Tumor Biol. DOI 10.1007/s13277-015-3415-1
RESEARCH ARTICLE
Interference with the β-catenin gene in gastric cancer induces changes to the miRNA expression profile Li Dong 1 & Jun Deng 1 & Ze-Min Sun 1 & An-Ping Pan 1 & Xiao-Jun Xiang 1 & Ling Zhang 1 & Feng Yu 1 & Jun Chen 1 & Zhe Sun 1 & Miao Feng 1 & Jian-Ping Xiong 1
Received: 18 December 2014 / Accepted: 30 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Aberrant activation of the Wnt/β-catenin signaling pathway plays a major role in carcinogenesis and the progression of many malignant tumors, especially gastric cancer (GC). Some research has suggested that expression of the βcatenin protein is associated with clinicopathologic factors and affects the biological behaviors of GC cells. However, the mechanism of these effects is not yet clear. Studies show that the Wnt/β-catenin pathway regulates some miRNAs. We hypothesize that oncogenic activation of β-catenin signaling is involved in the formation of GC through regulating certain microRNAs (miRNAs). The results of the current study demonstrate that expression of the β-catenin protein is associated with many clinicopathologic characteristics including the degree of differentiation, depth of tumor invasion, tumor site, and 5-year survival rate. We found that silencing the expression of β-catenin with lentiviruses could delay cell proliferation, promote apoptosis, weaken the invasive power of GC cells, and increase the sensitivity of GC cells to 5fluorouracil in vitro. Using miRNA microarrays to detect changes in the miRNA transcriptome following interference with β-catenin in GC cells, we found that miR-1234-3p, miR135b-5p, miR-210, and miR-4739 were commonly upregulated and that miR-20a-3p, miR-23b-5p, miR-335-3p, miR-4235p, and miR-455-3p were commonly downregulated. These data provide a theoretical basis for the potential interaction between miRNA and the β-catenin signaling pathway in GC.
Li Dong and Jun Deng contributed equally to this work. * Jian-Ping Xiong [email protected] 1
Department of Oncology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China
Keywords Gastric cancer . β-Catenin . miRNA . Signaling pathway
Introduction Gastric cancer (GC) is an aggressive neoplasm associated with a very poor prognosis. Worldwide, GC is the fourth most prevalent malignancy and the second leading cancer cause of death. Almost half of all GC cases occur in China [1]. Although germ-line mutations in E-cadherin (CDH1) and infection with Helicobacter pylori are the major risks factors that contribute to the progress of GC, the specific molecular mechanisms remain unclear [2, 3]. Recent studies, however, have provided evidence that abnormal signaling pathways and aberrant expression of small molecules are contributors to gastric carcinogenesis. Abnormal activation of the β-catenin signaling pathway is common and is critical in the pathogenesis of GC. Under normal conditions, the β-catenin protein, the central regulatory molecule of the signaling pathway, is localized at the plasma membrane. Cytoplasmic levels of β-catenin are low in normal cells because the protein is degraded by the ubiquitin–proteasome system. When genes encoding Wnt/β-catenin components, such as the adenomatous polyposis coli (APC) gene product, are mutated, β-catenin will accumulate in the cytoplasm and translocate into the nucleus where it acts as an oncoprotein [4]. Extensive evidence implicates abnormal localization and overexpression of β-catenin as contributors to carcinogenesis, especially for gastroenteric tumors. In both benign and malignant stomach tumors, the APC gene, which serves as a suppressor of the β-catenin signal transduction pathway, has been found to contain mutations. For example, up to 25 % of gastric adenomas exhibit somatic mutations in this gene [5]. Moreover, a loss of membrane β-catenin
Tumor Biol.
staining and nuclear accumulation are common in GC cells, and the rate of β-catenin mutations occurring in both diffuseand intestinal-type GC are higher than in normal gastric mucosa [6, 7]. All of these changes will contribute to the activation of downstream target genes. MicroRNAs (miRNAs) are often regulated by signaling pathways which are the key to cancer development [8]. miRNAs are ∼22 base single-stranded, non-coding RNAs that generally reside in eukaryotes and often function as negative post-transcriptional regulators [9]. Alterations in miRNA expression are involved in the occurrence and development of malignant tumors [10]. Notably, recent research has shown that β-catenin exerts its biological effects by regulating the expression of miRNAs. In early Xenopus embryos, activation of β-catenin signaling is accompanied by the decreased expression of miR-15/-16. In contrast, knockdown of β-catenin leads to increased endogenous levels of miR-15/-16 [11]. Other studies showed that inhibition of the β-catenin signaling pathway by treatment with Dickkopf-1 can reduce the levels of endogenous miR-29a and miR-29c [12, 13]. Moreover, miRNA-122a and miRNA-30e are regulated directly by the Wnt/β-catenin signaling pathway in colorectal cancers [14, 15]. Based on this information, we hypothesized that the βcatenin signaling pathway is involved in the formation of GC by regulating certain miRNAs. The aim of the current study was to identify miRNAs that may be affected by β-catenin. The results will provide a basis for further study of the connection between β-catenin signaling and miRNAs in the context of GC.
Materials and methods Western blotting and immunofluorescence to detect β-catenin AGS, MKN-45, SGC-7901, BGC-803, and GES-1 cell lines were obtained from The First Affiliated Hospital of Nanchang University. BGC-823, MGC803, and HGC-27 cell lines were purchased from the cell bank of the Chinese Academy of Sciences. All cells were cultured in RPMI 1640 medium with 10 % fetal bovine serum (FBS) and incubated at 37 °C in the presence of 5 % CO2. The expression of β-catenin, Bcl-2, Bax, cyclinD1, c-Myc, and β-actin (Cell Signaling Technologies, Danvers, MA, USA) proteins were quantified using Western blots. All cells were lysed on ice in lysis buffer. Proteins were separated by SDS–polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The membranes were then blocked with 10 % milk for 1 h at room temperature and incubated overnight with the primary antibody. After washing three times, membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit IgG
secondary antibody (1:1000) (Zhongshan Biotech, China) for 2 h, washed three times, and finally developed with an ECL kit. Immunofluorescence staining was performed to observe the location of β-catenin in the cells. All cell lines were cultured on glass slips, washed three times with PBS, and then fixed with ice-cold acetone for 5 min. The glass slips were washed three times with 0.1 % bovine serum albumin (BSA) and then blocked with goat serum for 1 h at 37 °C. Cells were then incubated overnight with the rabbit β-catenin primary antibody (1:50) in 0.1 % BSA at 4 °C. After rinsing with PBS, the cells were incubated for 2 h with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:50) in the dark at 37 °C. The cells were counterstained with DAPI and fluorescence images acquired using an Olympus fluorescence microscope equipped with a camera.
Immunohistochemistry to detect β-catenin in human tissue samples Seventy-six patients were enrolled, all of whom had undergone surgery and postoperative adjuvant chemotherapy in the First Affiliated Hospital of Nanchang University between June 2006 and June 2008. Postoperative pathology was used to confirm the presence/ absence of lymph node metastases (LNM), tumor differentiation, and tumor size. Postoperative paraffinembedded cancer tissue specimens were obtained from the pathology department. Thirty-eight cases of paraffinembedded para-carcinoma tissue specimens were obtained for comparison. Patients were divided into groups according to the pathological staging, the presence of LNM, tumor differentiation, invasion depth, tumor size, age, gender, and 5-year survival rate. None of the patients received any treatment related to the tumor before surgery, but all were given 5-fluorouracil (5-FU) for more than 4 weeks after the operation. This study was approved by Ethical Committee of The First Affiliated Hospital of Nanchang University, and written informed consent was obtained from all patients. Paraffin sections (4-mm thick) were deparaffinized with xylene and rehydrated in graded ethanol. Antigens were retrieved with the high-pressure method. After blocking endogenous peroxidase with 3 % hydrogen peroxide, tissue sections were incubated overnight at 4 °C with the primary β-catenin antibody (1:100). After washing three times with PBS, a biotinylated secondary antibody (Zhongshan Biotech, China) was added (1:100) for 30 min at 37 °C. Tissue sections were developed with 3,3′-diaminobenzidine and counterstained with hematoxylin.
Tumor Biol.
Lentivirus-infected cells The lentiviral vectors, β-catenin-RNAi-433-LV (433 denotes the interference sequence), and β-catenin-negative LV were synthesized by GenePharma (Shanghai, China) and used to infect the AGS and MGC803 cell lines. All stable cell lines were screened with puromycin and three groups were created: (1) the experimental group (infected with β-catenin-RNAi lentivirus: AGS-RNAi-433 and MGC803-RNAi-433), (2) the negative control group (infected with β-catenin-negative LV lentivirus: AGS-Neg and MGC803-Neg), and (3) the blank control group (untreated AGS and MGC803 cells). Quantitative real-time PCR and Western blot analysis of β-catenin Total RNA was isolated from the cell lines with TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The purity of total RNA was determined by spectrophotometry. RNA was reversetranscribed into cDNA in a total volume of 20 μL using a TransScript® reverse transcription kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. The PCR reaction conditions were initial denaturation at 50 °C for 2 min, then denaturation at 95 °C for 2 min followed by 40 cycles of annealing at 95 °C for 15 s, and 60 °C for 30 s, and a final incubation at 72 °C for 10 min. The relative amounts of β-catenin were calculated using the formula 2-ΔΔCt. Western blots for β-catenin was performed as described above. MTT assay to assess cell sensitivity to 5-FU Cells (AGS, AGS-Neg, AGS-RNAi-433, MGC803, MGC803-Neg, and MGC803-RNAi-433) were plated at a density of 1×104 cells/ml in quadruplicate in 96-well plates and treated with seven different concentrations of 5-FU (20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 μg/ml) for 24, 48, or 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 20 μl of a 5 mg/ml solution) was added to each well. After incubation for 4 h, the solution was removed from each well and 150 μl DMSO were added. The absorbance of each well at 490 nm was measured using a microplate spectrophotometer. Assessment of invasion and migration of AGS and MGC803 cells by the transwell assay A transwell chamber (6.5 mm in diameter with a polycarbonate membrane, 8.0-μm pore size) was placed into each well of a 24-well plate. Cells (1×105) were suspended in RPMI 1640 media containing 5 % FBS and added to the upper wells of a
Matrigel invasion chamber. RPMI 1640 medium with 20 % FBS was placed in the lower wells. After incubation for 24 h, cells in the upper chamber were scratched with a cotton swab, and the cells that remained on the filter were fixed with an ethanol-based crystal violet solution for 15 min and then washed three times with PBS. Cells were manually counted in nine random ×400 magnification fields using an inverted fluorescence microscope.
Flow cytometric cell cycle and apoptosis assays Annexin V-FITC (1 μl of a 2 mg/ml solution) was added to 500 μl of Annexin V binding buffer in which cells (1×105) were resuspended. After incubation for 15 min in the dark, 5 μl 7-aminoactinomycin D were added and the cells were incubated for an additional 15 min in the dark. The extent of apoptosis was analyzed by flow cytometry according the manufacturer’s instructions. For cell cycle analysis, 400 μl propidium iodide was added at 4 °C and cells were incubated in the dark for 30 min. Cells were then analyzed by flow cytometry.
miRNA chip analysis The lentiviral vectors β-catenin-RNAi-433-LVand β-cateninRNAi-769-LV were used to infect the AGS and MGC803 cell lines, respectively. AGS-Neg and MGC803-Neg cells were infected with the nonsense sequence lentiviral vector as a control. Stably infected cell lines were screened with puromycin. Total RNA was isolated from the cells as described above. Total RNA was quantified using a NanoDrop ND-2000 (Thermo Scientific) spectrophotometer, and RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies). miRNA expression profiling was performed using the 2100 Bioanalyzer assay (Agilent Human miRNA V19.0) that is capable of detecting 2006 unique human miRNAs. The detection process and data analysis were conducted by Oebiotech (Shanghai, China).
qRT-PCR for validation of the microarray assay Total RNA was isolated from the cell lines as described above and reverse-transcribed using a miScript II Reverse Transcription Kit. The expression of hsa-miR-135b-5p, hsa-miR-210, and miR-455-3p was detected by quantitative real-time PCR (qRT-PCR) using a LightCycler® 480 SYBR Green I Master Kit according to the manufacturer’s instructions and normalized to U6 small nuclear RNA levels. The relative amounts of miR-135b-5p, miR-210, and miR-455-3p were calculated using the formula 2-ΔΔC.
Tumor Biol. Fig. 1 β-Catenin is overexpressed and localized abnormally in gastric cancer cells. a Western blot and b quantification showing that the gastric cancer cells AGS, HGC27, MGC803, and BGC-803 exhibit higher differential expression levels of β-catenin compared with normal gastric mucosa GES-1 and gastric cancer MKN-45 cells. c Immunofluorescence staining shows that β-catenin accumulates abnormally in the cytoplasm/ nucleus in AGS, HGC-27, and MGC803 cells. In GES-1 and MKN-45 cells, β-catenin is mainly localized in membranes and only minimally expressed in the cytoplasm/nucleus. d βCatenin-RNAi-433-LV could significantly inhibit β-catenin protein expression in AGS and MGC803 cells
Statistical analysis IBM SPSS Statistics, V18.0 software (IBM, Armonk, NY, USA) was used for data analysis. Quantitative
Fig. 2 The positive expression of β-catenin protein is evident as a medium brown or brownish yellow stain in the cytoplasm and/ or nucleus of tumor tissues. a Low magnification, ×200 and b high magnification, ×400. In gastric cancer tissues, β-catenin is mainly localized in cytoplasm and/or nucleus. c Low magnification, ×200 and d high magnification, ×400. In paracarcinoma tissues, β-catenin was mainly localized in membranes
data are presented as means ± standard deviation (SD). Using the Student’s t test for independent groups, P < 0.05 was considered statistically significant.
Tumor Biol. Table 1 The expression of βcatenin protein in gastric cancer and para-carcinoma tissues
Tissue types
Cancer Para-carcinoma
Number
n=76 n=38
Expression intensity −∼∼+
++∼+++
29 29
47 9
β-Catenin in the cytoplasm and nucleus To detect the expression of β-catenin protein, a variety of GC cell lines (AGS, HGC-27, SGC-7901, MGC803, BGC-803, MKN-45, and BGC-823) and normal gastric mucosa cells (GES-1) were studied. Western blot analyses revealed that AGS, HGC-27, MGC803, and BGC-803 cells exhibited higher expression levels of β-catenin compared with GES-1 and gastric cancer MKN-45 cells (Fig. 1a, b). Immunofluorescence staining showed that β-catenin accumulated abnormally in the cytoplasm and nucleus in AGS, HGC-27, and MGC803 cells but was seldom expressed in the cytoplasm of GES-1 and
Clinicopathologic indicators
Gender Male Female Age ≥55
RESEARCH ARTICLE
Interference with the β-catenin gene in gastric cancer induces changes to the miRNA expression profile Li Dong 1 & Jun Deng 1 & Ze-Min Sun 1 & An-Ping Pan 1 & Xiao-Jun Xiang 1 & Ling Zhang 1 & Feng Yu 1 & Jun Chen 1 & Zhe Sun 1 & Miao Feng 1 & Jian-Ping Xiong 1
Received: 18 December 2014 / Accepted: 30 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Aberrant activation of the Wnt/β-catenin signaling pathway plays a major role in carcinogenesis and the progression of many malignant tumors, especially gastric cancer (GC). Some research has suggested that expression of the βcatenin protein is associated with clinicopathologic factors and affects the biological behaviors of GC cells. However, the mechanism of these effects is not yet clear. Studies show that the Wnt/β-catenin pathway regulates some miRNAs. We hypothesize that oncogenic activation of β-catenin signaling is involved in the formation of GC through regulating certain microRNAs (miRNAs). The results of the current study demonstrate that expression of the β-catenin protein is associated with many clinicopathologic characteristics including the degree of differentiation, depth of tumor invasion, tumor site, and 5-year survival rate. We found that silencing the expression of β-catenin with lentiviruses could delay cell proliferation, promote apoptosis, weaken the invasive power of GC cells, and increase the sensitivity of GC cells to 5fluorouracil in vitro. Using miRNA microarrays to detect changes in the miRNA transcriptome following interference with β-catenin in GC cells, we found that miR-1234-3p, miR135b-5p, miR-210, and miR-4739 were commonly upregulated and that miR-20a-3p, miR-23b-5p, miR-335-3p, miR-4235p, and miR-455-3p were commonly downregulated. These data provide a theoretical basis for the potential interaction between miRNA and the β-catenin signaling pathway in GC.
Li Dong and Jun Deng contributed equally to this work. * Jian-Ping Xiong [email protected] 1
Department of Oncology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China
Keywords Gastric cancer . β-Catenin . miRNA . Signaling pathway
Introduction Gastric cancer (GC) is an aggressive neoplasm associated with a very poor prognosis. Worldwide, GC is the fourth most prevalent malignancy and the second leading cancer cause of death. Almost half of all GC cases occur in China [1]. Although germ-line mutations in E-cadherin (CDH1) and infection with Helicobacter pylori are the major risks factors that contribute to the progress of GC, the specific molecular mechanisms remain unclear [2, 3]. Recent studies, however, have provided evidence that abnormal signaling pathways and aberrant expression of small molecules are contributors to gastric carcinogenesis. Abnormal activation of the β-catenin signaling pathway is common and is critical in the pathogenesis of GC. Under normal conditions, the β-catenin protein, the central regulatory molecule of the signaling pathway, is localized at the plasma membrane. Cytoplasmic levels of β-catenin are low in normal cells because the protein is degraded by the ubiquitin–proteasome system. When genes encoding Wnt/β-catenin components, such as the adenomatous polyposis coli (APC) gene product, are mutated, β-catenin will accumulate in the cytoplasm and translocate into the nucleus where it acts as an oncoprotein [4]. Extensive evidence implicates abnormal localization and overexpression of β-catenin as contributors to carcinogenesis, especially for gastroenteric tumors. In both benign and malignant stomach tumors, the APC gene, which serves as a suppressor of the β-catenin signal transduction pathway, has been found to contain mutations. For example, up to 25 % of gastric adenomas exhibit somatic mutations in this gene [5]. Moreover, a loss of membrane β-catenin
Tumor Biol.
staining and nuclear accumulation are common in GC cells, and the rate of β-catenin mutations occurring in both diffuseand intestinal-type GC are higher than in normal gastric mucosa [6, 7]. All of these changes will contribute to the activation of downstream target genes. MicroRNAs (miRNAs) are often regulated by signaling pathways which are the key to cancer development [8]. miRNAs are ∼22 base single-stranded, non-coding RNAs that generally reside in eukaryotes and often function as negative post-transcriptional regulators [9]. Alterations in miRNA expression are involved in the occurrence and development of malignant tumors [10]. Notably, recent research has shown that β-catenin exerts its biological effects by regulating the expression of miRNAs. In early Xenopus embryos, activation of β-catenin signaling is accompanied by the decreased expression of miR-15/-16. In contrast, knockdown of β-catenin leads to increased endogenous levels of miR-15/-16 [11]. Other studies showed that inhibition of the β-catenin signaling pathway by treatment with Dickkopf-1 can reduce the levels of endogenous miR-29a and miR-29c [12, 13]. Moreover, miRNA-122a and miRNA-30e are regulated directly by the Wnt/β-catenin signaling pathway in colorectal cancers [14, 15]. Based on this information, we hypothesized that the βcatenin signaling pathway is involved in the formation of GC by regulating certain miRNAs. The aim of the current study was to identify miRNAs that may be affected by β-catenin. The results will provide a basis for further study of the connection between β-catenin signaling and miRNAs in the context of GC.
Materials and methods Western blotting and immunofluorescence to detect β-catenin AGS, MKN-45, SGC-7901, BGC-803, and GES-1 cell lines were obtained from The First Affiliated Hospital of Nanchang University. BGC-823, MGC803, and HGC-27 cell lines were purchased from the cell bank of the Chinese Academy of Sciences. All cells were cultured in RPMI 1640 medium with 10 % fetal bovine serum (FBS) and incubated at 37 °C in the presence of 5 % CO2. The expression of β-catenin, Bcl-2, Bax, cyclinD1, c-Myc, and β-actin (Cell Signaling Technologies, Danvers, MA, USA) proteins were quantified using Western blots. All cells were lysed on ice in lysis buffer. Proteins were separated by SDS–polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The membranes were then blocked with 10 % milk for 1 h at room temperature and incubated overnight with the primary antibody. After washing three times, membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit IgG
secondary antibody (1:1000) (Zhongshan Biotech, China) for 2 h, washed three times, and finally developed with an ECL kit. Immunofluorescence staining was performed to observe the location of β-catenin in the cells. All cell lines were cultured on glass slips, washed three times with PBS, and then fixed with ice-cold acetone for 5 min. The glass slips were washed three times with 0.1 % bovine serum albumin (BSA) and then blocked with goat serum for 1 h at 37 °C. Cells were then incubated overnight with the rabbit β-catenin primary antibody (1:50) in 0.1 % BSA at 4 °C. After rinsing with PBS, the cells were incubated for 2 h with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:50) in the dark at 37 °C. The cells were counterstained with DAPI and fluorescence images acquired using an Olympus fluorescence microscope equipped with a camera.
Immunohistochemistry to detect β-catenin in human tissue samples Seventy-six patients were enrolled, all of whom had undergone surgery and postoperative adjuvant chemotherapy in the First Affiliated Hospital of Nanchang University between June 2006 and June 2008. Postoperative pathology was used to confirm the presence/ absence of lymph node metastases (LNM), tumor differentiation, and tumor size. Postoperative paraffinembedded cancer tissue specimens were obtained from the pathology department. Thirty-eight cases of paraffinembedded para-carcinoma tissue specimens were obtained for comparison. Patients were divided into groups according to the pathological staging, the presence of LNM, tumor differentiation, invasion depth, tumor size, age, gender, and 5-year survival rate. None of the patients received any treatment related to the tumor before surgery, but all were given 5-fluorouracil (5-FU) for more than 4 weeks after the operation. This study was approved by Ethical Committee of The First Affiliated Hospital of Nanchang University, and written informed consent was obtained from all patients. Paraffin sections (4-mm thick) were deparaffinized with xylene and rehydrated in graded ethanol. Antigens were retrieved with the high-pressure method. After blocking endogenous peroxidase with 3 % hydrogen peroxide, tissue sections were incubated overnight at 4 °C with the primary β-catenin antibody (1:100). After washing three times with PBS, a biotinylated secondary antibody (Zhongshan Biotech, China) was added (1:100) for 30 min at 37 °C. Tissue sections were developed with 3,3′-diaminobenzidine and counterstained with hematoxylin.
Tumor Biol.
Lentivirus-infected cells The lentiviral vectors, β-catenin-RNAi-433-LV (433 denotes the interference sequence), and β-catenin-negative LV were synthesized by GenePharma (Shanghai, China) and used to infect the AGS and MGC803 cell lines. All stable cell lines were screened with puromycin and three groups were created: (1) the experimental group (infected with β-catenin-RNAi lentivirus: AGS-RNAi-433 and MGC803-RNAi-433), (2) the negative control group (infected with β-catenin-negative LV lentivirus: AGS-Neg and MGC803-Neg), and (3) the blank control group (untreated AGS and MGC803 cells). Quantitative real-time PCR and Western blot analysis of β-catenin Total RNA was isolated from the cell lines with TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The purity of total RNA was determined by spectrophotometry. RNA was reversetranscribed into cDNA in a total volume of 20 μL using a TransScript® reverse transcription kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. The PCR reaction conditions were initial denaturation at 50 °C for 2 min, then denaturation at 95 °C for 2 min followed by 40 cycles of annealing at 95 °C for 15 s, and 60 °C for 30 s, and a final incubation at 72 °C for 10 min. The relative amounts of β-catenin were calculated using the formula 2-ΔΔCt. Western blots for β-catenin was performed as described above. MTT assay to assess cell sensitivity to 5-FU Cells (AGS, AGS-Neg, AGS-RNAi-433, MGC803, MGC803-Neg, and MGC803-RNAi-433) were plated at a density of 1×104 cells/ml in quadruplicate in 96-well plates and treated with seven different concentrations of 5-FU (20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 μg/ml) for 24, 48, or 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 20 μl of a 5 mg/ml solution) was added to each well. After incubation for 4 h, the solution was removed from each well and 150 μl DMSO were added. The absorbance of each well at 490 nm was measured using a microplate spectrophotometer. Assessment of invasion and migration of AGS and MGC803 cells by the transwell assay A transwell chamber (6.5 mm in diameter with a polycarbonate membrane, 8.0-μm pore size) was placed into each well of a 24-well plate. Cells (1×105) were suspended in RPMI 1640 media containing 5 % FBS and added to the upper wells of a
Matrigel invasion chamber. RPMI 1640 medium with 20 % FBS was placed in the lower wells. After incubation for 24 h, cells in the upper chamber were scratched with a cotton swab, and the cells that remained on the filter were fixed with an ethanol-based crystal violet solution for 15 min and then washed three times with PBS. Cells were manually counted in nine random ×400 magnification fields using an inverted fluorescence microscope.
Flow cytometric cell cycle and apoptosis assays Annexin V-FITC (1 μl of a 2 mg/ml solution) was added to 500 μl of Annexin V binding buffer in which cells (1×105) were resuspended. After incubation for 15 min in the dark, 5 μl 7-aminoactinomycin D were added and the cells were incubated for an additional 15 min in the dark. The extent of apoptosis was analyzed by flow cytometry according the manufacturer’s instructions. For cell cycle analysis, 400 μl propidium iodide was added at 4 °C and cells were incubated in the dark for 30 min. Cells were then analyzed by flow cytometry.
miRNA chip analysis The lentiviral vectors β-catenin-RNAi-433-LVand β-cateninRNAi-769-LV were used to infect the AGS and MGC803 cell lines, respectively. AGS-Neg and MGC803-Neg cells were infected with the nonsense sequence lentiviral vector as a control. Stably infected cell lines were screened with puromycin. Total RNA was isolated from the cells as described above. Total RNA was quantified using a NanoDrop ND-2000 (Thermo Scientific) spectrophotometer, and RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies). miRNA expression profiling was performed using the 2100 Bioanalyzer assay (Agilent Human miRNA V19.0) that is capable of detecting 2006 unique human miRNAs. The detection process and data analysis were conducted by Oebiotech (Shanghai, China).
qRT-PCR for validation of the microarray assay Total RNA was isolated from the cell lines as described above and reverse-transcribed using a miScript II Reverse Transcription Kit. The expression of hsa-miR-135b-5p, hsa-miR-210, and miR-455-3p was detected by quantitative real-time PCR (qRT-PCR) using a LightCycler® 480 SYBR Green I Master Kit according to the manufacturer’s instructions and normalized to U6 small nuclear RNA levels. The relative amounts of miR-135b-5p, miR-210, and miR-455-3p were calculated using the formula 2-ΔΔC.
Tumor Biol. Fig. 1 β-Catenin is overexpressed and localized abnormally in gastric cancer cells. a Western blot and b quantification showing that the gastric cancer cells AGS, HGC27, MGC803, and BGC-803 exhibit higher differential expression levels of β-catenin compared with normal gastric mucosa GES-1 and gastric cancer MKN-45 cells. c Immunofluorescence staining shows that β-catenin accumulates abnormally in the cytoplasm/ nucleus in AGS, HGC-27, and MGC803 cells. In GES-1 and MKN-45 cells, β-catenin is mainly localized in membranes and only minimally expressed in the cytoplasm/nucleus. d βCatenin-RNAi-433-LV could significantly inhibit β-catenin protein expression in AGS and MGC803 cells
Statistical analysis IBM SPSS Statistics, V18.0 software (IBM, Armonk, NY, USA) was used for data analysis. Quantitative
Fig. 2 The positive expression of β-catenin protein is evident as a medium brown or brownish yellow stain in the cytoplasm and/ or nucleus of tumor tissues. a Low magnification, ×200 and b high magnification, ×400. In gastric cancer tissues, β-catenin is mainly localized in cytoplasm and/or nucleus. c Low magnification, ×200 and d high magnification, ×400. In paracarcinoma tissues, β-catenin was mainly localized in membranes
data are presented as means ± standard deviation (SD). Using the Student’s t test for independent groups, P < 0.05 was considered statistically significant.
Tumor Biol. Table 1 The expression of βcatenin protein in gastric cancer and para-carcinoma tissues
Tissue types
Cancer Para-carcinoma
Number
n=76 n=38
Expression intensity −∼∼+
++∼+++
29 29
47 9
β-Catenin in the cytoplasm and nucleus To detect the expression of β-catenin protein, a variety of GC cell lines (AGS, HGC-27, SGC-7901, MGC803, BGC-803, MKN-45, and BGC-823) and normal gastric mucosa cells (GES-1) were studied. Western blot analyses revealed that AGS, HGC-27, MGC803, and BGC-803 cells exhibited higher expression levels of β-catenin compared with GES-1 and gastric cancer MKN-45 cells (Fig. 1a, b). Immunofluorescence staining showed that β-catenin accumulated abnormally in the cytoplasm and nucleus in AGS, HGC-27, and MGC803 cells but was seldom expressed in the cytoplasm of GES-1 and
Clinicopathologic indicators
Gender Male Female Age ≥55
