TMA construction and IHC analysis

A tissue microarray system (Quick-Ray, UT06, UNITMA, and Korea) was used as described previously in the Department of Clinical Pathology, Nantong University Hospital, Jiangsu, China [22]. The immunohistochemical analysis was performed as previously described [23]. The IHC analysis to evaluate the protein expression of NNMT in gastric tissues was conducted using anti-NNMT mouse monoclonal antibody (3D8) [NBP2-00537].

NNMT immunostaining was scored by blinded observers according to the intensity and percentage of positive cells. The staining intensity was scored according to 4 grades: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (intense staining). The product of the percentage of positive cells and the respective intensity scores was used as the final staining score, with a minimum value of 0 and a maximum value of 300. A cutoff value of 120 was found to be statistically significant using the X-tile software program (the Rimm Lab at Yale University; http://www.tissuearray.org/rimmlab/) [24].

RNA preparation, reverse transcription, and real-time quantitative PCR

Total RNA was isolated from tissue samples and cultured cells using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. RNA was reverse transcribed into cDNA using the Prime Script RT reagent Kit (Takara, China). Quantitative real-time PCR was performed using the SYBR Prime-Script RT-PCR kit (Takara, Japan) and QuantStudio^TM 6 Flex Real-Time PCR System. Relative NNMT expression was calculated and was normalized to β-actin using the 2^-ΔΔCt method. The primers used in the quantitative real-time PCR analysis are presented in Table 4. All experiments were conducted at least three times.

Cell culture and transfection

The gastric carcinoma cell lines MKN28, SGC7901, MGC803 and BGC823 were purchased from American Type Culture Collection (ATCC, Netherlands) and the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA) medium containing 10% fetal bovine serum (FBS, GIBCO) at 37°C in humidified air containing 5% CO₂. Small interfering RNA (siRNA) transfection. NNMT siRNA (si1 and si2) and negative control siRNA were purchased from Gene Pharma (Shanghai, China). Cells (5×10⁴ cells/well) were seeded into 6-well plates and were transfected with 10 nM siRNA in phosphate-buffered saline (PBS) using Lipofectamine-2000 (Invitrogen) according to the manufacturer’s instructions. The siRNA sequences are presented in Table 5.

Western blot assay

The cells were harvested for protein extraction, and the extracted proteins were quantified as previously described using a 12% or 4~20% polyacrylamide gradient sodium dodecyl sulfate (SDS) gel [23]. After the proteins were separated in the gel and transferred to a polyvinylidene difluoride (PVDF) membrane, the membrane was blocked in 2% BSA in TBST for 1 h. Then, the membranes were incubated overnight (4°C) with anti-NNMT mouse monoclonal antibody (3D8) [NBP2-00537]. After incubation with Alexa Fluor 488-labeled Goat Anti-Mouse IgG (Beyotime Biotechnology, China) at room temperature for 2 h, the relative protein levels were calculated using GAPDH (Abcam, ab181602) as a loading control. Each assay was repeated three times.

Cell proliferation

The cell proliferation assay was performed using Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan) at 48 h after transfection. Briefly, 2000 cells were plated per well in 96-well plates and were cultured for 24 h. Then, 10 μl of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 h. The absorbances at 450 nm (A450) and 650 nm (A650) were measured using a Synergy 2 microplate reader (BioTek, Winooski, VT, USA) and were detected in quadruplicate at various time points for 6 days. The final absorbance was calculated as A650 minus A450, and cell viability was normalized using the following formula: (final absorbance treated/final absorbance control) × 100%. All experiments were performed at least three times.

Flow cytometry

The cells were collected and washed three times with PBS and maintained in ethanol for at least 24 h at -20°C. Then, the cells were incubated in propidium iodide (PI) staining solution (RNase A 100 ug/ml and PI 500 ug/ml) for 30 min at 4°C. Next, the cells were analyzed using a FACScan flow cytometer (BD Biosciences), and the cell cycle distributions were calculated using the Cell Quest software package (BD Biosciences) according to the manufacturer’s protocol. The percentage of cells in G1, S, and G2 phase were counted and compared. Each assay was repeated three times.

Migration and invasion

The migration and invasion assays were performed using 24-well Transwell chambers (8 mm pores, Millipore, Billerica, MA). For the migration assay, tumor cells were resuspended in serum-free RPMI-1640 medium; 2×10⁵ cells were seeded into the upper chambers, and 0.5 mL RPMI-1640 containing 10% FBS was added to the bottom chambers. Following a 24 h-incubation, the cells on the upper surface of the membrane were scrubbed off, and the migrated cells were fixed with 95% ethanol, stained with 0.1% crystal violet, and counted under a light microscope. The invasion assay protocol was similar to the migration assay protocol, except that the upper chambers were first covered with 1 mg/ml Matrigel. All experiments were conducted at least three times.

Xenograft experiment

MGC803 cells were transfected with sh-NNMT or negative control siRNAs (si1 and siNC, respectively) using Lipofectamine 2000 (Invitrogen). NNMT mRNA and protein levels were determined by qRT-PCR and Western blot, respectively. The cells were collected and injected into either side of the posterior flank of the same BALB/c nude mouse after 48 h. Tumor volume and weight were measured every 2 days. Tumor volume was measured as length × width² × 0.5. At 25 days after injection, the mice were sacrificed, and the final tumor weights were measured. All experimental manipulations were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Statistical analysis

All statistical analyses were performed using SPSS 21.0 statistical software (SPSS Inc. Chicago, IL) [25]. The relationships between NNMT expression and clinicopathological attributes were analyzed using the x² test. Survival rates were calculated using the Kaplan-Meier method and were compared by the log rank test. The univariate and multivariate analyses followed the Cox proportional hazards regression model. A P-value of less than 0.05 was considered to indicate a statistically significant difference.

Association of NNMT expression with clinicopathological characteristics in gastric cancers

The relationship between NNMT expression and the clinicopathological attributes of gastric cancer are presented in Table 2. High NNMT positive staining within the cytoplasm and interstitium was significantly associated with primary tumor size (Pearson χ²=33.468, P<0.001), lymph node metastasis (Pearson χ²=33.486, P<0.001), distant metastasis (Pearson χ²=9.130, P=0.002) and TNM stage (Pearson χ²=46.134, P<0.001). NNMT expression was not associated with any other clinical parameters, including gender, age, histological type, differentiation, carcinoembryonic antigen (CEA) status and CA199 status.

Prognostic value of NNMT protein expression in gastric cancer

We examined possible prognostic factors for gastric cancer using both univariate and multivariate Cox regression analyses. The univariate analyses showed that increased expression of NNMT, age, differentiation, primary tumor size, lymph node metastasis, distant metastasis, TNM stage, CEA status and CA199 status were associated with the prognoses of gastric carcinoma patients in terms of overall survival rate. The prognostic indicators identified in the univariate analyses were included in the multivariate analyses, which further indicated that NNMT expression was associated with poor overall survival (hazard ratio (HR), 1.446; P=0.028; 95% confidence interval (CI), 1.041-2.065) as was primary tumor size, lymph node metastasis, distant metastasis and CEA status. The detailed findings are presented in Table 3. Kaplan-Meier survival curves revealed that low expression levels of NNMT and CEA and early cancer stage were associated with an enhanced rate of survival and that patients positive for the two markers NNMT and CEA had a significantly worse prognosis (Figure 2). These Kaplan-Meier survival curves will help us analyze the five-year survival rate of patients with gastric cancer (Figure 2A, ​,2B,2B, ​,2F2F).

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Figure 2

Survival curves of gastric carcinoma generated by the Kaplan-Meier method and the log-rank test. A. Overall survival curves of NNMT+ (green line, High) and NNMT- tissue samples (purple line, Low). B. Overall survival curves by primary tumor size: Tis (pink line, Tis), T1 (purple line, T1), T2 (orange line, T2), T3 (green line, T3) and T4 (blue line, T4). C. Overall survival curves by stage of lymph node metastasis: node-negative (blue line, N0), L1 (green line, L1), L2 (orange line, L2), and L3 (purple line, L3). D. Overall survival curves by distant metastasis: negative (green line, M0) and positive (light blue line, M1). E. Overall survival curves by preoperative CEA status: high (green line, ≤5) and low (blue line, >5). F. Overall survival curves by CEA and NNMT status: CEA-/NNMT- (purple line, 0), CEA-/NNMT+ (blue line, 2), CEA+/NNMT- (green line, 3) and CEA+/NNMT+ (orange line, 4).

Knockdown of NNMT inhibits cellular proliferation, invasion and migration in vitro

To better demonstrate the role of NNMT in gastric carcinoma, we first measured the expression of NNMT in four gastric cancer cell lines (MKN28, SGC7901, MGC803 and BGC823). The MGC803 and BGC823 cell lines exhibited increased NNMT expression levels compared with the other two cell lines (Figure 3A), and these cell lines were used for NNMT silencing experiments conducted using two siRNA molecules (Table 5). The NNMT mRNA and protein levels were analyzed to determine the specific effects of siRNA treatment on NNMT expression. Compared with the negative control, both NNMT mRNA and protein levels were significantly reduced in the siRNA groups. NNMT mRNA expression was approximately 4-fold reduced in the siRNA groups compared with the negative control group (Figure 3B).

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Figure 3

Knockdown of NNMT inhibited the proliferation, migration and invasion of gastric carcinoma cell lines in vitro. A. NNMT expression in MKN28, SGC7901, MGC803, and BGC823 cell lines. β-actin was used as the internal control in qRT-PCR, and GAPDH was used as the internal control in Western blot. B. In MGC803 and BGC823 cells, NNMT mRNA and protein levels were significantly reduced after transfection with NNMT siRNA (si1 and si2). C. NNMT promotes proliferation of gastric carcinoma cells. Cell proliferation assay using the CCK-8 kit: compared with negative control (NC), siRNA-mediated silencing of NNMT significantly inhibited cell proliferation in MGC803 and BGC823 cell lines. D. NNMT alters the cell cycle of gastric carcinoma cell lines. MGC803 and BGC823 cells transfected with NC (siRNA negative control) or NNMT siRNA (si1 and si2). Thirty-six hours after transfection, cells were stained and analyzed by flow cytometry. The cell cycle was arrested at the G2 phase. E. NNMT promotes migration of gastric carcinoma cell lines (MGC803 and BGC823). Compared with negative control (NC), cells transfected with NNMT siRNA (si1 and si2) showed weaker migratory and invasive abilities.

Induction of cell cycle arrest is an important mechanism for controlling tumor growth. We assessed the role of NNMT in regulating gastric carcinoma cell cycle progression to determine the effect of NNMT on gastric carcinoma cell growth. In MGC803 cells, NNMT downregulation caused a significant increase in the percentage of G2 phase cells and a concomitant decrease in the percentage of S phase cells, thus indicating the occurrence of G2 cell cycle arrest (Figure 3D). CCK-8 assays in MGC803 and BGC823 cell lines revealed that knockdown of NNMT reduced cell proliferation compared with the negative controls (Figure 3C).

To investigate the effect of NNMT on migration and invasion, two specific small interference RNAs, si1 and si2, and a negative control (si-NC) were transfected into MGC803 and BGC823 cells. Transwell migration assays indicated that NNMT knockdown led to a significant decrease in cell migration in MGC803 and BGC823 cell lines. To further assess the role of NNMT in the pathogenesis of gastric carcinoma, we also assessed whether NNMT knockdown inhibits invasion in MGC803 and BGC823 cell lines. Consistent with the previous results, knockdown of NNMT inhibited invasion in both cell lines (Figure 3E).

Knockdown of NNMT inhibited cell proliferation in vivo

To test whether NNMT regulates cell proliferation in vivo, we established xenograft tumor models in nude mice using MGC803 cells transfected with sh-NC or sh-NNMT. All nude mice developed xenograft tumors at the injection site, and the xenograft tumors were harvested 7 days after injection. The average tumor volume and weight in the sh-NNMT group was significantly reduced compared with the negative control group (Figure 4D, ​,4E).4E). And the IHC staining was performed on xenograft tumors, and the Ki-67 staining signal was weaker in the sh-NNMT group compared with the sh-NC group (Figure 4C). These data demonstrate that NNMT may inhibit tumor growth in vivo.

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Figure 4

Knockdown of NNMT inhibited the proliferation of gastric carcinoma cell line in vivo. A, B. MGC803 cells were transfected with either Si-NNMT or sh-NC.The injection sites are indicated by arrows. D, E. The xenograft tumor weight and volume in the sh-NNMT group were significantly reduced compared with the sh-NC group. C. IHC staining was performed on xenograft tumors, and the Ki-67 staining signal was weaker in the sh-NNMT group compared with the sh-NC group. (Single asterisk indicates P<0.05; double asterisks indicate P<0.01; triple asterisks indicate p<0.001; error bars indicate means ± SEM).

This work was supported by the Major Scientific and Technological Special Project for “significant new drugs creation” (No. 2012ZX09103301-040), the Innovative Team of Jiangsu Province (No. LJ201126) and the National Natural Science Foundation (No. 81572390).