MED30 Regulates the Proliferation and Motility of Gastric Cancer Cells

Cell cultures and transfection

Gastric cancer cell lines (SNU1, SNU16, SNU216, SNU620, SNU638, and N87) were purchased from the Korean Cell Line Bank (Seoul). Cells were cultured in RPMI1640 supplemented with 25 mM HEPES, 10% fetal bovine serum (FBS), and 100 μg/ml of penicillin/streptomycin (1 × P/S) in 5%CO₂/95% air at 37°C. Cells were transfected with siRNA using DharmaFECT reagent 1 or 3 (Dharmacon, Lafayette, CO), according to the manufacturer’s instructions. The sequences of siRNA used were as follows: MED30 siRNA (Bioneer, Daejeon, Korea), 5’-CGA GCA ACU UAU UCC AUA U(dTdT)-3’, 5’-GCU GCC AAA UGG UGU CAC U(dTdT)-3’, and 5’-CGA GAA AUU GCU GAA GUA A(dTdT)-3’; scrambled (SCR) siRNA (Dharmacon, Lafayette, CO), 5’- GAU CCG CAA AAG AGC GAA A(dTdT)-3’.

MED30 overexpression

In order to construct MED30-over expression vector, we used pLenti6.3-V5/DEST lentiviral vector (Invitrogen, Carlsbad, CA). Briefly, MED30 cDNA was cloned into pLenti6.3-V5/DEST vector using the in vivo recombination-based Gateway cloning system (Invitrogen). The donor vector (pDONR221) containing the coding sequence of MED30 (MED30 cDNA) was purchased from Ultimate^TM ORF Clones (Invitrogen), and recombined with the counter-selectable ccdB gene of the Gateway destination vector pLenti6.3-V5/DEST using the LR clonase enzyme mixture (Invitrogen). The empty vector pLenti6.3/V5-DEST was used as a mock control. Recombinant lentiviruses were produced in 293FT cells, and used to infect SNU638 cells according to the manufacturer’s instructions (ViraPower Lentiviral Expression System; Invitrogen). Stable cell lines were established by selection with blasticidin (7.5 μg/ml) (Invitrogen).

Real-time PCR

Gastric cancer tissues were obtained after obtaining written informed consent from patients who underwent surgical resection at Pusan National University Hospital or Pusan National University Yangsan Hospital. The study was approved by the Pusan National University Hospital-Institutional Review Board (PNUH-IRB) and the Pusan National University Yangsan Hospital-Institutional Review Board (PNUYH-IRB). Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen) or the RNeasy Mini kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. cDNA was synthesized using MMLV reverse transcriptase (Promega, Madison, WI), dNTP, and oligo-dT primers. The sequences of the primers used were as follows: MED30, 5’- ACC GGT TAA CAA AGC TAC AGG A -3’ (sense) and 5’- TAA GTT GCT CGA CTG GAA TGG G -3’ (antisense); CDH1, 5’- TGG GCC AGG AAA TCA CAT CC -3’ and 5’- CTC AGC CCG AGT GGA AAT GG -3’; CDH2, 5’- CAC TGT GGA GCC TGA TGC CA -3’ and 5’- TCC CCA ATG TCT CCA GGG TG -3’; CDH3, 5’- CCC CCA GAA GTA CGA GGC CC -3’ and 5’- ACG CCA CGC TGG TGA GTT GG -3’; TWIST1, 5’- CGG GAG TCC GCA GTC TTA -3’ and 5’- TGG ATC TTG CTC AGC TTG TC -3’; TWIST2, 5’- CTT ATG TTT GGG GGG AGG TT -3’ and 5’- TAG CCA AGC AAT CAC GGA GA -3’; VIM, 5’- TGA GTA CCG GAG ACA GGT GCA G -3’ and 5’- TAG CAG CTT CAA CGG CAA AGT TC -3’; SNAI1, 5’- GAG GCG GTG GCA GAC TAG -3’ and 5’- GAC ACA TCG GTC AGA CCA G -3’; SNAI2, 5’- TAG GAA GAG ATC TGC CAG AC -3’ and 5’- CCC CAA GGC ACA TAC TGT TA -3’; GAPDH, 5’- GCA GCC TCC CGC TTC GCT CT -3’ and 5’- TGG TGA CCA GGC GCC CAA TAC G -3’. Real-time PCR was performed using a LightCycler^TM 96 Real-time PCR system (Roche, Nutley, NJ, USA) with FastStart Essential DNA Green Master (Roche), according to the manufacturer’s instructions. GAPDH was used as the internal control.

Western blot analysis

Cells were lysed with RIPA buffer, protease inhibitor cocktail was added, and protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Samples (80 μg/well) were subjected to SDS-PAGE and then transferred to PVDF membranes. Anti-MED30 (1:500, Protein Tech #16787-1-AP, Chicago, IL), anti-E-cadherin (1:1000, BD Bioscience #610181, San Jose, CA), anti-N-cadherin (1:1000, BD Bioscience #610920), anti-P-cadherin (1:1000, BD Bioscience #610227), anti-Twist1/2 (1:750, Santa Cruz Biotechnology #sc-15393, Santa Cruz, CA), anti-vimentin (1:1000, DAKO #M7020, Carpentaria, CA) and anti-β-actin (1:2000, Santa Cruz Biotechnology #sc-47778) antibodies were diluted in 5% skim milk and incubated with membranes at 4°C overnight. The appropriate secondary antibody was applied (1:2000, horseradish peroxidase-conjugated anti-mouse or anti-rabbit) at room temperature for 1 hr. Chemiluminescence detection (GE Health Care, Little Chalfont, United Kingdom) was used to visualize MED30, E-cadherin, N-cadherin, P-cadherin, Twist1, vimentin, and β-actin. Western blot analysis was performed in triplicate.

Immunohistochemistry

Tissue microarray sections containing gastric cancer tissues from patients were obtained from SuperBiochip (Seoul). Histopathologic diagnoses were performed at the Department of Pathology, Pusan National University Hospital, and clinicopathologic staging was performed using the TNM classification (AJCC, 7th ed.). All patients had histologically confirmed gastric cancer, and tumor samples were checked to ensure that tumor tissue composed more than 80% of samples. For immunohistochemistry, slides were deparaffinized and rehydrated, treated with 0.3% hydrogen peroxide for 30 min to quench endogenous peroxidase activity, and blocked with 10% normal donkey serum (NDS) and 1% BSA in 1 × phosphate buffered saline (PBS). Slides were then incubated overnight at 4°C in blocking buffer containing primary antibody (anti-human MED30; 1:50, Proteintech). Secondary antibody (HRP-conjugated) binding was performed at a dilution of 1:200 in blocking buffer for 2 hr at RT. Slides were then stained for HRP (Vector Laboratories) and counterstained with hematoxylin staining buffer (Sigma-Aldrich, St. Louis, MO) for 1 min. Percentages of cells that stained for MED30 in sections were determined and sections were then graded using a 1–4 scale (1, <24%; 2, 25–49%; 3, 50–74%; 4, 75–100%). Intensities of tumor cell staining were graded as 0, 1, 2, or 3, which corresponded to basal, weak, moderate, and strong respectively. Overall staining was then represented by composite scores, which were calculated by multiplying these two grades. Accordingly, overall stained was graded from 0 to 12.

Cell proliferation assay

To examine the effect of siRNA on the proliferation of gastric cancer cells, culture media were replaced with 1% FBS medium one day after the siRNA transfection. Five days after the transfection, 10 μl of Ez-Cytox (ITSBIO, Seoul) was added and cells were incubated for 0.5 to 2 hr under normal culture conditions. Then the absorbance was measured at 450 nm using an ELISA reader (TECAN, Mannedorf, Switzerland). To examine the effect of MED30 overexpression, cells were seeded in 1% FBS medium. Three days after seeding 10 μl of Ez-Cytox was added.

Boyden chamber assay

The bottom chamber of a transwell chamber was filled with medium containing 10% FBS. One day after transfection with scrambled (SCR) or MED30 siRNA, gastric cancer cells were seeded into the upper chamber at a density of 5 × 10⁵ cells/ml in 50 μl of serum-free medium. To examine the effects of MED30 overexpression, cells (mock and MED30-over) were seeded at a density 1 × 10⁵ cells/ml. To eliminate proliferation associated effects, mitomycin C (0.01 μg/ml, Sigma-Aldrich) was added. Cells were allowed to migrate for four or six hrs. Membranes were then fixed and stained using Diff-quik solution (Sysmex, Kobe, Japan) and washed with distilled water. Cell numbers in 10 randomly chosen fields were counted using a light microscope.

Matrigel invasion assay

The ability of gastric cancer cells to invade was investigated using 24-well BioCoat^TM Matrigel^TM chamber inserts (BD Bioscience, San Jose, CA, USA). The top surface of inserts in invasion chambers were coated with 0.5 mg/ml growth factor-reduced Matrigel (BD Bioscience) and the bottom surface with 0.5 mg/ml of fibronectin (Sigma-Aldrich). One day after transfection with SCR or MED30 siRNA, cells were seeded at a density of 5 × 10⁴ cells/ml in serum-free medium into 8-μm porous BioCoat Matrigel chamber inserts, and placed in wells filled with 750 μl of medium supplemented with 10% FBS as a chemoattractant. To examine the effects of MED30 overexpression, cells (mock and MED30-overexpressing cells) were seeded at a density 1 × 10⁴ cells/ml. To remove proliferation-associated effects, mitomycin C (0.01 μg/ml, Sigma-Aldrich) was added. After incubation for 24 hrs, non-invading cells on top surfaces of membranes were removed by scraping. Invading cells on bottom surfaces were fixed, and stained with Diff-quik solution. Experiments were performed in triplicate, and at least 5 fields were counted per experiment.

Xenograft assay

The SNU638 cells were transfected with SCR or MED30 siRNA. After 2 days, cells were collected by trypsinization, washed twice with PBS, and were injected subcutaneously (1 × 10⁶ cells in 100 μl PBS) into severe combined immunodeficiency (SCID) mice. Tumor volumes were calculated every week from week three to week seven after the injection using the following formula: tumor volume (mm³) = (a × b ²)/2, where a = length in mm and b = width in mm. At seven weeks, mice were sacrificed and tumor volumes and weights were recorded. This experiment was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals issued by the National Institute of Health. The Pusan National University Institutional Animal Care and Use Committee (PNUIACUC) approved the experimental protocol.

Statistical analysis

Results are presented as means±SDs. The nonparametric Mann-Whitney U-test or the Student’s t-test (unpaired) was used to determine the significances of differences between the mean values of two groups, and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons was used to analyze three or more groups. * indicates a P value of <0.05. Survival time was defined as the time elapsed between treatment and death or until the last objective follow-up information was obtained. The Kaplan-Meier method was used to determine the relation between survival and MED30 expression. Curves were compared using the log-rank test at a significance level of 95%. P values of <0.05 were considered to be statistically significant. Data were analyzed using SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA).

Overexpression of MED30 in gastric cancer tissues

To investigate roles played by MED30 in gastric cancer, we first examined the expression status of MED30 in the tumor tissues of 23 gastric cancer patients. MED30 protein was found to be widely overexpressed in cancerous tissue regions (Fig 1A–1D), and was obviously overexpressed in invading gastric cancer cells (Fig 1C) and in metastatic cancer cells inside affected lymph nodes (Fig 1D). In order to validate our immunohistochemistry results, we performed quantitative real-time PCR on gastric cancer tissues using MED30-specific primers. As shown in Fig 1F, DPY30 was overexpressed in 10 cases (10/23, 43%). We also examined the expression pattern of MED30 in gastric cancer cell lines by real-time PCR, and found it to be overexpressed in five of the gastric cancer cell lines tested (except SNU1) versus normal gastric tissues (Fig 1E). To investigate the correlation between MED30 expression and clinical characteristics, we performed immunohistochemistry using 41 gastric cancer tissue samples (Table 1). Interestingly, the expression of MED30 was positively correlated with N stage (Fig 1G) but not with patient survival (data not shown).

Roles of MED30 in the proliferation, migration, and invasion of gastric cancer cells

To examine roles of MED30 in gastric carcinogenesis, we examined the effects of MED30 knockdown or overexpression on the proliferation, migration, and invasion of the six gastric cancer cell lines (SNU1, SNUI16, SNU216, SNU620, SNU638, and NCI-N87 cells). Real-time PCR and western blot were used to analyze the knockdown and overexpression of MED30 in SNU216 and SNU638 cells (Fig 2A–2C). When these cells were transfected with MED30 siRNA (100 nM), MED30 expression diminished at the protein and mRNA levels by about 80% two days later versus SCR siRNA, and MED30 overexpression by cDNA transfection increased MED30 mRNA and protein levels versus empty control vector-transfected cells (mock cells) by more than 3-fold.

We next investigated the role of MED30 in the proliferation of cancer cells. Proliferation assays were performed 5 days after transfection with SCR or MED30 siRNA. Knockdown of MED30 inhibited the proliferations of all gastric cancer cells tested (SNU16, SNU216, SNU620, and SNU638) versus SCR by 18%, 68%, 98%, and 42%, respectively (Fig 2D). Similar results were observed when we performed proliferation assays two or three days after transfection (data not shown). Consistently, MED30 overexpression enhanced the growths of SNU216 and SNU638 cells by 1.9 and 2.2 fold, respectively, versus mock cells.

To determine the role played by MED30 in the migration of gastric cancer cells, we used a Boyden chamber assay. Knockdown of MED30 decreased FBS-induced migrations of SNU216 and SNU638 cells by 90% and 52%, respectively, versus SCR transfected cells (Fig 3A and 3C). Moreover, MED30 overexpression increased the FBS-induced migration of SNU216 and SNU638 cells by 3.2 and 2.8 fold, respectively, versus mock cells (Fig 3B and 3C). These results led us to examine the role of MED30 in the invasion of gastric cancer cells. In a Matrigel invasion assay, MED30 siRNA inhibited FBS-induced invasions of SNU216 and SNU638 cells versus SCR siRNA by 64% and 47%, respectively (Fig 4A and 4C), and MED30 overexpression accelerated the FBS-induced invasions of SNU216 and SNU638 cells versus mock cells by 2.5 and 2.2 fold, respectively (Fig 4B and 4C).

Effect of MED30 knockdown on in vivo tumorigenicity

To examine the effect of MED30 on tumor growth, we subcutaneously injected SNU638 cells transfected with SCR or MED30 siRNA into SCID mice, and then monitored tumor growth for seven weeks (Fig 5A). Palpable tumors were detected within three weeks in the SCR control sides. However, cancer cells transfected with MED30 siRNA exhibited slower growth. Seven weeks after injection, mice were sacrificed, and tumor volumes and weights were measured (Fig 5B and 5C). The results showed MED30 knockdown significantly reduced tumor volumes and weights.

Regulation of EMT pathway by MED30

To determine the mechanism underlying the promotion of gastric carcinogenesis by MED30, we investigated the expression patterns of genes involved in epithelial-mesenchymal transition (EMT: a key process in metastasis and invasion) by real-time PCR. Knockdown of MED30 slightly increased the level of E-cadherin (CDH1) mRNA in SNU638 cells versus SCR transfection, but decreased expressions of N-cadherin (CDH2), P-cadherin (CDH3) and vimentin (VIM) by 42%, 36%, and 41%, respectively (Fig 6A). Consistently, MED30 overexpression decreased CDH1 mRNA levels by 35% versus mock cells, but increased the expressions of N-cadherin (CDH2), P-cadherin (CDH3), twist family bHLH transcription factor 1 and 2 (TWIST1/2), and vimentin (VIM) by 2.9, 3.3, 3.4, 2.4, 2.2, and 4.2 fold, respectively (Fig 6A). The mRNA levels of snail family zinc finger 1 and 2 (SNAI1/2) were unchanged. We then confirmed that MED30 overexpression also increased the protein levels of E-cadherin, N-cadherin, P-cadherin, Twist1, and vimentin (Fig 6B). An examination of the morphology of SNU638 cells revealed that MED30 overexpression triggered a transition from a cobblestone-like to an elongated fibroblast-like morphology (Fig 6C), whereas MED30 knockdown did not alter morphology (data not shown). These results suggest that MED30 positively regulates EMT.