for 4 h. Next, cell lysis buffer [10% sodium dodecyl sulfate (SDS) in 0.01 M HCL] was added to the wells to dissolve the formazan crystals produced by MTT. The optical density (550 nm) at each time point (day 0, 1, 3, 5, and 7) is presented as the mean ± standard error of the mean (n=6).

Colony formation assay

Colony formation assays were performed by seeding 500 cells of each line in 12-well plates. After 14 days, cells were fixed/stained with 3.7% formaldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing 0.2 % (wt/vol) crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature and number of colonies were imaged and counted manually. Bar graphs represent the number of stained colonies. Data are presented as the mean ± standard error of the mean (n=3).

Western blotting

Cells were lysed in radioimmunoprecipitation assay buffer (Wako Pure Chemical Industries, Ltd.) supplemented with a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) for 30 min at 4°C. Insoluble material was removed by centrifugation at 15,400 × g for 10 min at 4°C. Protein content was determined using a DC protein assay kit (5000112JA; Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. Protein lysates were mixed with loading buffer and boiled for 5 min. A total of 10 µg protein was separated by electrophoresis on 10% PAGE gels (TEFCO, Tokyo, Japan). Proteins were then transferred to Immobilon-P membranes (EMD Millipore, Billerica, MA, USA) and blocked in 5% skim milk in Tris-buffered saline with 0.01% Tween-20 (TBS-T) for 1 h at room temperature. All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Membranes were incubated with primary antibodies [PTEN, 1:3,000, #9559; AKT1, 1:1,000, #9272; phosphorylated (p)-AKT, 1:1,000, #9271; phosphorylated (p)-Rb, 1:3,000, #9307; cyclin D1, 1:3,000, #2922; CDC2, 1:3,000, #9112; CDK2, 1:3,000, #2546; CDK4, 1:3,000, #2906; CDK6, 1:1,000, #3136; CDK7, 1:1,000, #2916 and GAPDH 1:5,000, #5174] overnight at 4°C. After three washes with TBS-T, membranes were incubated with anti-mouse, #7076/rabbit, #7074, horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000) for 1 h at room temperature followed by a final wash. Proteins were detected by applying ImmunoStar LD (Wako Pure Chemical Industries, Ltd.) to the membrane and signals were quantified using ImageQuant LAS-4000 (GE Healthcare Life Sciences, Little Chalfont, UK) according to the manufacturer's protocol.

RNA extraction

For microarray analysis, reverse transcription (RT)-polymerase chain reaction (PCR) and quantitative (q) PCR, total RNA was extracted from cell lines with the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. RNA extraction for miRNA analysis was performed with the MiRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol.

Microarray analysis

Expression profiling analysis of mRNA was performed using the Agilent Oligo Microarray Kit (8×60 K; G4852B; Agilent Technologies, Inc., Santa Clara, CA, USA). Nucleic acid labeling and hybridization were performed with the One-Color Microarray-Based Gene Expression Analysis kit (Agilent Technologies, Inc.) according to the manufacturer's protocol. Briefly, 100 ng of total RNA was amplified and labeled using the Low Input Quick Amp Labeling Kit. After labeling, RNA was purified with RNeasy Mini Kit (Qiagen GmbH). The RNA fragmentation reaction was performed at 60°C for 30 min, after which the samples were collected on ice for 1 min, and 2X Hi-RPM Hybridization Buffer was added to stop the reaction. Samples were further mixed, centrifuged at 13,000 × g for 1 min at room temperature, placed on ice, and then loaded onto the array. The arrays were hybridized at 65°C for 17 h. The microarray slides were then washed with Gene Expression Wash Buffers I and II and scanned with the Agilent Microarray Scanner-G2505C (Agilent Technologies, Inc.). Feature Extraction Software Version 11.0.1.1 (Agilent Technologies, Inc.) was used to extract and analyze the signals and signal intensities were normalized as previously described (17).

The background signals were normalized and microarray expression data were rank-ordered according to the expression levels of the ΔPTEN/mock cells. Differentially expressed genes between ΔPTEN and mock cells were considered relevant if there was ≥10-fold change.

Functional enrichment analyses of differentially expressed genes were carried out using the Panther web-based tool (18).

To confirm gene expression, RT-qPCR was performed using the ABI 7900 HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) and calculated using the 2−ΔΔCq method (19). Total RNA from the three cell lines were converted to cDNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The Tet methylcytosine dioxygenase 1 (Tet1), twist family BHLH transcription factor 2 (Twist2), arginase 1 (Arg1), C-fos induced growth factor (Figf), Wingless-Type MMTV Integration Site Family, Member 3 (Wnt3), prostaglandin reductase 1 (Ptgr1), polypeptide N-acetylgalactosaminyltransferase 14 (Galnt14), and GAPDH (reference) genes were amplified and detected using SYBR-Green (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR reactions were prepared in a final volume of 20 µl, with 17.6 µl of SYBR-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 2.0 µl of DNA, and 0.2 µl each of the 10 pmol/µl forward and reverse primers. Thermal cycler settings included DNA polymerase activation at 95°C for 2 min followed by 55 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, and extension at 70°C for 20 sec. Each measurement was performed in triplicate. The quality of the PCR products was monitored by using the post-PCR melt-curve analysis.

In order to visualize PCR Products, PCR was carried out changing the number of cycles to 30 cycles. The PCR products were electrophoresed in 1.5% agarose gel, and stained with GelRed Nucleic Acid Stain (41003-T; Biotium, Inc., Fremont, CA, USA) and photographed. Primer sequences are listed in Table I.

Table I.

Primers used for polymerase chain reaction.

Table I.

Primers used for polymerase chain reaction.

Gene nameAccessionRelative intensity of KO/mockForward primerReverse primer
Tet1NM_00125385732 ACACAGTGGTGCTAATGCAG AGCATGAACGGGAGAATCGG
Twist2NM_00785513 TACAGCAAGAAATCGAGCGAAG GCTGAGCTTGTCAGAGGGG
Arg1NM_00748212 CTCCAAGCCAAAGTCCTTAGAG GGAGCTGTCATTAGGGACATCA
FigfNM_01021610 AACAGATCCGAGCAGCTTCTA TTTTGAGCTTCAACCGGCATC
Wnt3NM_00952110 AGCGTAGCAGAAGGTGTGAAG CCAGGTGGCCCCTTATGATG
Galnt14NM_027864−19 CTCATCAAACTGCTCCCACA GCTCTGGATCACCGTACTGC
Ptgr1NM_025968−30 CAATCGTTCCTTTTGGGAAG CATGAGAGTTGCAGCCAAAA

[i] Tet1, Tet methylcytosine dioxygenase 1; Twist2, twist family BHLH transcription factor 2; Arg1, Arginase 1; Figf, c-fos induced growth factor; Wnt3, Wint family member 3; Ptgr1, prostaglandin reductase 1; Galnt14, polypeptide N-acetylgalactosaminyltransferase 14.

miRNA array analysis

miRNA profiling analysis was performed using the Agilent Mouse miRNA kit (8×60 K; G4872A-070155). miRNA labeling, hybridization, and washing were carried out according to the manufacturer's protocol (Agilent Technologies, Inc.). Hybridized microarrays were scanned with a DNA microarray scanner (Agilent Microarray Scanner; G2505C) and features were extracted using the Agilent Feature Extraction image analysis tool (version 11.0.1.1). Briefly, average values of the spots of each miRNA were background-subtracted and subjected to further analysis. miRNA array expression data were rank-ordered according to the expression levels of the ΔPTEN/mock cells. To confirm miRNA expression level, RT-qPCR was performed. Total RNA from the three cell lines were converted to cDNA with the Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. Realtime PCR was performed using Taqman MicroRNA Assays (has-miR-210, RT:000512 and snoRNA234; RT:001234 as control; Applied Biosystems) according to the manufacturer's protocol.

Statistical analysis

At least three replications per experiment and three independent experiments were performed. The results are expressed as the mean ± standard error of the mean. Independent-samples Student's t-test and one-way analysis of variance, followed by the least significant difference post-hoc test were performed to analyze data using JMP Ver.13.2.1 (SAS Institute Inc; Tokyo, Japan). P<0.05 was considered to indicate a statistically significant difference (*P<0.05; **P<0.01; ***P<0.001).

Results

Establishment of PTEN-knockout cells using the CRISPR/Cas9 System

The present study performed genome editing (targeting sequence presented in Fig. 1A) using the CRISPR/Cas9 system and obtained multiple clones (ΔPTEN). Fig. 1B and C reveal DNA sequencing of target sites in the resultant clones. These results demonstrated that targeting PTEN with the CRISPR/Cas9 system effectively generated mutant-PTEN clones. Next, the present study assessed the effects of PTEN inactivation in the resulting clones. Overall, the general appearance was similar between parental, mock, and ΔPTEN cells; however, marginal differences were observed in the morphological appearance of ΔPTEN cells such as heterogeneity of the cell shape (Fig. 2).

Using western blot analysis, the present study verified the effective knockout of PTEN in ΔPTEN cells. Fig. 3 shows that the expression of PTEN was present in the parental strain and mock-transfected cells but was effectively downregulated in ΔPTEN cells. Accordingly, Akt phosphorylation levels increased in the absence of PTEN. Activation of the PI3K/Akt pathway promotes the cellular proliferation of transformed cells; thus, the expression of molecules involved in proliferation was then assessed. In ΔPTEN cells, the upregulation of Akt phosphorylation was associated with the elevated expression of cyclin D1, cyclin dependent kinase (CDK)4, and decreased expression of CDK7. In addition, increased phosphorylation of the retinoblastoma tumor-suppressor protein (RB) was observed, a known regulator of cellular proliferation, in ΔPTEN cells. As the expression of molecules involved in the cell cycle was demonstrated to be enhanced in ΔPTEN cells, the present study then assessed the effects of PTEN inactivation on cell viability. Fig. 4 shows that the inactivation of the PTEN function in ΔPTEN cells results in increased cell growth and enhanced colony formation.

Microarray and miRNA array analysis of PTEN-knockout cells

Using gene microarrays, the present study assessed changes in gene expression following the loss of the PTEN function and focused on genes that were differentially expressed in ΔPTEN cells relative to mock-transfected cells and the parental strain (Fig. 5). Overall, 111 genes were upregulated by at least 10-fold, including one with a 74-fold increase in ΔPTEN cells (Table II). In addition, 23 genes were identified to be substantially downregulated by ≥10-fold, one with a 139-fold decrease (Table III).

Table II.

Genes that are upregulated 10-fold or more in phosphatase and tensin homolog knockout cells.

Table II.

Genes that are upregulated 10-fold or more in phosphatase and tensin homolog knockout cells.

Gene symbolAccessionUniGeneFunctionRelative intensity of KO/mock
Sprr2dNM_011470Mm.87820Small proline-rich protein 2D74
Serpinb6bNM_011454Mm.36526Serine (or cysteine) peptidase inhibitor, clade B, member 6b36
LifrNM_013584Mm.149720Leukemia inhibitory factor receptor36
Tet1NM_001253857Mm.491965Tet methylcytosine dioxygenase 132
Sprr2eNM_011471Mm.261596Small proline-rich protein 2E29
Dppa2NM_028615Mm.27857Developmental pluripotency associated 229
BB612626BB612626Mm.463155Not indicated26
Gap43NM_008083Mm.1222Growth associated protein 4325
Sprr1bNM_009265Mm.140151Small proline-rich protein 1B23
Tspan10NM_145363Mm.209875Tetraspanin 1021
Tex15NM_031374Mm.280624Testis expressed gene 1520
Prl7a2NM_011168Mm.153999Prolactin family 7, subfamily a, member 219
Gm9519XR_391191Mm.389803Not indicated19
Nlrc5NM_001033207Mm.334720NLR family, CARD domain containing 519
Gm26579XR_380816Mm.356554Not indicated19
Aqp9NM_001271843Mm.335570Aquaporin 918
Sp110NM_175397Mm.335802Sp110 nuclear body protein18
Gm29824XR_870586Mm.135986ncRNA17
Sdr16c6BC125450Mm.298546Short chain dehydrogenase/reductase family 16C, member 617
Prl7a1NM_008930Mm.196424Prolactin family 7, subfamily a, member 117
Cfhr2NM_001025575Mm.439660Complement factor H-related 217
Fa2hNM_178086Mm.41083Fatty acid 2-hydroxylase17
Zfp352NM_153102Mm.214642Zinc finger protein 35216
Cyp7a1NM_007824Mm.57029Cytochrome P450, family 7, subfamily a, polypeptide 116
Tvp23aNM_001013778Mm.325732Trans-golgi network vesicle protein 23A15
Mmel1NM_013783Mm.116944Membrane metallo-endopeptidase-like 115
IvlNM_008412Mm.207365Involucrin15
Sprr2hNM_011474Mm.10693Small proline-rich protein 2H15
AV154423AV154423Mm.486971Not indicated15
Phf11dNM_199015Mm.479285PHD finger protein 11D15
Col8a1NM_007739Mm.130388Collagen, type VIII, alpha 114
Evi2aNM_001033711Mm.439665Ecotropic viral integration site 2a14
Samd15NM_001290288Mm.302304Sterile alpha motif domain containing 1514
Gm13119NM_001034101Mm.389596Not indicated14
Trp63NM_011641Mm.20894Transformation related protein 6314
AA623943XM_006534607not indicatedExpressed sequence AA62394314
Defb3NM_013756Mm.103651Defensin beta 314
Gm30692XR_863174not indicatedncRNA14
Gm32204XM_006497575not indicatedNot indicated14
Serpinb2NM_011111Mm.271870Serine (or cysteine) peptidase inhibitor, clade B, member 214
Rsad2NM_021384Mm.24045Radical S-adenosyl methionine domain containing 214
Gm31115XR_390648not indicatedNot indicated14
Slco4a1NM_148933Mm.133687Solute carrier organic anion transporter family, member 4a114
Sod3NM_011435Mm.2407Superoxide dismutase 3, extracellular13
Ccdc153NM_001081369Mm.347681Coiled-coil domain containing 15313
Lce1dNM_027137Mm.176243Late cornified envelope 1D13
Twist2NM_007855Mm.9474Twist basic helix-loop-helix transcription factor 213
Plcb1NM_019677Mm.330607Phospholipase C, beta 113
Olfr376NM_001172686Mm.236410Olfactory receptor 37613
Gm19619NR_040428Mm.125059Not indicated13
Dnm3NM_172646Mm.441620Dynamin 313
Chst5NM_019950Mm.432506Carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 513
BF119155BF119155Mm.432156Not indicated12
Arg1NM_007482Mm.154144Arginase, liver12
Cdh7AK034096Mm.487119Cadherin 7, type 212
Ugt1a6bNM_201410Mm.300095UDP glucuronosyltransferase 1 family, polypeptide A6B12
Chrna5NM_176844Mm.103778Cholinergic receptor, nicotinic, alpha polypeptide 512
Phf11aNM_172603Mm.254918PHD finger protein 11A12
Sh3tc2NM_172628Mm.262320SH3 domain and tetratricopeptide repeats 212
Armc2NM_001034858Mm.211320Armadillo repeat containing 212
Fgf21NM_020013Mm.143736Fibroblast growth factor 2112
Ccdc109bNM_025779Mm.31056Coiled-coil domain containing 109B12
Klk7NM_011872Mm.251227Kallikrein related-peptidase 7 (chymotryptic, stratum corneum)12
Sh3gl3NM_017400Mm.432002SH3-domain GRB2-like 311
Gm17202XR_389494not indicatedNot indicated11
Iigp1NM_021792Mm.261140Interferon inducible GTPase 111
AF067061NM_199060Mm.247428cDNA sequence AF06706111
Plce1NM_019588Mm.34031Phospholipase C, epsilon 111
TprgNM_175165Mm.126851Transformation related protein 63 regulated11
Spint3NM_001177401Mm.234248Serine peptidase inhibitor, Kunitz type, 311
LOC102634459XR_387206not indicatedNot indicated11
Gm4340NM_001177535Mm.339215Not indicated11
Arhgap9NM_001285785Mm.227198Rho GTPase activating protein 911
Aldh1a3NM_053080Mm.140988Aldehyde dehydrogenase family 1, subfamily A311
Adh7NM_009626Mm.8473Alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide11
Prl3d2NM_172155Mm.458451Prolactin family 3, subfamily d, member 111
Lama1AK051116Mm.303386Laminin, alpha 111
Gm7978NM_001270457Mm.380154Not indicated11
BB114814XM_006508432Mm.304207Expressed sequence BB11481411
D5Ertd577eNM_177187Mm.348793DNA segment, Chr 5, ERATO Doi 577, expressed11
Lrrn4NM_177303Mm.386903Leucine rich repeat neuronal 411
Trav12-1BC038136Mm.333502T cell receptor alpha variable 12-111
CV783874CV783874Mm.441595Not indicated11
Igfbp5NM_010518Mm.405761Insulin-like growth factor binding protein 511
Gm3259NM_001270456Mm.402477Not indicated10
Gm4665AK132957Mm.437523Not indicated10
Sox11NM_009234Mm.41702SRY (sex determining region Y)-box 1110
Tcstv1NM_018756Mm.3021752-Cell-stage, variable group, member 110
Sprr3NM_011478Mm.57092Small proline-rich protein 310
PodnNM_001285956Mm.74710Podocan10
Ifit3bNM_001005858Mm.271850Interferon-induced protein with tetratricopeptide repeats 3B10
Gabra1NM_010250Mm.439668Gamma-aminobutyric acid (GABA) 1 A receptor, subunit alpha10
Ifi44NM_133871Mm.30756Interferon-induced protein 4410
aNM_015770Mm.315593Nonagouti10
Rd3lXM_006515764Mm.134213Retinal degeneration 3-like10
FigfNM_010216Mm.297978Vascular endothelial growth factor D10
B430212C06RikNR_033214Mm.491997Not indicated10
AK034098AK034098Mm.446266Not indicated10
Bank1NM_001033350Mm.30832B cell scaffold protein with ankyrin repeats 110
Sel1l3NM_172710Mm.235020Sel-1 suppressor of lin-12-like 310
Sgsm1NM_172718Mm.200203Small G protein signaling modulator 110
Krt13NM_010662Mm.4646Keratin 1310
TpoNM_009417Mm.4991Thyroid peroxidase10
Tnfrsf18NM_009400Mm.491989Tumor necrosis factor receptor superfamily, member 18
Klra2NM_008462Mm.4783Killer cell lectin-like receptor, subfamily A, member 2
Pcdh7NM_018764Mm.332387Protocadherin 710
Cbr2NM_007621Mm.21454Carbonyl reductase 210
Wnt3NM_009521Mm.159091Wingless-type MMTV integration site family, member 3
AK042637AK042637not indicatedNot indicated10
CO811058CO811058Mm.421039Not indicated10
St18NM_173868Mm.234612Suppression of tumorigenicity 1810

Table III.

Genes that are downregulated 10-fold or more in phosphatase and tensin homolog knockout cells.

Table III.

Genes that are downregulated 10-fold or more in phosphatase and tensin homolog knockout cells.

Gene symbolAccessionUniGeneFunctionRelative intensity of mock/KO
WtipNM_207212Mm.422738WT1-interacting protein139
Gm32139XR_001782443not indicatednot indicated38
Gm38426NR_103491Mm.437155not indicated37
Ptgr1NM_025968Mm.34497prostaglandin reductase30
Pla2g16NM_139269Mm.274810phospholipase A2, group XVI29
Galnt14NM_027864Mm.271953 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1419
Dhrs4NM_030686Mm.27427 dehydrogenase/reductase (SDR family) member 419
Fam124aNM_001243857Mm.291864family with sequence similarity 124, member A18
Robo1NM_019413Mm.310772roundabout guidance receptor 116
Evc2NM_145920Mm.25506Ellis van Creveld syndrome 215
Spats2lNM_144882Mm.159989spermatogenesis associated, serine-rich 2-like14
Ttc12NM_172770Mm.177413tetratricopeptide repeat domain 1214
Gxylt2NM_198612Mm.272037glucoside xylosyltransferase 214
Tmem173NM_028261Mm.45995transmembrane protein 17313
Gm2030NM_001100445Mm.411645not indicated13
Gm16404NM_001220497Mm.380174not indicated13
Gm1993NM_001102677Mm.484626not indicated12
Gm10487NM_001100609Mm.483167not indicated12
Gm5168NM_001025607Mm.370361not indicated12
SlxNM_001136476Mm.489202Sycp3 like X-linked12
Scml2NM_001290651Mm.159173sex comb on midleg-like 211
Gm14625NM_001220498Mm.477978not indicated11
PpicNM_008908Mm.4587peptidylprolyl isomerase C10

The mRNA expression levels of Tet1, Twist2, Arg1, Figf, Wnt3, Ptgr1, and Galnt14 were measured by RT-qPCR to confirm the results of the microarray analysis (Fig. 6A). The expression patterns of the RT-PCR analysis were parallel to those in the microarray, thereby verifying the results (Fig. 6B).

Next, the present study performed gene enrichment profiling to gain better insight into the genes differentially expressed after PTEN-knockout in the model. The analysis using the Panther annotation database revealed that 64 of the 111 upregulated genes corresponded to 15 pathways (Fig. 7).

Furthermore, the expression of non-coding RNAs, in particular miRNAs, were assessed to further delve into the impact of PTEN inactivation and altered gene expression. Notably, the miRNA expression analysis in this study revealed that only the mmu-miR-210-3p expression increased (≥10-fold) due to PTEN-knockout. Corroborating our initial observation, the RT-qPCR analysis presented an evident increase of mmu-miR-210-3p in ΔPTEN cells compared with the parental strain and mock-transfected cells (Fig. 8).

Discussion

The present study investigated the changes in carcinogenesis-associated genes caused by PTEN deficiency in a mouse prostate cancer model. A PTEN-deficient mouse prostate cancer isogenic cell line was established using the CRISPR/Cas9 system. The phosphorylation of Akt and the expression of cyclin D1 were elevated in PTEN-deficient cells. Although the expression of CDK7 wa srevealed to be decreased, the reason remains unknown. The expression levels of cyclinD1 and CDK4 that increased as demonstrated by western blotting, were not recognized as much different from that of parental cell or mock cell in microarray analysis. The reason for this increase in the expression currently remains unclear. Furthermore, microarray and miRNA arrays were used to assess the changes in gene expression. These results suggested that the PTEN expression is essential for normal gene expression.

The present study observed alterations in the expression levels of various genes due to the loss of the PTEN gene, which may be attributed to an increase in the expression of TET1. Demethylation is increased in several genes due to the enhanced expression of TET1, a dioxygenase that catalyzes the conversion of the modified genomic base 5-methylcytosine (5 mc) into 5-hydroxymethylcytosine (5hmc). Reportedly, Nanog is an example of a gene induced by Tet1 expression. Not only does Nanog have a role in the maintenance of mouse ES cells (20), but also in the maintenance of cancer stem cells, suppression of apoptosis, promotion of cancer progression and metastasis, and angiogenesis (21). These findings suggest that PTEN deficiency drives TET1 and TET1-gene regulation during carcinogenesis; however, the underlying reasons for an increase in the TET1 expression in PTEN deficiency remain unclear.

In this model, we established the upregulation of Twist2, Figf, Wnt3, and Arg1 in response to Pten-knockout; these genes play vital roles in the development and progression of cancer and promote the immune suppression in the surrounding tumor microenvironment.

Twist2, a member of the basic helix-loop-helix (BHLH) transcription factors, is overexpressed in several cancer types. Previously, studies have reported the correlation of Twist expression with head and neck squamous cell carcinomas, cervical carcinomas, and ovarian cancer (2224). Yang et al reported that hypoxia inducible factor-1 (HIF-1) promotes EMT through direct regulation of Twist expression (25). Twist is a master regulator of gastrulation and mesoderm specification and is implicated to be essential in the mediation of cancer metastasis (25). In this study, the expression of Twist was enhanced by the absence of functional PTEN, suggesting transcriptional regulation by mechanisms mediated by proteins other than HIF-1.

Figf, also termed vascular endothelial growth factor (VEGF)-D is a member of the platelet-derived growth factor (PDGF)/VEGF family and is active in angiogenesis and endothelial cell growth. Reportedly, the normal VEGF-D expression is detected in the lung, heart, skeletal muscle, skin, adrenal gland, and gastrointestinal tract (2628). In addition, VEGF-D is upregulated in glioblastoma (29), melanoma (30), colorectal carcinoma (31), breast carcinoma (32), and cervical intraepithelial neoplasia (33). Tian et al reported that PTEN suppresses VEGF expression in HCC through both phosphatase-dependent and -independent mechanisms (34). The present study also implicated that PTEN regulates VEGF expression through both phosphatase- and HIF-1a- -independent mechanisms. Kaushal et al (35) reported that VEGF-D expression is observed in all prostate cancer tissues and that it is increased in samples at advanced stages compared with those at the early stage; however, they did not report the correlation between VEGF-D expression and PTEN. In 20% of prostate cancers, PTEN is deleted (36). PTEN-deficient prostate cancers express VEGF-D at an early stage; therefore, it is imperative to compare the expression between tumors with and without PTEN.

The Wingless-type MMTV integration site family genes comprise structurally related genes that encode a secreted signaling protein implicated in oncogenesis and several developmental processes, including the regulation of cell fate and patterning during embryogenesis (37,38). In normal tissues, Wnt3 RNA is primarily detected in the testis, skin, and brain. Reportedly, Wnt3 is associated with cancer progression, invasion, and malignant conversion in cancer of the gastric (39), lungs (40,41), and hepatocellular carcinoma (42,43). In ΔPTEN cells, the cyclin D1 expression was upregulated. In addition, PTEN loss has been reported to augment cyclin D1 expression, although the underlying mechanism remains only partially understood. However, Zang et al reported that the Wnt/b-catenin pathway stimulates increased cyclin D1 levels in lymph node metastasis in papillary thyroid cancer (44). These results suggest an association between the PTEN-loss-induced Wnt3 expression and the subsequent upregulation of cyclin D1.

In mammals, two arginase isoforms (Arg1 and Arg2) have been identified. Arg1 is known as the liver type and is expressed in hepatocytes (45). In mice, Arg1 can also be expressed in myeloid cells under the stimulation of T helper 2 cytokines interleukin (IL)-4 and IL-13 (46), transforming growth factor-b (47), macrophage-stimulating protein (48), or GM-CSF (49). Although immune responses are controlled by amino acid metabolism, Rodriguez et al reported that arginase produced by tumor-infiltrating macrophages repressed the expression of the T-cell receptor CD3z, resulting in the suppression of antigen-specific T-cell responses (46). In this study, it was observed that Arg1 expression increased following Pten deficiency; however, to the best of the author's knowledge, there are no reports demonstrating that Arg1 is highly expressed in any cancer other than liver cancer. Recent cancer treatment strategies have targeted arginine metabolism (50,51), thereby making Arg1 an attractive therapeutic approach for PTEN-deficient tumors.

The present study also analyzed differential expression of miRNA and observed an increased expression of mmu-miR-210-3p in the PTEN-deficient cell line. Reportedly, the expression of miR-210 is induced by HIF-1, which is known to be upregulated in PTEN-deficient cells (52). The expression levels of the target genes of miR-210 were not observed to be altered in the present study. Studies have suggested that miR-210 is involved in multiple processes, including angiogenesis, metastasis, oncogenesis, decreased cell division, and tumor suppression (53,54). However, the exact function remains unclear and requires further investigation.

In conclusion, the present study established a mouse prostate cancer cell model of PTEN deficiency and provided evidence of altered expression of genes associated with the deregulation of signaling processes implicated in human cancers. Gene expression profiling suggested enhanced regulation cancer hallmarks, including cell proliferation, angiogenesis, metastasis, and immunosuppression. However, further studies are warranted to dissect and elucidate molecular mechanisms involved in promoting PTEN-deficient cancer progression.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

AT, KY, SK, AO, HU, MADV, YK, SS, RU, TN and YH equally took part in the conception and design of the study, acquisition and interpretation of data, drafting the article and final approval of the version to be published.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors state that they have no competing interests.

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November-2018
Volume 40 Issue 5

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Online ISSN:1791-2431

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Spandidos Publications style
Takao A, Yoshikawa K, Karnan S, Ota A, Uemura H, De Velasco MA, Kura Y, Suzuki S, Ueda R, Nishino T, Nishino T, et al: Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling. Oncol Rep 40: 2455-2466, 2018.
APA
Takao, A., Yoshikawa, K., Karnan, S., Ota, A., Uemura, H., De Velasco, M.A. ... Hosokawa, Y. (2018). Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling. Oncology Reports, 40, 2455-2466. https://doi.org/10.3892/or.2018.6683
MLA
Takao, A., Yoshikawa, K., Karnan, S., Ota, A., Uemura, H., De Velasco, M. A., Kura, Y., Suzuki, S., Ueda, R., Nishino, T., Hosokawa, Y."Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling". Oncology Reports 40.5 (2018): 2455-2466.
Chicago
Takao, A., Yoshikawa, K., Karnan, S., Ota, A., Uemura, H., De Velasco, M. A., Kura, Y., Suzuki, S., Ueda, R., Nishino, T., Hosokawa, Y."Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling". Oncology Reports 40, no. 5 (2018): 2455-2466. https://doi.org/10.3892/or.2018.6683