High expression of TMEM40 is associated with the malignant behavior and tumorigenesis in bladder cancer

BCa tissues and adjacent normal tissues were obtained from 12 patients, who underwent bladder surgical resection without preoperative systemic therapy in Nanfang Hospital of Southern Medical University between October 2014 and September 2016. After surgical removal, the tissues were immediately frozen using liquid nitrogen. All the patients signed informed consent and the study was approved by the Ethics Committee of Nanfang Hospital of Southern Medical University.

BCa EJ, J82, BIU-87, UMUC3, SW780, 5637, T24 and normal urothelial cell line SV-HUC-1 cells used in this study were obtained from laboratory preservation. The 5637, EJ, SW780, J82 and BIU87 cells were cultured in RPMI-1640 Medium (Invitrogen, Carlsbad, CA, USA), the UMUC3, T24 cells were cultured in Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA, USA), SV-HUC-1 cells were cultured in F12K (Invitrogen, Carlsbad, CA, USA). All cells were plus 10% fetal bovine serum (FBS) and 1% ampicillin (100 units/mL) and streptomycin (100 units/mL) and then placed at 37 °C with a humidified atmosphere of 95% air and 5% CO₂ in incubator.

The specific small interfering RNA of TMEM40 and a scrambled negative control were purchased from RiboBio (Guangzhou, China). The overexpression vector pEZ-M98 and a scrambled negative control empty vector were also purchased from GeneCopoeia (Guangzhou, China). The shRNA vector and a scrambled negative control vector were obtained from Genechem (Shanghai, China). The sequence of siRNA was 5′-GGAUGAGCUUCAACUCUAUTT-3′; NC was 5′-UUCUCCGAACGUGUCACGUTT-3′; shRNA was 5′-GUGGACGCCUCUCAGUUAA-3′; NC was 5′-TTCTCCGAACGTGTCACGT-3′;

After cells were cultured 24 h, the cells were transiently transfected with corresponding siRNA or plasmid DNA using Lipofectamine 2000 reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, cells transfected with siRNA or shRNA and plasmid were harvested for qRT-PCR or western blot detection.

All total RNA was extracted from BCa tissues or cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Qualified total RNA was then reverse transcribed to complementary DNA using a PrimeScript RT reagent Kit (Takara, Dalian, China). The quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green PCR kit (Takara, Dalian, China) following the manufacturer’s instructions. GAPDH was measured as an internal control. The primer sequences were listed as follows: TMEM40 primers: (forward, 5′-GCGGTAGGGGTGTACGGT-3′; reverse, 5′-CCGGACACGCTGAACT TGT-3′); P53 primers: (forward, 5-AAGATCCGCGGGCGTAA-3; reverse, 5′-CATCCTTTAACTCTAAG GCCTCATTC-3′); c-myc primers: (forward, 5′-CTTCTCTCCGTCCTCGGATTCT-3′; reverse, 5′-GAA GGTGATCCAGACTCTGACCTT-3′); JNK2 primers: (forward, 5′-TACGTGGTGACACGGTACTACC-3′; reverse, 5′-CACAACCTTTCACCAGCTCTCC-3′); RB primers: (forward, 5′-ATCAAGGGTCATTATGG GTTAGGC-3′; reverse, 5′-TAGGTGTAGGGGAGGGGAGAAGC-3′); GAPDH primers: (forward, 5′-CG CTCTCTGCTCCTCCTGTTC-3′; reverse, 5′-ATCCGTTGACTCCGACCTTCAC-3′). The reactions were carried out on an ABI PRISM 7500 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA) in triplicate. The average value in each triplicate was used to calculate the relative amount of TMEM40 using 2^−ΔΔCt methods.

The BCa cells and tumor tissue samples lysates were extracted with RIPA cell lysis buffer, and the protein concentration in the lysates was quantified using an enhanced bicinchoninic acid protein assay kit (Thermo Fisher Scientific, MA, USA) with bovine serum albumin as a standard. The total protein extract will be used for western blot analysis. Equal amounts of total protein of tissues or cells were subjected to 10% SDS-PAGE and transferred to a PVDF membrane followed by immunoblotting using the following primary polyclonal antibodies: mouse anti-TMEM40 (Santa Cruz, CA, USA), rabbit anti-p53 (Proteintech Group, INC, USA), rabbit anti-p21 (Proteintech Group, INC, USA), mouse anti-CCND1 (Proteintech Group, INC, USA), rabbit anti-Caspase-3 (Cell Signaling Technology, MA, USA), rabbit anti-Caspase-9 (Proteintech Group, INC, USA), rabbit anti-PARP (Proteintech Group, INC, USA), rabbit anti-c-MYC (Proteintech Group, INC, USA) and GAPDH (Cell Signaling Technology, MA, USA). Specific proteins were detected with enhanced chemiluminescence (ECL, Millipore, USA). Band density was measured (ImageJ software) and normalized to GAPDH.

A scratch wound healing assay was adapted to evaluate the ability of cell migration. Cells at a density of 80–90% confluence in 12-well plates were scratched manually with a sterile 200 µL plastic pipette tip, cultured for 24 h and photographed under a light microscope. The width of the wound area was quantitated, using the Image J. For the invasion assay, a transwell chamber was placed into a 24-well plate and was coated with 30 μL Matrigel and incubated for 60 min at 37 °C. Cells were cultured in normal medium and transfected with corresponding siRNA or plasmid DNA. 24 h after transfection, 5 × 10⁴ cells were first starved in 200 μL serum free medium and then placed in the uncoated dishes. The lower chamber was filled with 750 μL of complete medium. The cells were incubated for 48 h at 37 °C in a 5% CO₂ incubator, and then the cells that had migrated to the bottom surface of the filter membrane were stained with 0.1% crystal violet staining solution and photographed in five preset fields per insert. The results represented the average of three independent experiments.

Cell proliferation was determined using Cell Counting Kit-8 (Beyotime Inst Biotech, China). In short, 5 × 10³ cells/well were seeded in a 96-well flat-bottomed plate for 24 h, then transfected with corresponding siRNA or plasmid DNA cultured in normal medium. At 0, 24, 48 and 72 h after transfection, 10 μL of CCK-8 (5 mg/mL) was added to each well and the cells were cultured for 2 h then determined the absorbance at a wavelength of 450 nm using a microplate reader (Tecan, Infinite^®M200, Austria). Experiments were repeated at least three times.

Cell proliferation was also determined by 5-Ethynyl-2′-deoxyuridine incorporation assay using an EdU Apollo DNA in vitro kit (RiboBio, Guangzhou, China) following the manufacturer’s instructions. Briefly, 5637 and EJ cells were plated in confocol dish plates at a density of 4 × 10³–1×10⁵ cells and incubated with 100 μL of 50 μM EdU per dish for 2 h at 37 °C, at 48 h after transfected with corresponding siRNA or plasmid DNA, respectively. Then, the cells were fixed for 30 min at room temperature using 100 μL of fixing buffer (4% polyformaldehyde containing PBS). Subsequently, the cells were incubated with 100 μL of 2 mg/mL glycine for 5 min followed by washing with 100 μL of PBS (Phosphate Buffered Saline). After permeabilization with 100 μL of 0.5% Triton X-100 for 10 min, the cells were reacted with 100 μL of 1× Apollo solution for 30 min at room temperature in the dark. After that, cells were incubated with 100 μL of 1× Hoechst 33342 solution for 30 min at room temperature in the dark followed by washing with 100 μL of PBS. The cells were then visualized under a fluorescence microscopy. Experiments were repeated at least three times.

The BCa cells were plated in 6-well plates at a density of 1 × 10⁶ cells every well cultured for 24 h, then transfected with siRNA and plasmid DNA. After 48 h, cells were then treated with trypsin, fixed in 70% frozen ethanol, followed by overnight incubation and washed with PBS, nucleus was stained by 450 μL Propidium Iodide (PI) and 50 μL RNAase. The percentages of 5637/EJ cells in the G0/G1, S, and G2/M phases were examined by Becton–Dickinson FACSVerse™ flow cytometry (San Jose, CA, USA). All experiments were performed three times.

Briefly, BCa cells were plated in 6-well plates at a density of 1–5×10⁵ cells every well cultured for 24 h. After transfection for 48 h, cells were harvested by trypsinization and washed with PBS and suspended in 500 μL Annexin V binding buffer. The percentage of cells actively undergoing apoptosis was determined by double staining with 5 μL AnnexinV-APC and 5 μL 7-AAD apoptosis detection kits (KeyGEN Biotech, Nanjing, China), and analyzed using a Becton–Dickinson FACSVerse™ flow cytometry (San Jose, CA, USA). At least 10,000 cells were obtained from each sample.

Five-week-old female/male BALB/c-nu athymic nude mice (Experimental Animal Center of Southern Medical University, Guangzhou, China) were subcutaneously injected in the right flank with 1.0 × 10⁷ cells in 0.2 mL of PBS. Once tumors were formed, tumor volume (V) was measured daily by caliper and calculated using the formula V = (L × W²)/2, where L was the length and W was the width of the tumor. The mice were randomly divided into two groups (n = 6) for inoculation of shRNA transfected EJ cells and negative control (NC) for 27 days. Growth curves were plotted using average tumor volume within each experimental group every 3 days. 4 weeks later, the mice were euthanized, and the dissected tumors were collected. All animal experiments were approved by the animal center of the Southern Medical University.

All statistical analyses were performed using the IBM SPSS 20.0 software package (SPSS, Inc., Chicago, IL, USA). Group difference was assessed using Student’s t test. Data are presented as the mean ± SD based on at least three repeats. Differences were considered to be statistically significant at P < 0.05.

From the expression of TMEM40 RNAseq data of the Cancer Genome Atlas (TCGA), we found that TMEM40 expression levels was significantly increased in BCa and other organ cancers compared with their controls (Fig. 1a, N = 30). Furthermore, from the Gene Expression Profiling Interactive Analysis (GEPIA), we also found that the level of TMEM40 in the tumor tissues (N = 404) was significantly higher than that in the controlled bladder tissues (N = 28) (Fig. 1b, P < 0.05). To investigate the expression status of TMEM40 in BCa, western blotting and quantitative RT-PCR analyses were performed in seven BCa cell lines (i.e. EJ, T24, 5637, SW780, UMUC3, J82 and BIU-87), one normal uroepithelial cell line SV-HUC-1 and 12 cases fresh BCa tissues paired with their adjacent non-neoplastic bladder tissues. The results showed that TMEM40 were significantly upregulated in five BCa cell lines (i.e. EJ, T24, 5637, UMUC3 and J82) compared with normal uroepithelial cell line SV-HUC-1 both on protein and mRNA levels (Fig. 1c, e, P < 0.05). Similarly, TMEM40 were considerably higher in BCa tissue specimens when compared with their paired normal bladder tissues (Fig. 1d, f, P < 0.05).

To investigate the functions of TMEM40 in BCa, overexpression vector containing green fluorescence protein (GFP) (Fig. 2a) were used to increase the expression of TMEM40 in 5637 and EJ cells. Then transfected with the vector and tested the efficiency of fluorescence expression (Fig. 2b). The data showed that pEZ-M98 vector could upregulate both TMEM40 mRNA (Fig. 2e, f) and protein (Fig. 2c) expressions in BCa cells. Meanwhile, we inhibited TMEM40 expression in BCa cells 5637 and EJ by transfecting them with its specific siRNA. Then we performed qRT-PCR and western blot to measure its expression levels at 48 h post-transfection. The data showed that TMEM40 expression could be effectively suppressed in BCa cells (Fig. 2d, g, h, P < 0.05).

Based on the aforementioned results, we further evaluated whether TMEM40 could affect the biologic activities of BCa cells. Wound healing assay was used to assess the capability of cell motility, and the results showed that 5637 and EJ cells transfected with siRNA underwent a slower closing of scratch wounds compared with the negative control groups (Fig. 3a, b, P = 0.001; P = 0.002). Then transwell assay was applied to evaluate its impact on invasion ability in BCa cells, and the findings indicated that silencing TMEM40 significantly repressed the capability of invasion of BCa cells (Fig. 3c, d, P < 0.001). Meanwhile, the Cell Counting Kit-8 (CCK-8) (Fig. 4a–d, P < 0.05) and 5-Ethynyl-2′-deoxyuridine (EdU) (Fig. 4e–h, P < 0.05) assays demonstrated that suppression of TMEM40 attenuated cell proliferation in 5637 and EJ cells. Furthermore, results from the cell cycle analysis suggested that TMEM40 knockdown induced G1 phase arrest both in 5637 and EJ cells (Fig. 5a, b, P < 0.05). Finally, the effect of TMEM40 on cellular apoptosis was explored. FACS analysis showed that there was a significantly higher percentage of Annexin V-positive cells in TMEM40-silenced 5637 and EJ cells compared with control cells (Fig. 5c, d, P < 0.01). This indicated that TMEM40 knockdown induced cellular apoptosis. In contrast, overexpressing TMEM40 had proliferation, migration and invasion promoting effect as determined by the wound healing, CCK-8, EdU and transwell assay (Figs. 3, 4, 5, P < 0.05). From these results, we speculated that TMEM40 may play some important roles in BCa cells.

The short hairpin RNA (shRNA) (Fig. 6a) was introduced into 5637 and EJ cell lines, which exhibit low TMEM40 expression, whereas exogenous TMEM40 was stably introduced into 5637 cell, which shows relative high TMEM40 expression (Fig. 6b–d, P < 0.05). To confirm the oncogenic efficiency of TMEM40 in vivo, we constructed a BALB/c nude mouse xenograft model by EJ cells. Our results demonstrated the tumor weight and volume of tumors in nude mice treated with shRNA were markedly suppressed (38.5% of decrease in tumor weight) relative to that of treated with empty vector (Fig. 6f, P < 0.05). These data indicated that knockdown of TMEM40 markedly inhibited the tumorigenicity of EJ cells in the nude mouse xenograft model (Fig. 6e, g, P < 0.05). Moreover, we checked the expression levels of oncogenes (i.e. c-myc), as well as tumor suppressor genes (i.e. p53, Rb and JNK2), all of which were well known to be involved in urothelial tumorigenesis, in NC versus siRNA cells. TMEM40 silencing resulted in significant downregulation of the expression of c-myc as well as significant upregulation of the expression of p53, Rb and JNK2 (Fig. 7a, P < 0.05).

To comprehensively find mechanistic insight into the role of TMEM40 in regulating BCa cell proliferation and apoptosis, we measured the alteration of proliferation-related signals (p53, p21, c-MYC, cyclin D1) and apoptosis-related proteins (p53, activated caspase-3, activated caspase-9, activated PARP) by western blotting. Knockdown of TMEM40 increased the expression levels of p53 and p21, activated caspase-3, caspase-9, and PRAP and decreased the levels of c-MYC and cyclin D1 in 5637 and EJ cells. Together, all the above suggested that TMEM40 knockdown reduced the proliferation and enhanced apoptosis of BCa cells (Fig. 7b, c, P < 0.05).