Tumor Biol. DOI 10.1007/s13277-015-3381-7
RESEARCH ARTICLE
Decreased MT1-MMP in gastric cancer suppressed cell migration and invasion via regulating MMPs and EMT Wenfeng Li 1 & Shouzhi Li 1 & Liang Deng 1 & Shibin Yang 1 & Mingzhe Li 1 & Shuo Long 1 & Sile Chen 1 & Fuxiang Lin 1 & Longbin Xiao 1
Received: 14 October 2014 / Accepted: 24 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Membrane type 1-matrix metalloproteinase (MT1MMP) has been identified to play a significant role in several types of cancers, but little is known about the significance of MT1-MMP in gastric cancer patients. The purpose of this study is to investigate the involvement of MT1-MMP in tumor progression of gastric cancer. MT1-MMP expression levels were examined in gastric cancer tissues and cells, and normal gastric tissues and cells. The effects and molecular mechanisms of MT1-MMP expression on cell proliferation, migration, and invasion were also explored. In our results, MT1MMP messenger RNA (mRNA) and protein expression levels were significantly increased in gastric cancer tissue. Moreover, the overexpression of MT1-MMP was positively associated with the status of clinical stage and lymph node metastasis through real-time PCR. Furthermore, knocking down MT1-MMP expression significantly suppressed the cell migration and invasion in vitro and regulated the expression of MMPs and epithelial-mesenchymal transition (EMT)-associated genes. In conclusions, our study demonstrates that MT1MMP was overexpressed in gastric cancer tissue, and reduced expression of MT1-MMP suppressed cell migration, invasion, and through regulating the expression of MMPs and the process of EMT in gastric cancer. Keywords MT1-MMP . Gastric cancer . Biomarker . Metastasis . Epithelial-mesenchymal transition Wenfeng Li and Shouzhi Li are co-first authors. * Longbin Xiao [email protected] 1
Department of Gastrointestinal Surgery of the Eastern Hospital of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510700, Guangdong, China
Introduction Gastric cancer is one of the common malignancies worldwide and represents the third leading cause of cancer-related death [1, 2]. Although there has been a steady decline in the incidence and mortality risk of gastric cancer over several decades in most countries [3, 4], the clinical outcome of gastric cancer patients remains unsatisfactory. Most patients present with unresectable or metastatic disease at the time of diagnosis [4]. Thus, development of novel diagnostic and therapeutic approaches for gastric cancer is urgently needed. Membrane type 1-matrix metalloproteinase (MMP) (MT1MMP), which is a member of the MMPs family, has been implicated in multiple biological processes for its extracellular matrix degrading and accelerating angiogenesis [5]. Present studies indicated that a selective MT1-MMP inhibitor reduced cancer cell motility and tumor growth in human melanoma, fibrosarcoma, tongue squamous cell carcinoma, oral carcinoma, and breast carcinoma cell line [6]. Furthermore, increased plasma membrane localization of MT1-MMP can facilitate prostate cancer cell invasion and metastasis [7]. Interestingly, recent studies showed that upregulation of MT1-MMP was associated with the transcriptional changes during tumor formation because gene mutations have not been observed for MT1-MMP in cancer [5]. MicroRNAs, an abundant class of small and non-coding RNAs, are significantly regulating factor for MT1-MMP. In lung cancer and esophageal cancer, MT1-MMP was a functional target of miR-133a and that miR-133a suppresses proliferation, migration, and invasion by targeting MT1-MMP [8, 9]. In this context, MT1-MMP has been implied to involve in the development and progression of human cancer, but the accurate role of MT1-MMP in gastric cancer remains unknown. The aim of this study was to identify the pathological roles of MT1-MMP in gastric cancer.
Tumor Biol.
Materials and methods
siRNA transfection
Cell culture
MT1-MMP small interfering RNA (siRNA) (si-MT1-MMP) and non-targeting siRNA (si-control) were purchased from GenePharma (Shanghai, China) and used at 20 mM. OptiMEM transfection media and Lipo2000 (both from Invitrogen, USA) were used to transfect the cells once they reached 60 % confluency. Knockdown was assessed by Western blotting after 48 h of transfection.
MKN-28 (well differentiated), NCI-N87 (well differentiated), MKN-74 (moderately differentiated), SGC-7901 (moderately differentiated), MKN-45 (poorly differentiated), AGS (poorly differentiated), and GES-1 (a normal gastric epithelial cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified CO2 (5 %) atmosphere. Collection of clinical specimens Forty-six fresh gastric cancer tissues and 23 fresh adjacent normal tissues were obtained at the time of diagnosis before any therapy from the First Affiliated Hospital of Sun Yat-sen University. Adjacent non-tumor tissues were located more than 5 cm away from the tumor tissues and verified by pathological examination. All fresh samples were obtained from surgical biopsy and immediately preserved in liquid nitrogen. The clinical processes were approved by the Ethics Committees of the First Affiliated Hospital of Sun Yat-sen University. The patients provided informed consents. The clinical staging was based on the seventh AJCC Cancer Staging Manual. Real-time PCR To quantitate messenger RNA (mRNA) expression, total RNA was extracted from clinical samples with RNAiso Plus (TaKaRa, Japan). The isolated total RNA was reverse transcribed using the PrimeScript RT Master Mix (Perfect Real Time) (TaKaRa, Japan) for MT1-MMP, according to the manufacturer’s instructions. Relative expression was calculated via the comparative cycle threshold (Ct) method and was normalized to the expression of GAPDH. The sequence-specific forward and reverse primers sequences for MT1-MMP mRNA were 5′-GGATACCCAATGCCCATTGGCCA-3′ and 5′-CCATTGGGCATCCAGAAGAGAGC-3′, respectively. Forward and reverse primers sequences for GAPDH mRNA were 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′TGGTGAAGACGCCAGTGGA-3′, respectively. qPCR was performed using SYBR Premix Ex TaqTM II (TaKaRa, Japan) on a LightCycler (Roche Diagnostics, USA). Relative quantification of miRNA expression was calculated by using the 2−△△Ct method. The raw data were presented as the relative quantity of target mRNA, normalized with GAPDH, and relative to a calibrator sample. All qRT-PCR reactions were performed in triplicate.
Cell proliferation assays Cell proliferation was analyzed using MTT assay. Briefly, 1× 103 cells were seeded into a 96-well plate with quadruplicate repeat for each condition. For si-MT1-MMP and si-control, the cells were incubated for 1, 2, 3, and 4 days. Twenty microliters of MTT (5 mg/ml) (Sigma, USA) was added to each well and incubated for 4 h. At the end of incubation, the supernatants were removed and 150 μl of DMSO (Sigma, USA) was added to each well. The absorbance value (OD) of each well was measured at 490 nm. Experiments were performed three times. Cell migration and invasion assays In vitro cell migration and invasion assays were examined according to previous study [10]. Briefly, 1×105 cells were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning, Corning, USA). After the cells were incubated for 12 h, Giemsa-stained cells adhering to the lower surface were counted under a microscope in five predetermined fields (×100). For the cell invasion assay, the procedure was similar to the cell migration assay, except that the transwell membranes were pre-coated with 24 mg/ml Matrigel (Corning, USA). Western blot Western blot was carried out according as described [10] with anti-MT1-MMP antibody (1:1000; Chemicon, USA); antiMMP1, MMP3, MMP7, MMP8, MMP10, MMP11, and MMP13 (1:1500, Abcam, USA); and anti-MMP2, MMP9, Zo-1, E-cadherin, Vimentin, Snail, Slug, and ZEB1 (1:1000; Cell Signaling Technology, USA). An HRP-conjugated antirabbit/mouse IgG antibody was used as the secondary antibody (1:2000; Cell Signaling Technology, USA). Signals were detected using enhanced chemiluminescence reagents (Pierce, USA). Statistical analysis All data were analyzed for statistical significance using SPSS 13.0 software. Two-tailed Student’s t test was used for
Tumor Biol.
comparisons of two independent groups. One-way ANOVA was used to determine cell growth in vitro. A P value of less than 0.05 was considered statistically significant.
Results MT1-MMP is expressed at high levels in gastric cancer Using real-time PCR to measure the expression of MT1-MMP transcripts, we found that the MT1-MMP expression level was significantly increased with an average increase of 4.52fold in gastric cancer tissues in comparison to adjacent normal tissues (P
RESEARCH ARTICLE
Decreased MT1-MMP in gastric cancer suppressed cell migration and invasion via regulating MMPs and EMT Wenfeng Li 1 & Shouzhi Li 1 & Liang Deng 1 & Shibin Yang 1 & Mingzhe Li 1 & Shuo Long 1 & Sile Chen 1 & Fuxiang Lin 1 & Longbin Xiao 1
Received: 14 October 2014 / Accepted: 24 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Membrane type 1-matrix metalloproteinase (MT1MMP) has been identified to play a significant role in several types of cancers, but little is known about the significance of MT1-MMP in gastric cancer patients. The purpose of this study is to investigate the involvement of MT1-MMP in tumor progression of gastric cancer. MT1-MMP expression levels were examined in gastric cancer tissues and cells, and normal gastric tissues and cells. The effects and molecular mechanisms of MT1-MMP expression on cell proliferation, migration, and invasion were also explored. In our results, MT1MMP messenger RNA (mRNA) and protein expression levels were significantly increased in gastric cancer tissue. Moreover, the overexpression of MT1-MMP was positively associated with the status of clinical stage and lymph node metastasis through real-time PCR. Furthermore, knocking down MT1-MMP expression significantly suppressed the cell migration and invasion in vitro and regulated the expression of MMPs and epithelial-mesenchymal transition (EMT)-associated genes. In conclusions, our study demonstrates that MT1MMP was overexpressed in gastric cancer tissue, and reduced expression of MT1-MMP suppressed cell migration, invasion, and through regulating the expression of MMPs and the process of EMT in gastric cancer. Keywords MT1-MMP . Gastric cancer . Biomarker . Metastasis . Epithelial-mesenchymal transition Wenfeng Li and Shouzhi Li are co-first authors. * Longbin Xiao [email protected] 1
Department of Gastrointestinal Surgery of the Eastern Hospital of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510700, Guangdong, China
Introduction Gastric cancer is one of the common malignancies worldwide and represents the third leading cause of cancer-related death [1, 2]. Although there has been a steady decline in the incidence and mortality risk of gastric cancer over several decades in most countries [3, 4], the clinical outcome of gastric cancer patients remains unsatisfactory. Most patients present with unresectable or metastatic disease at the time of diagnosis [4]. Thus, development of novel diagnostic and therapeutic approaches for gastric cancer is urgently needed. Membrane type 1-matrix metalloproteinase (MMP) (MT1MMP), which is a member of the MMPs family, has been implicated in multiple biological processes for its extracellular matrix degrading and accelerating angiogenesis [5]. Present studies indicated that a selective MT1-MMP inhibitor reduced cancer cell motility and tumor growth in human melanoma, fibrosarcoma, tongue squamous cell carcinoma, oral carcinoma, and breast carcinoma cell line [6]. Furthermore, increased plasma membrane localization of MT1-MMP can facilitate prostate cancer cell invasion and metastasis [7]. Interestingly, recent studies showed that upregulation of MT1-MMP was associated with the transcriptional changes during tumor formation because gene mutations have not been observed for MT1-MMP in cancer [5]. MicroRNAs, an abundant class of small and non-coding RNAs, are significantly regulating factor for MT1-MMP. In lung cancer and esophageal cancer, MT1-MMP was a functional target of miR-133a and that miR-133a suppresses proliferation, migration, and invasion by targeting MT1-MMP [8, 9]. In this context, MT1-MMP has been implied to involve in the development and progression of human cancer, but the accurate role of MT1-MMP in gastric cancer remains unknown. The aim of this study was to identify the pathological roles of MT1-MMP in gastric cancer.
Tumor Biol.
Materials and methods
siRNA transfection
Cell culture
MT1-MMP small interfering RNA (siRNA) (si-MT1-MMP) and non-targeting siRNA (si-control) were purchased from GenePharma (Shanghai, China) and used at 20 mM. OptiMEM transfection media and Lipo2000 (both from Invitrogen, USA) were used to transfect the cells once they reached 60 % confluency. Knockdown was assessed by Western blotting after 48 h of transfection.
MKN-28 (well differentiated), NCI-N87 (well differentiated), MKN-74 (moderately differentiated), SGC-7901 (moderately differentiated), MKN-45 (poorly differentiated), AGS (poorly differentiated), and GES-1 (a normal gastric epithelial cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified CO2 (5 %) atmosphere. Collection of clinical specimens Forty-six fresh gastric cancer tissues and 23 fresh adjacent normal tissues were obtained at the time of diagnosis before any therapy from the First Affiliated Hospital of Sun Yat-sen University. Adjacent non-tumor tissues were located more than 5 cm away from the tumor tissues and verified by pathological examination. All fresh samples were obtained from surgical biopsy and immediately preserved in liquid nitrogen. The clinical processes were approved by the Ethics Committees of the First Affiliated Hospital of Sun Yat-sen University. The patients provided informed consents. The clinical staging was based on the seventh AJCC Cancer Staging Manual. Real-time PCR To quantitate messenger RNA (mRNA) expression, total RNA was extracted from clinical samples with RNAiso Plus (TaKaRa, Japan). The isolated total RNA was reverse transcribed using the PrimeScript RT Master Mix (Perfect Real Time) (TaKaRa, Japan) for MT1-MMP, according to the manufacturer’s instructions. Relative expression was calculated via the comparative cycle threshold (Ct) method and was normalized to the expression of GAPDH. The sequence-specific forward and reverse primers sequences for MT1-MMP mRNA were 5′-GGATACCCAATGCCCATTGGCCA-3′ and 5′-CCATTGGGCATCCAGAAGAGAGC-3′, respectively. Forward and reverse primers sequences for GAPDH mRNA were 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′TGGTGAAGACGCCAGTGGA-3′, respectively. qPCR was performed using SYBR Premix Ex TaqTM II (TaKaRa, Japan) on a LightCycler (Roche Diagnostics, USA). Relative quantification of miRNA expression was calculated by using the 2−△△Ct method. The raw data were presented as the relative quantity of target mRNA, normalized with GAPDH, and relative to a calibrator sample. All qRT-PCR reactions were performed in triplicate.
Cell proliferation assays Cell proliferation was analyzed using MTT assay. Briefly, 1× 103 cells were seeded into a 96-well plate with quadruplicate repeat for each condition. For si-MT1-MMP and si-control, the cells were incubated for 1, 2, 3, and 4 days. Twenty microliters of MTT (5 mg/ml) (Sigma, USA) was added to each well and incubated for 4 h. At the end of incubation, the supernatants were removed and 150 μl of DMSO (Sigma, USA) was added to each well. The absorbance value (OD) of each well was measured at 490 nm. Experiments were performed three times. Cell migration and invasion assays In vitro cell migration and invasion assays were examined according to previous study [10]. Briefly, 1×105 cells were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning, Corning, USA). After the cells were incubated for 12 h, Giemsa-stained cells adhering to the lower surface were counted under a microscope in five predetermined fields (×100). For the cell invasion assay, the procedure was similar to the cell migration assay, except that the transwell membranes were pre-coated with 24 mg/ml Matrigel (Corning, USA). Western blot Western blot was carried out according as described [10] with anti-MT1-MMP antibody (1:1000; Chemicon, USA); antiMMP1, MMP3, MMP7, MMP8, MMP10, MMP11, and MMP13 (1:1500, Abcam, USA); and anti-MMP2, MMP9, Zo-1, E-cadherin, Vimentin, Snail, Slug, and ZEB1 (1:1000; Cell Signaling Technology, USA). An HRP-conjugated antirabbit/mouse IgG antibody was used as the secondary antibody (1:2000; Cell Signaling Technology, USA). Signals were detected using enhanced chemiluminescence reagents (Pierce, USA). Statistical analysis All data were analyzed for statistical significance using SPSS 13.0 software. Two-tailed Student’s t test was used for
Tumor Biol.
comparisons of two independent groups. One-way ANOVA was used to determine cell growth in vitro. A P value of less than 0.05 was considered statistically significant.
Results MT1-MMP is expressed at high levels in gastric cancer Using real-time PCR to measure the expression of MT1-MMP transcripts, we found that the MT1-MMP expression level was significantly increased with an average increase of 4.52fold in gastric cancer tissues in comparison to adjacent normal tissues (P
