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 PSA^Cre;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.

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′.

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).

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 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).

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.

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.

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.

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.

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).

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.

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).

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).