MED30 Regulates the Proliferation and Motility of Gastric Cancer Cells

MED30 is an essential member of the mediator complex that forms a hub between transcriptional activators and RNA polymerase II. However, the expressions and roles of MED30 have been poorly characterized in cancer. In this study, we examined the functional roles of MED30 during gastric cancer progression. It was found that MED30 was overexpressed in gastric cancer tissues and cell lines. Moreover, MED30 overexpression increased the proliferation, migration, and invasion of gastric cancer cells, whereas MED30 knockdown inhibited these effects. Furthermore the knockdown significantly inhibited tumorigenicity in SCID mice. MED30 also promoted the expressions of genes related to epithelial-mesenchymal transition and induced a fibroblast-like morphology. This study shows MED30 has pathophysiological roles in the proliferation, migration, and invasion of gastric cancer cells and suggests that MED30 should be viewed as a potent therapeutic target for malignant gastric carcinoma


Introduction
Gastric cancer is one of the leading causes of cancer death worldwide [1,2]. Approximately 95% of gastric cancers are adenocarcinomas and in epidemiological studies gastric cancer has been classified by anatomic site as cardia/proximal or noncardia/distal [3] and by histological phenotype as intestinal, diffuse, or mixed/unclassifiable as described by Lauren [4]. Furthermore, patients with proximal gastric cancer have poorer survival independent of TNM stage [5]. Helicobacter pylori infection has been demonstrated to be an etiologic agent of gastric cancer, particularly of cancers found in the distal stomachs of elderly males, which are usually of the intestinal type. More recently, several molecular classifications of gastric cancer have been proposed based on the findings of whole-genome gene expression studies and/or gene copy number studies [6][7][8][9][10].
Transcriptional regulation is a crucial step that controls cell identity, growth, differentiation, and development. Human mediator (MED) complex, which contains~30 proteins, is a key coactivator/activator of the expressions of RNA polymerase II (Pol II)-transcribed genes [11]. MED complex facilitates the pre-initiation complex (PIC) assembly by interaction with Pol II and gene specific transcription factors (TFs), such as, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and  TFIIH [12,13]. MED complex consists of three distinct structural submodules (head, middle, and tail). The head module directly interacts with Pol II, whereas the elongated tail module interacts with gene-specific regulatory proteins [12,13], and the middle module acts in regulatory signal transfer at a post-binding stage [13]. Although the mechanism is not fully understood, MED complex tightly binds to Pol II, changes its conformation and affects the transcription initiation process [14,15]. Since MED complex is an essential component of the transcription machinery, most of subunits in the core of MED are required for embryonic growth and cell viability [16].
Cancer genome sequencing studies have reported mutations or alterations in the RNA transcription machinery components contained in MED subunits, and correlations between some of these changes in MED subunits, (MED1, MED12, MED19, MED23, MED28, CDK8, and cyclin C) and cancer progression have been reported for various cancers, although mechanisms responsible for these correlations are unknown [17].
Recently, it was reported that a MED19 can participate in gastric cancer progression, as its knockdown significantly inhibited cell proliferation and colony-formation capacity, and induced G1 phase cell-cycle arrest in two human gastric cancer cell lines (SGC7901 and MGC803) [18]. However, the functional roles and pathological relevance of other MED subunits in gastric cancer remain unclear.
In the present study, to reveal the functional importance of MED30 during gastric cancer progression, we examined its roles in proliferation, migration, invasion and tumorigenicity of gastric cancer cells. Before the functional examination, we checked the expression pattern of MED30 in gastric cancer cells and tissues.

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:

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 × 10 5 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 × 10 5 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 × 10 4 cells/ml in serumfree 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 × 10 4 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 × 10 6 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 (mm 3 ) = (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. Ã 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 Ncadherin (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.

Discussion
The functional roles of the MED subunit MED30 (also known as TRAP25) are poorly understood. In a recent report, MED30 was found to play a significant role in regulating mitochondrial functions and integrity in homozygous mice [19]. In the present study, we examined the functional roles of MED30 during gastric cancer progression. It was found MED30 regulated the proliferation, migration, and invasion of gastric cancer cells and their in vivo tumorigenicity. After ensuring knockdown and overexpression efficiency by quantitative real-time PCR and western blot analysis (Fig 2), we found that MED30 knockdown remarkably suppressed the proliferation, migration, and invasion of gastric cancer cells, and that MED30 overexpression had the opposite effects (Figs 2-4). Moreover MED30 knockdown reduced in vivo tumorigenicity ( Fig 5). Furthermore, we also found that MED30 was overexpressed in gastric cancer tissues and gastric cancer cell lines (Fig 1). These results suggest that MED30 might play important pathophysiological roles during gastric carcinogenesis.
Summarizing, the present study supports the notion that MED30 acts as an oncogene during gastric carcinogenesis and suggests MED30 might regulate EMT in gastric cancer. Although further studies are required to determine how MED30 triggers EMT, our results suggest that MED30 be considered a novel molecular target for the treatment of gastric cancer.