ONCOMINE (http://www.oncomine.org), is an online cancer microarray database, and was used in the present study to analyze the transcription levels of the TRPM family in different types of human cancers. The mRNA expression of the TRPM family in clinical cancer specimens were compared with that in normal controls. The present study set the thresholds as follows: The gene rank, P-value and fold change were defined as 10%, 0.01 and 2, respectively.

The CCLE (https://portals.broadinstitute.org/ccle/home) provides public access to genomic data, analysis and visualization for ~1,000 cell lines. ‘TRPM8’ was searched in the CCLE database and the expression data of TRPM8 from the available cancer cell lines in a series of cancers was verified by using cell line data from CCLE.

The survival data were downloaded from CGGA (http://www.cgga.org.cn), a newly developed interactive web server for analyzing the gene expression data of 325 patients (203 males and 122 females). The samples were categorized into two groups (high and low TRPM8 expression levels) using the median gene expression value and were compared using a Kaplan-Meier survival plot. The investigators established overall survival (OS) time as the prognostic value of patients with glioblastoma. Survival analysis with P<0.05 was considered to indicate a statistically significant difference.

The human glioblastoma U251 cells were kindly gifted by G.F. Vande Woude, (Van Andel Research Institute, Grand Rapids, MI, USA). The human glioblastoma U251 cells were cultured in DMEM containing 10% fetal bovine serum and placed in a humidified cell incubator with 5% CO₂ at 37°C.

Cell monolayers (at 70% confluency) were transfected with the pEGFP-C1-TRPM8 plasmids and the enhanced green fluorescent protein plasmid-C1 (pEGFP-C1) vector using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. shRNA targeting TRPM8 was designed and inserted into the pGPU6/GFP/Neo vector (Thermo Fisher Scientific, Inc.). shRNA-TRPM8 was also transfected into human glioblastoma U251 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cDNA Synthesis kit (Takara Biotechnology Co., Ltd.) was used for the synthesis of cDNA according to the manufacturer's protocol. RT-qPCR was performed using The PrimeScript™ II Reverse Transcriptase kit obtained from Takara Biotechnology Co., Ltd. The 2^−ΔΔCq method was used to calculate the expression level (defined as the fold change) of TRPM8 compared with GAPDH expression. The primer sequences were as follows: TRPM8 sense chain, 5′-TATCTTACTGAACACCTGTAGTCCCAG-3′ and antisense chain, 5′-TGAGTTTAGTGTATTCAAAGCTGAGAAA-3′ (256 bp); GAPDH sense chain, 5′-AGTGAAGTCGTCGTCAAC-3′ and antisense chain, 5′-CGCTCCTGAGATGTGAT-3′ (32 bp). The experiment was repeated 3 times.

Cells were lysed in RIPA buffer (KeyGen Biotech. Co., Ltd., Nanjing, China) and protein in supernatant extracts was quantified using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). A total of 50 µg per lane of total cell lysates was resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore). The blots were incubated with a primary antibody overnight at 4°C. The blots were subsequently incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibody (dilution 1:3,000; cat. nos. STAR71D800GA and STAR117D800GA; Bio-Rad Laboratories, Inc.). Immunoreactivity was detected using enhanced chemiluminescence (Amersham; GE Healthcare). Densitometric analysis was performed using Quantity One software (Bio-Rad Laboratories, Inc.). GAPDH was used as a loading control. TRPM8 (dilution 1:1,000; cat. no. sc-169688), ERK (dilution 1:1,000; cat. no. sc-271270), cyclin D1 (dilution 1:1,000; cat. no. sc-8396), Bcl-2 (dilution 1:1,000; cat. no. sc-70411), and GAPDH (dilution 1:1,000; cat. no. sc-51907) antibodies were purchased from Santa Cruz Biotechnology, Inc.

For the activity test on the TRPM8 channel, patch-clamp recordings were performed in whole-cell configuration at room temperature using Axonpatch 200B (Molecular Devices, LLC) or HEKA EPC10 (HEKA Elektronik GmbH) amplifier. U251 cells were seeded on coverslips prior to recording the TRPM8 currents via electrophysiology with the standard extracellular solution (ECS) containing (in mM): 130 NaCl, 5 KCl, 10 D-glucose, 10 HEPES, 1.2 MgCl₂ and 1.5 CaCl₂, pH 7.4. For specificity evaluation of characteristics of the TRPM8 channel of U251 cells, menthol (100 µM) was added in ECS to activate TRPM8, and AMTB Hydrate (50 µM) was applied in ECS to inhibit TRPM8. Ca²⁺ measurements by fluorescence imaging of Fluo-4am were subsequently performed for further evaluation. U251 cells grown on 10-mm glass coverslips were incubated with Fluo-4 am for 0.5 h in the dark at 37°C for the Ca²⁺ measurements. The cells were then washed with HBSS containing calcium and placed on a confocal microscope. The intracellular calcium images were recorded using Evolve 512 EMCCD (Teledyne Photometrics) and menthol using RSC-200 Rapid Solution Changer (Bio-Logic Science Instruments). Then the fluorescence intensities were analyzed using ImageJ software (version 1.8.0; National Institutes of Health). Data were presented as the traces of average fluorescence intensities values.

Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Laboratories, Inc.). Transfection of TRPM8 or the non-specific control was performed in 96-well plates in quadruplicate. Cell culture medium was replaced at 24 h following transfection. CCK-8 (10 µl) was added to each well, which also contained 100 µl medium. Following a 2-h incubation with the CCK-8 solution, the absorbance at 450 nm was measured at 48 h following transfection. Each experiment was performed in triplicate.

Apoptosis was determined using the Annexin V-PE/7AAD apoptosis kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. Briefly, after 72 h of culture, the cells were collected and washed twice in PBS and re-suspended at a density of 1×10³ cells/ml. The transfected cells were subsequently stained with Annexin V and propidium iodide in the dark and 7-ADD for 20 min and analyzed using a FACSCalibur instrument.

The cells were seeded on coverslips and incubated for 24 h under normoxic conditions. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature and permeabilized with 0.2% Triton X-100 for 10 min. The cells were subsequently washed with PBS and blocked in PBS containing 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 90 min. The cells were washed with PBS and the primary antibodies were diluted with PBS containing 2% BSA as follows: Rabbit anti-phospho-specific ERK (cat. no. sc-271270), Bcl-2 (cat. no. sc-70411), phospho-specific c-Jun N-terminal protein kinase (JN; cat. no. sc-7345), caspase-3 (cat. no. sc-271759), phospho-specific p38 mitogen-activated protein kinase (MAPK) (cat. no. sc-271759) antibodies (dilution 1:200; Santa Cruz Biotechnology, Inc.) and incubated overnight at 4°C. The cells were then washed with PBS and incubated for 2 h with an anti-mouse fluorescent secondary antibody (cat. no. A10036; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature. Finally, DAPI (Beijing ComWin Biotech Co., Ltd.) was added to each sample for nuclear counterstaining. The coverglass was examined and photographed to reveal representative cells using an Olympus BX61WI-FV1200MPE confocal microscope (Olympus Corp.).

Transwell invasion was performed with 24-well Matrigel-coated chambers (8-µM pore size) from BD Biosciences. Briefly, cells were permitted to grow to ~75-80% confluence and were starved for 24 h by serum. Approximately 1×10⁵ cells resuspended in 200 µl serum-free medium were plated in the upper Transwell chamber. Then, 600 µl of medium with 10% FBS was added to the bottom wells of the chambers. At 48 h following incubation, the Transwell insert was washed twice with PBS and fixed with 5% glutaraldehyde for 5 min. The invading cells on the bottom surface of the membrane were stained for 20 min with 0.1% crystal violet. Then the membranes were observed at an ×100 magnification using a light microscope (Olympus Corp.). The membrane was then dissolved by 10% acetic acid, and a multifunctional microplate reader was employed to measure the optical density (OD) value of each well at 570 nm. The invasion rate = OD 570 post-crystal violet staining/OD 490 at the time of inoculation. Each experiment was performed in triplicate.

All data in the present study are presented as the mean ± standard deviation of at least three independent experiments. Statistical tests were performed using SPSS version 19.0.0 software (IBM Corp.). A two-tailed Student's t-test was used for comparisons between two groups. One-way analysis of variance (ANOVA) test and Bonferroni post hoc test were used for evaluating differences among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To assess the differences in mRNA expression of TRPM family members in human cancers and normal tissues in a variety of cancer types, the present study performed an analysis using the ONCOMINE database (Fig. 1). The results of the ONCOMINE analysis indicated that TPRM8 was elevated in cancer of the brain and CNS when compared with the normal tissue groups.

By searching the CCLE (https://portals.broadinstitute.org/ccle/home), the results revealed that TRPM8 was highly expressed in all GBM cell lines (Fig. 2). These results indicated that TRPM8 may serve a unique role in the development of GBM, which was consistent with the results of the ONCOMINE analysis.

To validate the influence of TRPM8 on the prognosis of glioma patients, an overall survival (OS) analysis was performed using the Kaplan-Meier method via CGGA datasets. The results indicated that the high expression of TRPM8 mRNA was associated with a shorter overall survival (OS) time in all patients with GBM (P<0.0001) (Fig. 3). The survival analysis indicated that over-expression of TRPM8 was associated with worse survival in patients with GBM, suggesting TRPM8 serves a tumor-promoting role in GBM.

Menthol is a specific agonist against TRPM8, and AMTB hydrate is a TRPM8 blocker (11). To evaluate the characteristics of the TRPM8 channel, the whole cell membrane current of U251 cells was investigated by whole-cell patch-clamp recordings. Fig. 4A and B illustrate the whole cell membrane current of U251 cells at voltage ramps from −80 to 80 mV. The current-time curve of the TRPM8 channel after treatment with two consecutive 100-µM menthol activations ruled out the effects of switching the drug delivery tube (Fig. 4A). When the transmembrane voltage was positive, the outward current was markedly increased. The whole cell membrane currents were significantly increased when menthol (100 µM) was added in ECS to activate TRPM8 (Fig. 4B), and was reversed by AMTB hydrate (50 µM), further confirming that AMTB hydrate functions as a blocker of TRPM8. In addition, the fluorescence imaging results revealed that the ion channel activity of TRPM8 may be attributable to increased [Ca²⁺]_i following the addition of menthol. Menthol increased U251 [Ca²⁺]_i, and this increase could be reversed following the removal of menthol (Fig. 4C). The ability of menthol to activate TRPM8 ion channels in the glioma cells was examined by overexpressing the channel in the U251 cells. The vehicle of pEGFP-C1 as control groups were also transfected into the U251 cells. The Ca²⁺ influx was monitored using Fluo-4am, and there was a slight increase following the overexpression of TRPM8. Following treatment with 100 µM menthol, a significant increase in fluorescence was detected for TRPM8, whereas no fluorescence increase was observed for the control groups (Fig. 4D).

Firstly, by detecting the gene and protein expression levels of TRPM8 via RT-qPCR and western blot assay, the present study confirmed that transfection with shRNA-TRPM8 could significantly inhibit the mRNA and protein expression of TRPM8 (P<0.01). Conversely, the overexpression of TRPM8 was significantly increased following transfection with the pEGFP-C1-TRPM8 plasmid (P<0.05). These results indicated that the downregulated and upregulated TRPM8 U251 cell lines were successfully constructed (named U251/shRNA and U251/TRPM8, respectively) (Fig. 5). As revealed in Fig. 6A, inactivation of TRPM8 expression could significantly increase the apoptosis rate (P<0.01), indicating that the modulation of cell apoptosis was significantly affected by the expression of TRPM8. The expression levels of p-ERK, Bcl-2 were significantly decreased when TRPM8 was upregulated and increased when TRPM8 was downregulated in human glioblastoma U251 cells as revealed in Fig. 6B. The present study also confirmed these results via cellular immunofluorescence (Fig. 7). In addition, cellular immunofluorescence also revealed that the expression of phospho-specific JNK, caspase-3 and phospho-specific p38 MAPK were increased when TRPM8 was downregulated (Fig. 7).

The results of the CCK-8 assay indicated that the proliferative ability of U251 cells in the U251/TRPM8 group was higher than that in the U251 and U251/Con groups (P<0.05); however, there was no significant difference between the U251 and U251/Con groups (P>0.05). The details of the proliferation capacity are presented in Table I. In addition, the expression level of cyclin D1 was markedly altered when TRPM8 was regulated in human glioblastoma U251 cells (Fig. 6B). Collectively, the results demonstrated that the proliferation ability of U251 cells was significantly affected by the expression of TRPM8.

The invasion rate is a crucial cancer cell property that accurately reflects the invasive ability of human glioblastoma U251 cells. As illustrated in Fig. 8, the Transwell invasion assay revealed that the U251 cells in the U251/TRPM8 group had an increased number of invasive cells when compared with the U251 and U251/Con groups. According to the OD value, the transfer rate indicated that the U251/TRPM8 group had a higher invasion rate than the U251 and the U251/Con groups (P<0.05); however, the difference between the U251 and U251/Con groups was not statistically significant (P>0.05) (Table I). Collectively, these results indicated that the invasion of human glioblastoma U251 cells was positively associated with the expression level of TRPM8.