Hongchao He, Jun Dai, Xiaoqun Yang, Xiaojing Wang, Fukang Sun and Yu Zhu* Silencing of MED27 inhibits adrenal cortical carcinogenesis by targeting the Wnt/B-catenin signaling pathway and the epithelial-mesenchymal transition process
https://doi.org/10.1515/hsz-2017-0304
Received December 5, 2017; accepted March 25, 2018; previously published online March 30, 2018
Abstract: This study aimed to explore the effect of MED27 on the expression of epithelial-mesenchymal transition (EMT)-related proteins and ß-catenin in adrenal cortical carcinoma (ACC). The functional mechanism of MED27 on ACC processes was also explored. The expression of MED27 was assessed by quantitative real-time poly- merase chain reaction (qRT-PCR). siRNA was utilized to knockdown the expression of MED27. CCK8 assays were performed to evaluate SW-13 cell proliferation. Transwell assays were performed to assess the invasion ability, and wound healing assays were utilized to detect migration. A tumor xenograft mouse model was established to investi- gate the impact of silencing MED27 on tumor growth and metastasis. MED27 was highly expressed in ACC tissues and cells. Down-regulation of MED27 induced ACC cell apoptosis, and significantly attenuated ACC cell prolifera- tion, invasion and metastasis in vivo and in vitro. MED27 knockdown regulated the expression of EMT-related proteins and Wnt/ß-catenin signaling pathway-related proteins. Our study investigated the function and mecha- nism of MED27 and validated that MED27 plays a nega- tive role in ACC occurrence and progression and could be utilized as a new therapeutic target in ACC prevention and treatment.
Keywords: adrenal cortical carcinoma; epithelial-mesen- chymal transition; MED27; Wnt/ß-catenin.
*Corresponding author: Yu Zhu, Department of Urology, Shanghai Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, No. 197, Ruijin Er Road, Shanghai 200025, China, e-mail: zyyyhyq@126.com
Hongchao He, Jun Dai, Xiaojing Wang and Fukang Sun: Department of Urology, Shanghai Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, No. 197, Ruijin Er Road, Shanghai 200025, China
Xiaoqun Yang: Department of Pathology, Shanghai Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
Introduction
Adrenocortical tumors (ACTs) include adrenal cortical adenoma (ACA) and adrenal cortical carcinoma (ACC) (Fassnacht et al., 2013). ACC is a rare malignant carci- noma. Its annual incidence is one case per million people (Varghese and Habra, 2017). Drugs such as rottlerin and gene targets such as sphingosine kinase 1 inhibitors can be used to inhibit the proliferation of ACC cells (Xu et al., 2016; Zhu et al., 2017). Despite the ability to inhibit ACC cell proliferation, advanced ACC is frequently accom- panied with a poor prognosis, and effective treatment options remain scarce. Thus, new therapeutic targets are urgently needed.
The mediator complex, consisting of 28 elements, par- ticipates in gene transcription in combination with RNA polymerase II. It plays a vital role in transcriptional elon- gation, epigenetic regulation, mRNA processing, noncod- ing RNA activation and termination, and super enhancer formation (Yin and Wang, 2014). MED works as a signal transducer of transcriptional regulators and is likely to participate in the evolutionary diversification of eukary- otes. MED 23-30 are the subunits of the mediator complex that only exist in mammals (Bourbon, 2008). MED27 is an enzyme that is ubiquitously expressed in humans and is required for transcription initiation (Tang et al., 2016). MED27 is a subunit of the CRSP (cofactor required for SP1 activation) complex, which interacts with promoters and mediates the assembly of regulatory proteins to initi- ate the transcription of SP1-driven gene expression (Ryu et al., 1999). MED27 was reported to belong to the five middle module factors group in the mediator complex, whose depletion significantly affects human immunode- ficiency virus 1 (HIV-1) replication within the host cells (Ruiz et al., 2014). In addition, MED27 knockdown leads to reduced cell proliferation, reduced cell cycle progression and increased apoptosis of melanoma cells (Tang et al., 2016). However, to date, the function of MED27 in tumor growth, let alone its regulatory mechanism, has rarely been explored.
The Wnt/B-catenin signaling pathway belongs to the Wnt pathway, and -catenin is a member of the catenin family (Yun et al., 2015). More importantly, the Wnt/ß-catenin signaling pathway plays a vital role in
adrenal cortex development (Kim et al., 2008). Active ß-catenin functions as an adrenal oncogene, triggering benign aldosterone-secreting tumor development and promoting malignancy (Berthon et al., 2010). The epithe- lial-mesenchymal transition (EMT) is a biological process, during which the phenotype of epithelial cells changes from epithelial to mesenchymal. This transition enhances the migratory capacity of epithelial cells and thus plays a pivotal role in the metastasis of tumor cells (Barasch, 2001). A recent study indicated that the dysregulation of Wnt/ß-catenin pathway-related molecules was involved in the development of cancer, and EMT was required for metastasis (Ghahhari and Babashah, 2015). In addition, MED12 loss induces an EMT-like phenotype, which is asso- ciated with chemotherapy resistance in colon cancer and lung cancer (Huang et al., 2012). As a result, we hypoth- esized that ß-catenin and components of the EMT process are the targets of MED27 in ACC.
The aim of this study was to explore the expression of MED27 and ß-catenin in ACC specimens. The effect of MED27 on ACC cells via the Wnt/ß-catenin signaling pathway and EMT process was also investigated. Our study might supplement the understanding of the molec- ular function of MED27 and provide new insights for the therapeutic regimen of ACC.
Results
MED27 is overexpressed in ACC tissues
The quantitative real-time polymerase chain reaction (qRT-PCR) results indicated that the level of MED27 mRNA was dramatically higher in ACC tissues than in adjacent tissues (p<0.01, Figure 1A). Western blot showed similar results as qRT-PCR that MED27 was overexpressed in ACC tissues (p<0.01, Figure 1B). In addition, immunohisto- chemistry also demonstrated that MED27 was upregulated in ACC tissues compared to in adjacent tissues (Figure 1C). High MED27 expression predicted a poor prognosis of ACC patients according to the overall survival (OS) results from the analysis of the TCGA data (p<0.01, Figure 1D). As a result, MED27 was overexpressed in ACC tissues, which may correlate with the low survival rate of ACC patients.
MED27 down-regulation inhibits proliferation and the cell cycle but promotes apoptosis
Two siRNA targeting MED27 were designed and tested by qRT-PCR to investigate their ability to down-regulate the
expression of MED27. The results showed that siRNA- MED27-1 and siRNA-MED27-2 significantly suppressed the expression of MED27 (Figure 2A). CCK8 assay results indicated that MED27 knockdown inhibited the prolif- eration of ACC cells (p<0.05, Figure 2B). Through flow cytometry assays, more SW-13 cells were arrested at the G0/G1 phase of the cell cycle in the siRNA-MED27 groups (65.20% and 65.00%) than in the siRNA-NC group (p <0.05, Figure 2C). The apoptosis rate was much higher in the siRNA-MED27 groups than in the control group (p<0.05, Figure 2D).
MED27 down-regulation decreases EMT-related protein expression in ACC cells
TTF-1-pcDNA3.1, which has been reported previously as a TGF-ß inhibitor (Saito et al., 2009), was transfected as a positive control EMT inhibitor to block the EMT process. According to the Transwell assays, invasion of ACC cells decreased dramatically in the siRNA-MED27 groups com- pared with the siRNA-NC group (p <0.05), while there was no notable difference between the siRNA-MED27 groups and the positive control group (p>0.05, Figure 3A,B). In addition, Western blot was performed to detect the expression of the EMT-related proteins E-Cadherin and Vimentin. The expression level of E-Cadherin was higher in the siRNA-MED27 groups, while the Vimentin expres- sion level was lower (both p<0.01). No significant dif- ferences were found between the siRNA-MED27 groups and the positive control group (p> 0.05, Figure 3C). The results suggested that MED27 could promote the EMT process, which therefore leads to the aggressiveness of ACC.
The expression of ß-catenin in ACC cells
To further investigate whether the effect of MED27 on tumor growth was correlated with the Wnt/B-catenin signaling pathway, Western blotting was performed on SW-13 cells from each group. The protein expression level of ß-catenin (total and nucleus fraction) was declined sig- nificantly after transfection of siRNA-MED27 compared with that in the control group (both p<0.01, Figure 4A). The expression levels of Wnt/ß-catenin signaling down- stream proteins, such as CyclinD1, CD44, MMP-7, PPAR-Y and VEGF, were evaluated with Western blot and showed the same trend as B-catenin (all p<0.05, Figure 4B). Therefore, the function of MED27 was correlated with the Wnt/ß-catenin signaling pathway.
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A
B
Adjacent
ACC
MED27
GAPDH
Relative MED27 mRNA expression
10
**
8
Relative MED27 protein expression
**
8
6
6
4
4
2
2
0
0
Adjacent
ACC
Adjacent
ACC
C
D
1.0
OS (p=0.03234)
MED27 expression
Low
High
0.8
Survival rate
0.6
0.4
Adjacent
ACC
0.2
#
0.0
0
1000
2000
3000
4000
5000
Time (days)
(A) The expression level of MED27 mRNA in the ACC group was higher than in the adjacent group, which was determined by qRT-PCR; n=15. (B) The expression level of MED27 protein in the ACC group was higher than in the adjacent group, which was determined by Western blot; n=15. (C) Immunohistochemistry staining revealed that MED27 protein was overexpressed in ACC tissues. (D) The relationship between MED27 and overall survival rate. High expression of MED27 predicted a poor prognosis of ACC patients according the OS analysis via the TCGA database. ** p <0.01, compared with the adjacent group.
MED27 knockdown inhibits ACC progression in xenograft mouse models
The oncogenic role of MED27 in ACC was further exam- ined in vivo through xenograft mouse models. Knock- down of MED27 by siRNA dramatically suppressed ACC tumor growth in weight and volume in comparison to the tumors in the siRNA-NC group (p<0.05, Figure 5A, B). In addition, the levels of Wnt/ß-catenin downstream proteins CyclinD1, CD44, MMP-7, PPAR-y and VEGF in the xenografted tumor tissues were significantly lower in the siRNA-MED27 groups than in the siRNA-NC group (p<0.05, Figure 5C), indicating that MED27 knockdown might suppress Wnt signaling. Thus, MED27 down-regu- lation was able to inhibit ACC progression by suppressing Wnt signaling.
Discussion
In our study, we discovered a high level of MED27 in ACC tissues and its negative relationship with the sur- vival rate of ACC patients. CCK8 experiments showed that the low level of MED27 significantly suppressed ACC cell proliferation. qRT-PCR and Western blotting demon- strated that the decrease in MED27 led to the expression changes in EMT-related proteins. In addition, ß-catenin expression was significantly suppressed in the experi- mental group transfected with siRNA-MED27. The mouse xenograft model experiments also confirmed that MED27 down-regulation led to the inhibition of tumor growth and metastasis. Therefore, MED27 may regulate ACC cell activities via the EMT process and Wnt/ß-catenin signal- ing pathway.
A
1.5
B
Relative MED27 mRNA expression
1.0
SiRNA-NC
1.0
si-MED27-1
OD values (450 nm)
0.8
+ si-MED27-2
*
0.6
0.5
*
0.4
*HTH*
0.2
0.0
0.0
Mock
SiRNA-NC
siRNA-MED27-1 siRNA-MED27-2
0
1
2
3
4
C
Time (day)
GO/G1: 56.24%
GO/G1: 65.20%
120
G0/G1
(ZZZZ]
S
G2/M
S: 20.50%
G2/M: 22.48%
S: 19.21%
280
Percentage of cells cycle (%)
400
G2/M: 16.20%
90
Number
210
Number
300
60
140
200
70
100
30
0
0
0
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Channels (FL 3 area-PE texas red)
Channels (FL 3 area-PE texas red)
siRNA-NC
siRNA-MED27-1 siRNA-MED27-2
siRNA-NC
siRNA-MED27-1
160
GO/G1: 65.00%
D
104
104
S: 15.22%
G2/M: 22.00%
Q1
Q2
Q1
Q2
120
0.79
5.03
2.29
7.76
103
103.
Number
80
102
PI
102
PI
40
101
101
Q4
0
Q3
Q4
Q3
10º
91.8
160
2.38
10º
86.0
3.95
0
40
80
120
Channels (FL 3 Lin-PE texas red)
10º
101
102
103
104
10º
101
102
103
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siRNA-MED27-2
Annexin V-FITC
Annexin V-FITC
siRNA-NC
SiRNA-MED27-1
15
NC
siRNA-MED27-1
siRNA-MED27-2
104
*
*
Q1
Q2
Cell apoptosis rate (%)
4.33
7.68
10
103
미 102
5
101
Q4
84.5
10º
0
SiRNA-NC
siRNA-MED27-1 siRNA-MED27-2
10º
101
102
103
104
Annexin V-FITC
siRNA-MED27-2
A
B
24 h
SİRNA-NC
siRNA-MED27-1
siRNA-MED27-2
TTF-1
0h
400
SiRNA-NC
siRNA-MED27-1
siRNA-MED27-2
TTF-1
**
ns
Invasion cell number
300
Cell migration distance (% of control)
1.5
**
200
ns
1.0
100
0.5
0
SiRNA-NC
siRNA-MED27-1
siRNA-MED27-2
TTF-1
0.0
siRNA-NC
siRNA-MED27-1
siRNA-MED27-2
TTF-1
C
siRNA-NC
siRNA-MED27-1
siRNA-MED27-2 TTF-1
2.0
E-Cadherin
☐ Vimentin
**
E-Cadherin
Relative protein expression
**
1.5
*
Vimentin
1.0
**
*
**
T
0.5
GAPDH
0.0
SiRNA-NC
siRNA-MED27-1
SÍRNA-MED27-2
TTF-1
The mediator complex is closely involved in gene transcription. Several studies have identified the altered expression of particular subunits in diverse human diseases. Nagalingam et al. (2012) demonstrated that MED1 overexpression was indispensable for acquired tamoxifen resistance, as ablation of MED1 reversed tamoxifen resistance. Yang et al. (2016) reported that the emergence of MED12 led to the development of uterine fibroids. It was also reported by Cani et al. (2015) that MED12 was mutated in phyllodes tumors. In addition, accumulated studies have demonstrated that the media- tor complex plays a key role in many human cancers. For instance, Shaikhibrahim et al. (2014) found that MED15 was overexpressed at a high frequency in castration- resistant prostate cancer. Similarly, Yang et al. (2012) revealed that lower MED23 expression predicted better survival in Ras-active lung cancer patients and xeno- graft mice. Yun and colleagues found that MED1 expres- sion in lung adenocarcinoma cell lines from females
was dramatically upregulated compared with that from males; therefore, MED1 may contribute to the difference in estrogenic responsiveness of lung adenocarcinomas (Yun et al., 2011). MED1 is also believed to be associated with muscle invasion, metastatic spread and shorter OS time in bladder cancer, as demonstrated by Klumper et al. (2017). Consistent with these studies, we identified a high expression level of MED27, another member of the mediator complex, in ACC tissues. We also found that MED27 was negatively correlated with the survival rate of ACC patients. However, there are few studies on the function of MED27 in cancer cells. Recently, Tang et al. demonstrated the potential oncogenic and migration- promoting effects of MED27 in melanoma cells. Knock- down of MED27 suppressed the growth and survival of melanoma cells, as well as induced their apoptosis (Tang et al. 2016). Our study was consistent with their findings. The outcome suggested that cell proliferation was evi- dently suppressed in the siRNA1-MED27 group compared
A
siRNA-NC siRNA-MED27-1 siRNA-MED27-2
ß-Catenin (nuclear)
Relative ß-catenin protein expression
1.5
Total
☐ Nuclear
Lamin B
1.0
**
**
siRNA-NC siRNA-MED27-1 siRNA-MED27-2
**
**
ß-Catenin (total)
0.5
GAPDH
0.0
siRNA-NC
siRNA-MED27-1
siRNA-MED27-2
B
siRNA-NC siRNA-MED27-1 siRNA-MED27-2
CyclinD1
1.5
siRNA-NC
siRNA-MED27-1
CD44
Relative protein expression
siRNA-MED27-2
1.0
MMP-7
*
PPAR-Y
0.5
VEGF
0.0
GAPDH
CyclinD1
CD44
MMP-7
PPAR-Y
VEGF
with that in the control group. On the other hand, the down-regulation of MED27 induced cell apoptosis and cell cycle arrest. Therefore, MED27 might play a modula- tory role in ACC cells.
The mechanism of MED27 regulation in ACC cells was investigated. Changes in the expression levels of two EMT- related proteins when MED27 was inhibited demonstrated that MED27 may regulate the EMT process, which might be the underlying mechanism. Salomon et al. (2015) revealed that the loss of ß-catenin inhibited cell growth and reversed EMT, indicating that EMT in correlated with ß-catenin. In addition, Liu-Chittenden et al. (2017) showed that retinoic acid receptor responder protein 2 (RARRES2) overexpres- sion in ACC cells inhibited the Wnt/B-catenin pathway by promoting ß-catenin phosphorylation and degradation. Arend et al. (2016) found that niclosamide and its analogs potentially inhibited Wnt/ß-catenin signaling in ovarian cancer. Therefore, we hypothesized that the ß-catenin signaling pathway is targeted by MED27 in ACC, which was confirmed by the fact that cells transfected with siRNA- MED27 showed lower ß-catenin expression than cells in the control group.
Our study revealed the molecular function of MED27, which may provide new insights for the therapeutic regimen of ACC. However, limitation still exists in the study. First, one cell line model was used in this study, causing potential experimental bias. Furthermore, the interactions between the EMT process and Wnt/B-catenin signaling are worth investigating to help determine the underlying mechanism of MED27 on ACC development.
We found that MED27 was overexpressed in ACC tissues and correlated with a poor survival rate of ACC patients. We also identified the facilitator role of MED27 on ACC cell activities, possibly via the EMT process and the Wnt/B-catenin signaling pathway. Our study may provide novel insights into a therapeutic target of ACC.
Materials and methods
Tissue specimens
Adrenal tissue samples (15 normal adrenal cortex and 15 ACTs) were collected from ACC surgical specimens at Shanghai Ruijin Hospital
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A
siRNA-MED27-1
siRNA-MED27-2
B
siRNA-NC
40
600
SİRNA-NC
siRNA-NC
SiRNA-MED27-1
SiRNA-MED27-1
Tumor volume (mm3)
- siRNA-MED27-2
Tumor weight (g)
30
+ siRNA-MED27-2
400
20
*
*
200
*
*
*
*
*
10
*
*
*
*
0
0
3
6
9
12
15
18
0
6
12
18
Days after the first injection (days)
Days after the first injection (days)
C
siRNA-NC
siRNA-MED27-1 siRNA-MED27-2
CyclinD1
1.5
Relative protein expression
siRNA-NC
si-1
si-2
CD44
1.0
**
*
*
*
MMP-7
**
**
T
T
T
**
**
**
T
**
T
0.5
PPAR-Y
VEGF
0.0
CyclinD1
CD44
MMP-7
PPAR-Y
VEGF
GAPDH
(A) Knockdown of MED27 dramatically suppressed ACC tumor volume in comparison to the control group. (B) The weight of ACC tumors showed a similar trend to the volume. (C) The Western blot results proved that silencing MED27 led to the decreased expression of the Wnt/B-catenin downstream proteins CyclinD1, CD44, MMP-7, PPAR-y and VEGF. * p<0.05 and ** p <0.01 compared with the siRNA-NC group; n=4 in each group; total n= 72.
Affiliated to Shanghai Jiaotong University School of Medicine. All patients from whom the specimens were collected were pathologi- cally diagnosed and had not received chemoradiotherapy before surgery. Informed consent was gathered from every patient, and the study was approved by the Ethics Board of Shanghai Ruijin Hospital Affiliated to Shanghai Jiaotong University.
RT-qPCR
Total RNA was extracted by Trizol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was carried out with a Transcriptor First Strand
| Gene | Primer sequence (5'->3) |
|---|---|
| MED27 forward | GAAGGTGACAGACCATGCCA |
| MED27 reverse | ACCAGGTCATGAAGGATCGG |
| GAPDH forward | AATCCCATCACCATCTTCC |
| GAPDH reverse | CATCACGCCACAGTTTCC |
cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Wilmington, DE, USA) following the manufacturer’s instructions. RT-qPCR was per- formed with a Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Fisher). The melting curve was analyzed, and the PCR product speci- ficity was analyzed by agarose gel electrophoresis. Data analysis was carried out by the 2-44Ct method. Primer sequences are shown in Table 1.
Western blotting
Total protein was isolated from normal adrenal cortex and tumor tissues by homogenization in T-PER lysis buffer (Pierce Chemical, Rockford, IL, USA). For nuclear ß-catenin detection, nuclear pro- teins were extracted using the NE-PERTM Nuclear and Cytoplasmic Extraction Reagents Thermo Fisher Scientific (Waltham, MA, USA). Protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the Novex NuPAGE system Invitro- gen (Carlsbad, CA, USA). The separated proteins were transferred to 0.45-Am nitrocellulose membranes. Membranes were blocked in TBST buffer containing 5% nonfat milk for 1 h and incubated overnight with the primary antibodies rabbit anti-human MED27, E-Cadherin,
Vimentin, ß-catenin, CyclinD1, CD44, MMP-7, PPAR-y, VEGF, GAPDH and Lamin B [1:800; Abcam (Cambridge, MA, USA)]. Membranes were washed using TBST and hybridized with the horseradish peroxi- dase (HRP)-linked antibody goat anti-rabbit IgG (1:800, Abcam) for 1 h. The membranes were washed with TBST and developed using a Supersignal West Dura chemiluminescence reagent Pierce (Rockford, IL, USA) following the manufacturer’s protocol.
Immunohistochemistry staining
Briefly, the tissue sections were deparaffinized and rehydrated. Then, the sections were immersed in dimethyl benzene and graded alcohols before they were washed 3 times in phosphate buffered saline (PBS) for 5 min. After being immersed in a 3% hydrogen peroxide-metha- nol solution, the sections were then blocked in 1000 ml of 0.01 mm sodium citrate buffer solution and Goat Serum Blocking Solution (Thermo Fischer) at room temperature for 1 h. The sections were then incubated overnight with a primary rabbit antibody against MED27 (1:50, Abcam). Then, the sections were incubated with 50 ul of sec- ondary antibody HRP-conjugated goat anti-rabbit IgG (1:800, Abcam) at 37℃ for 10 min. Specimens were stained with 3,3’-diaminobenzi- dine (DAB) for 3 min and counterstained with hematoxylin. The tar- get-positive cells were photographed using an Olympus microscope (Model BX40F4, Tokyo, Japan).
Cell culture
Human adrenal cortical carcinogenesis cell line SW-13 was purchased from ATCC (Manassas, VA, USA). SW-13 cells were cultivated in Leibo- vitz Medium (L-15; Gibco, Carlsbad, CA, USA) at 37℃ without CO2. The SW-13 cells in logarithmic growth phase were removed 24 h before transfection. After that, the cells were digested using 0.25% trypsin (Gibco) containing 0.02% EDTA and maintained in complete medium to establish the cell suspension system.
siRNA transient transfection
Cells (2×105) were added to 6-well plates with 2 ml/well of complete medium. The plates were cultured in an incubator for 18 h until the cell confluence reached 30-40%. Five microliters of siRNA (Sigma, St. Louis, MO, USA) were dissolved in serum-free Opti-MEM (Life Technologies, Gaithersburg, MD, USA), gently blended, and main- tained at room temperature for 5 min. Transfections with siRNA were executed by Lipofectamine 2000 (Invitrogen) at 37°℃ in 5% CO2. The
| Gene | Primer sequence (5'->3') |
|---|---|
| SİRNA-MED27- upstream | GGCUCCAAUUUGUCUAUAATT |
| SIRNA-MED27-1 downstream | UUAUAGACAAAUUGGAGCCTT |
| SIRNA-MED27-2 upstream | GGUGGCCAUAGUUCGAUAUTT |
| SIRNA-MED27-2 downstream | AUAUCGAACUAUGGCCACCTT |
| siRNA-NC upstream | GCAUTUCUAGUGCATUACUAU |
| siRNA-NC downstream | TUAGCAUCTACGUCAGACUCA |
medium was replaced with complete medium after a 6-h-transfec- tion. Primer sequences are shown in Table 2.
Lipofection
TTF1-pcDNA3.1 and pcDNA3.1 plasmids were purchased from GenePharma (Shanghai, China). The SW-13 cells in logarithmic growth phase were removed 24 h before transfection. The cells were digested in trypsin and maintained in medium for re-suspension. Cells were plated in 6-well plates at a density of 1x 106 cells/well and cultured in the incubator for 24 h at 37℃ with 5% CO2 until the cell confluence reached 80-90%. Transfections were executed by Lipo- fectamine 2000 (Invitrogen). The medium was replaced with com- plete medium after a 6-h transfection.
Cell counting kit-8 (CCK8)
Cell proliferation was assessed using a Cell Counting Kit-8 (Biotech Well, Shanghai, China). The suspended cells were prepared and transferred into 96-well plates, approximately 2×103 per well, then cultivated in a 37℃ incubator. After culture under adherent condi- tions for 24 h, 48 h, 72 h, or 96 h, 10 ul of CCK8 was added to each well and mixed gently. The mixture was incubated for 2 h, and the absorbance at 450 nm was measured.
Transwell cell invasion assay
One hundred microliters of Matrigel (BD Biosciences, San Jose, CA, USA) and 400 ul of serum-free culture medium were mixed and added into the Transwell chambers (40 ul per chamber). After being re-sus- pended with serum-free medium at a density of 0.5-2.5×106/ml, the cells were added into the chambers (200 ul per chamber). The lower chambers were filled with 500 ul of Dulbecco’s Modified Eagle Medium (DMEM) containing 20% FBS. After 48 h of incubation, non-invading cells were removed while invading cells were fixed with paraformalde- hyde for 20 min before staining with 0.1% crystal violet for 20 min and observation under a microscope (model E400; Nikon Inc., Melville, NY, USA). Five fields were randomly selected in each chamber.
Wound healing assay
Cells were cultivated in 6-well plates (4x105 cells/well). The mono- layer of cells was scratched until the cells reached 90% confluence. The distance between the scratches was measured under a micro- scope. After rinsing with PBS three times, the cells were placed into the medium with 1% FBS. An Olympus CK-2 inverted microscope (Olympus) was employed to observe the migrated cells after 24 h.
Cell apoptosis assay
Briefly, 48 h after siRNA transfection, cells were washed, resus- pended in chilled methanol, and kept overnight at 4℃. Cells
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were then collected into 15-ml centrifuge tubes, centrifuged and re-suspended. A FITC Annexin V Apoptosis Detection Kit (BD Bio- sciences) was used to stain the SW-13 cells, and the cells were cul- tured for 15 min at room temperature after that. The cell apoptosis was detected using a flow cytometer (EPICS XL; Beckman Coulter, Miami, FL, USA).
Cell cycle analysis
SW-13 cells were gathered, washed, and re-suspended at a density of 5×107/ml. The cells were then fixed in 75% cold ethanol at -20℃ for 24 h followed by Annexin V-FITC and propidium iodide (PI) stain using an Annexin V-FITC/PI-staining kit (Becton Dickinson, 5 ul each). The stained cells were placed in the dark for 30 min and then analyzed with a flow cytometer (EPICS XL). The fraction of the total cell population in each of the G0/G1, S and G2/M cell cycle phases was determined via ModFit LT software (Verity Software House, Inc., Topsham, MI, USA).
Tumor xenograft mouse model
Female athymic nude mice aged 3-4 weeks were purchased and housed in the Animal Center of Shanghai Ruijin Hospital Affili- ated with Shanghai Jiaotong University School of Medicine. All the operations were performed according to Shanghai Ruijin Hos- pital Affiliated to Shanghai Jiaotong University School of Medi- cine for the Care and Use of Laboratory Animals. The serum-free cell suspensions (1×107/ml) transfected with siRNA-MED27 and siRNA-NC were injected subcutaneously into the back of each mouse (0.2 ml). When the tumor size reached approximately 50- 100 mm3 after cell inoculation, the tumor size was measured once every 3 days using Vernier calipers, and the tumor volumes were calculated based on the formula V = (width2x length)/2. At the ter- mination of the experiment, mice were sacrificed, and the tumor was excised from each mouse. A portion of each tumor was used for Western blot analysis.
Statistical analyses
For experimental data analysis, the expression data of MED27 in 15 tissue samples are presented as the median and quantile range. Other data are presented as the mean ± standard deviation (SD). GraphPad prism 6.0 software (San Diego, CA, USA) was used to evaluate the significant differences. Statistical analysis was car- ried out using one-way analysis of variance (ANOVA) for cell exper- imental results, whereas nonparametric tests were conducted on non-normal distributed data (tissue experiments). For survival analysis, clinical data and expression data were downloaded from a TCGA database. The survival time and status of every patient were used, and survival and survplot (both are R packages) were used for analysis. The patients were grouped into a high MED27 level group and a low MED27 level group based on the mean value of MED27 expression. p-Values <0.05 were regarded as statistically significant.
Funding: This study was supported by the Medical and Technology Intercrossing Research Foundation of Shang- hai Jiaotong University (YG2016QN65).
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