Tumor Biol. DOI 10.1007/s13277-014-2898-5
RESEARCH ARTICLE
Involvement of miR-143 in cisplatin resistance of gastric cancer cells via targeting IGF1R and BCL2 Ming Zhuang & Qin Shi & Xiuwei Zhang & Yongbin Ding & Liuqun Shan & Xia Shan & Jiaqi Qian & Xin Zhou & Zebo Huang & Wei Zhu & Yin Ding & Wenfang Cheng & Ping Liu & Yongqian Shu
Received: 26 August 2014 / Accepted: 26 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract We investigated the possible role of miR-143 in the development of cisplatin resistance in human gastric cancer cell line. miR-143 was detected by quantitative real-time PCR. Cisplatin resistance changes of cells was tested via MTT assay. Target genes of miR-143 were verified by dualluciferase activity assay. Immunohistochemistry, immunofluorescence staining, Western blot, cell proliferation, and clonogenic and apoptosis assay were used to elucidate the mechanism of miR-143 in cisplatin resistance formation. Ming Zhuang and Qin Shi contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-2898-5) contains supplementary material, which is available to authorized users. M. Zhuang : J. Qian : X. Zhou : Z. Huang : W. Zhu (*) : P. Liu : Y. Shu (*) Department of Oncology, Clinical Medical College of Yangzhou University, No. 98 Nantong Western Road, Yangzhou 225001, People’s Republic of China e-mail: [email protected] e-mail: [email protected] Q. Shi : W. Cheng Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, People’s Republic of China X. Zhang : X. Shan Department of Respiration, The Affiliated Jiangning Hospital of Nanjing Medical University, 168 Gushan Road, Nanjing 211100, People’s Republic of China Y. Ding : L. Shan Department of General Surgery, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, People’s Republic of China Y. Ding State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, No. 22 Hankou Road, Nanjing 210093, People’s Republic of China
miR-143 was downregulated in gastric cancer tissues and cell lines. It was also downregulated in cisplatin-resistant gastric cancer cell line SGC7901/cisplatin (DDP), which was concurrent with the upregulation of IGF1R and BCL2, compared with the parental SGC7901 cell line, respectively. Overexpressed miR-143 sensitized SGC7901/DDP cells to cisplatin. The luciferase activity suggested that IGF1R and BCL2 were both target genes of miR-143. Enforced miR-143 reduced its target proteins, inhibited SGC7901/DDP cells proliferation, and sensitized SGC7901/DDP cells to DDPinduced apoptosis. Our findings suggested that hsa-miR-143 could modulate cisplatin resistance of human gastric cancer cell line at least in part by targeting IGF1R and BCL2. Keywords miR-143 . Cisplatin resistance . IGF1R . BCL2 Abbreviations miRNAs MicroRNAs DDP Cisplatin MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
Introduction Gastric cancer is still one of the major causes of cancer mortality worldwide. Most patients with advanced stages of cancer need chemotherapy. One of the greatest obstacles to effective chemotherapy is the development of drug resistance. Resistance to chemotherapy is a result from a lot of factors including individual differences in patients and genetic or epigenetic changes in tumors, even those from the same tissue of origin [1]. Drug resistance is often intrinsic to the cancer, but as treatment becomes more and more effective, acquired resistance has also become common. The main reason for
Tumor Biol.
acquired resistance to anticancer drugs is the expression of one or more energy-dependent transporters, such as Pglycoprotein (P-gp) and MDR-associated protein 1 (MRP1), which detect and eject anticancer drugs from cells, while other mechanisms of drug resistance such as defective drug-induced apoptosis, drug-detoxification induction, and alterations in cell cycle and proliferation may also take part in acquired drug resistance [2, 3]. Among these, miRNAs, as a class of small non-coding RNAs of 18–24 nucleotides, are posttranscriptional regulators binding complementary sequences of target mRNAs, usually resulting in translational repression or target degradation [4]. By regulating gene expression at the post-transcriptional level, miRNAs has been linked to pathways associated with cell differentiation, proliferation, survival, and apoptosis, and their abnormal expression has been shown to be involved in anticancer drug resistance [5]. Recently, a number of studies showed that miR-143 was downregulated in a range of human cancers, such as gastric cancer [6], colorectal cancer [7], lung cancer [8], cervical cancer [9], breast carcinoma [10], B-cell malignancies [11], and bladder cancer [12], suggesting that it might have potential roles as tumor-suppressor gene in these cancers mentioned above. In this study, we reported that miR-143 was significantly downregulated in both gastric cancer tissues and various gastric cancer cell lines. Moreover, it was downregulated in cisplatin-resistant gastric cancer cell line SGC7901/ cisplatin (DDP). We demonstrated that miR-143 might modulate cisplatin resistance of human gastric cancer cell line at least in part by targeting IGF1R and BCL2.
cisplatin (DDP, with final concentration of 1 μg/ml) was added to the culture media for SGC7901/DDP cells. Quantitative real-time PCR analysis for miRNA Both biopsy specimens and cells were isolated with Trizol reagent (Invitrogen, Carlsbad, CA) and miRNA fraction was further purified using a mirVanaTM miRNA isolation kit (Ambion, Austin, TX). The concentration and purity of the RNA samples were determined spectroscopically. Expression of mature miRNA was assayed using stem-loop RT followed by real-time PCR analysis. The primers of reverse transcription and polymerase chain reaction were purchased from RiboBio Co., Ltd., (Guangzhou, China) named BulgeLoopTM miRNA qRT-PCR Primer Set as previously described [13]. qRT-PCR was performed according to the protocol of the primer set. PCR product amplification was detected by the level of fluorescence emitted by SYBR Green (SYBR®Premix Ex Taq™ II, TaKaRa) which intercalated into double stranded DNA [13]. The U6 gene was used for normalizing each sample. The ΔCt method was used for miRNA expression analysis of biopsy specimens. First, the cycle number at the threshold level of fluorescence (Ct) for each sample was determined. Next, the ΔCt value was calculated. The ΔCt value was the difference between the Ct value of miR-143 and U6 (ΔCt=Ct (miR-143) −Ct (U6)). The fold-change for miRNA from cells relative to each control cells was calculated using the 2-ΔΔCt method [13], where ΔΔCt=ΔCt MKN45, MGC803, BGC823, SGC7901−ΔCt GES-1 or ΔΔCt=ΔCt SGC7901/DDP−ΔCt SGC7901. PCR was performed in triplicate.
Material and methods In vitro drug sensitivity assay Clinical samples Endoscopic biopsy specimens including 24 cases of gastric cancer and 30 cases of non-tumor gastric mucosa collected from the First Affiliated Hospital of Nanjing Medical University between March 2012 and December 2012 were included. All the samples were confirmed by pathological examination and stored in liquid nitrogen for miRNA analysis. Cell culture Human gastric epithelium cell line GES-1 and gastric adenocarcinoma cell lines MKN45, MGC803, BGC823, and SGC7901 were purchased from the National Institute of Cells (Shanghai, China). Cisplatin-resistant variant SGC7901/DDP was obtained from KeyGEN Biotechnology Company (Nanjing, China). All the cells were cultured in RPMI-1640 medium supplemented with 10 % fetal calf serum (Gibco BRL, Grand Island, NY) in a humidified atmosphere containing 5 % CO2 at 37 °C. To maintain the cisplatin-resistant phenotype,
SGC7901/DDP and SGC7901 cells were plated in 6-well plates (6×105 cells/well). One hundred nanometer of the miR-143 mimic or 100 nM of the miRNA mimic control were transfected in SGC7901/DDP cells, while 100 nM of the miR143 inhibitor or 100 nM of the miRNA inhibitor control were transfected in SGC7901 cells, using lipofectamine 2000 (Invitrogen, Long Island, NY, USA) according to the manufacturer’s protocol, respectively. The miR-143 mimic, miRNA mimic control, 2′-O-methyl (2′-O-Me)-modified miR-143 inhibitor, and miRNA inhibitor control were chemically synthesized by Shanghai GenePharma Company (Shanghai, China). The sequence of each was shown in Supplementary data 1. Twenty-four hours after transfection, cells were seeded into 96-well plates (5×103 cells/well) for next step experiment. After cellular adhesion, freshly prepared cisplatin (DDP) was added with the final concentration being 0.01, 0.1, 1, and 10 times of the human peak plasma concentration for cisplatin as previously described [14]. The peak serum concentration of cisplatin was 2.0 μg/ml [14]. Forty-eight hours after the
Tumor Biol.
addition of drugs, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The absorbance at 490 nm (A490) of each well was read on a spectrophotometer. The concentration, at which cisplatin produced 50 % inhibition of growth (IC50), was estimated by the relative survival curve. Three independent experiments were performed in quadruplicate.
0.1 for 1–9 %, 0.5 for 10–49 %, and 1.0 for 50 % or more. We multiplied the staining intensity by the proportion score of the percentage of positive cancer cells. Thus, we separated the patients into positive ones (the product >1) and negative ones (the product ≤1).
Dual-luciferase activity assay
GES-1, MKN45, MGC803, BGC823, SGC7901, and SGC7901/DDP cells were grown on glass coverslips and fixed with 4 % paraformaldehyde at 4 °C for 15 min and were further permeabilized and blocked with 0.5 % Triton X-100 and 5 % bovine serum albumin in phosphate buffered saline (PBS) for 30 min. The coverslips were then exposed to primary antibodies as mentioned above at 4 °C overnight, followed by the appropriate secondary antibodies. The preparations were visualized using an Olympus IX70 fluorescence microscope.
The 3′UTR of human IGF1R and BCL2 cDNA containing the putative target sites for the miR-143 (sequence shown in Supplementary data 2) was chemically synthesized and inserted at the XbaI site, immediately downstream of the luciferase gene in the pGL3-control vector (Promega, Madison, WI) by Integrated Biotech Solutions Co., Ltd (Shanghai, China), respectively. Twenty-four hours before transfection, cells were plated at 1.5×105 cells/well in 24-well plates. Two hundered nanogram of pGL3-IGF1R-3′-UTR or pGL3BCL2-3′-UTRplus 80 ng pRL-TK (Promega) were transfected in combination with 60 pmol of the miR-143 mimic or miRNA mimic control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol as described, respectively [14]. Luciferase activity was measured 24 h after transfection using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Three independent experiments were performed in triplicate. Immunohistochemistry Twenty cases of tissue samples were obtained from surgical specimens diagnosed with gastric cancer, from May 2011 to December 2012, at the First Affiliated Hospital of Nanjing Medical University. Tissue samples were formalin-fixed and paraffin-embedded, 4-μm-thick sections were cut and stained by using the avidin-biotin complex method. After that, the slides were pretreated with microwaves for antigen retrieval in 10 mM citrate buffer (pH 6.0) and incubated in the primary antibody at 4 °C overnight. The antibody of IGF1Rα (Catalog sc-271606) and BCL2 (Catalog BS1511) were purchased from Santa Cruz Biotechnology and Bioworld Technology, respectively. If the staining was uncertain, we repeated to confirm it. For the scoring of the above proteins, the slides were scored by two separate observers blinded to the clinical data. They evaluated the immunostainings of the slides under an optical microscope of a magnification of 400×. If there were any intra-observer differences, the slides were reevaluated to reach consensus. The staining intensity of the above protein expressions was scored on a scale of 1–3 as follows: 0 score for no staining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining. The percentage of positive cancer cells was scored as follows: 0 score for 0 %,
Immunofluorescence staining
Western blot analysis SGC7901/DDP cells were plated in 6-well plates (6×105 cells/well); 72 h after the transfection of miR-143 mimic or miRNA mimic control, cells were harvested and homogenized with lysis buffer. Total protein was separated by denaturing 10 % SDS—polyacrylamide gel electrophoresis. Total protein of GES-1, MKN45, MGC803, BGC823, SGC7901, and SGC7901/DDP was also extracted and separated. Western blot analysis was performed as described [14]. The primary antibodies for IGF1Rα (Catalog sc-271606), IGF1R (Catalog BS1183), BCL2 (Catalog BS1511), and GAPDH (Catalog BS6945) were purchased from Santa Cruz Biotechnology and Bioworld Technology, respectively. Protein levels were normalized to GAPDH. Fold changes were determined. Cell proliferation assay Six hours after the transfection of miR-143 mimic or miRNA mimic control, SGC7901/DDP cells were trypsinized and seeded into 96-well culture plates at a density of 5×103 cells/well in growth medium supplemented with 10 % serum. The MTT assay was performed 24, 48, and 72 h post-transfection, and absorbance was measured using a spectrophotometer at 490 nm. Each assay was performed in triplicate with three independent replicates. Clonogenic assay SGC7901/DDP cells were transfected with miR-143 mimic or miRNA mimic control as previously described and plated into 6-well plates at a density of 300 cells per well, incubated at 37 °C for 2 weeks, and fixed and stained with crystal violet. The mean±SEM, number of colonies containing>50 cells,
Tumor Biol.
were counted under a microscope from three independent replicates. Apoptosis assay SGC7901/DDP cells were plated in 6-well plates (6×105 cells/well). Twenty-four hours after the transfection of miR143 mimic or miRNA mimic control as previously described, cells were treated by DDP, with final concentration of 10 μg/ml, respectively. Forty-eight hours after the treatment of DDP, flow cytometry was used to detect apoptosis of the transfected SGC7901/DDP cells by determining the relative amount of annexin V-FITC-positive-PI-negative cells as previously described [14]. Statistical analysis Each experiment was repeated at least 3 times. Numerical data were presented as mean±SD. The difference between means was analyzed with Student’s t test. All statistical analyses were performed using SPSS11.0 software (Chicago, IL). Differences were considered significant when p
RESEARCH ARTICLE
Involvement of miR-143 in cisplatin resistance of gastric cancer cells via targeting IGF1R and BCL2 Ming Zhuang & Qin Shi & Xiuwei Zhang & Yongbin Ding & Liuqun Shan & Xia Shan & Jiaqi Qian & Xin Zhou & Zebo Huang & Wei Zhu & Yin Ding & Wenfang Cheng & Ping Liu & Yongqian Shu
Received: 26 August 2014 / Accepted: 26 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract We investigated the possible role of miR-143 in the development of cisplatin resistance in human gastric cancer cell line. miR-143 was detected by quantitative real-time PCR. Cisplatin resistance changes of cells was tested via MTT assay. Target genes of miR-143 were verified by dualluciferase activity assay. Immunohistochemistry, immunofluorescence staining, Western blot, cell proliferation, and clonogenic and apoptosis assay were used to elucidate the mechanism of miR-143 in cisplatin resistance formation. Ming Zhuang and Qin Shi contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-2898-5) contains supplementary material, which is available to authorized users. M. Zhuang : J. Qian : X. Zhou : Z. Huang : W. Zhu (*) : P. Liu : Y. Shu (*) Department of Oncology, Clinical Medical College of Yangzhou University, No. 98 Nantong Western Road, Yangzhou 225001, People’s Republic of China e-mail: [email protected] e-mail: [email protected] Q. Shi : W. Cheng Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, People’s Republic of China X. Zhang : X. Shan Department of Respiration, The Affiliated Jiangning Hospital of Nanjing Medical University, 168 Gushan Road, Nanjing 211100, People’s Republic of China Y. Ding : L. Shan Department of General Surgery, First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, People’s Republic of China Y. Ding State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, No. 22 Hankou Road, Nanjing 210093, People’s Republic of China
miR-143 was downregulated in gastric cancer tissues and cell lines. It was also downregulated in cisplatin-resistant gastric cancer cell line SGC7901/cisplatin (DDP), which was concurrent with the upregulation of IGF1R and BCL2, compared with the parental SGC7901 cell line, respectively. Overexpressed miR-143 sensitized SGC7901/DDP cells to cisplatin. The luciferase activity suggested that IGF1R and BCL2 were both target genes of miR-143. Enforced miR-143 reduced its target proteins, inhibited SGC7901/DDP cells proliferation, and sensitized SGC7901/DDP cells to DDPinduced apoptosis. Our findings suggested that hsa-miR-143 could modulate cisplatin resistance of human gastric cancer cell line at least in part by targeting IGF1R and BCL2. Keywords miR-143 . Cisplatin resistance . IGF1R . BCL2 Abbreviations miRNAs MicroRNAs DDP Cisplatin MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
Introduction Gastric cancer is still one of the major causes of cancer mortality worldwide. Most patients with advanced stages of cancer need chemotherapy. One of the greatest obstacles to effective chemotherapy is the development of drug resistance. Resistance to chemotherapy is a result from a lot of factors including individual differences in patients and genetic or epigenetic changes in tumors, even those from the same tissue of origin [1]. Drug resistance is often intrinsic to the cancer, but as treatment becomes more and more effective, acquired resistance has also become common. The main reason for
Tumor Biol.
acquired resistance to anticancer drugs is the expression of one or more energy-dependent transporters, such as Pglycoprotein (P-gp) and MDR-associated protein 1 (MRP1), which detect and eject anticancer drugs from cells, while other mechanisms of drug resistance such as defective drug-induced apoptosis, drug-detoxification induction, and alterations in cell cycle and proliferation may also take part in acquired drug resistance [2, 3]. Among these, miRNAs, as a class of small non-coding RNAs of 18–24 nucleotides, are posttranscriptional regulators binding complementary sequences of target mRNAs, usually resulting in translational repression or target degradation [4]. By regulating gene expression at the post-transcriptional level, miRNAs has been linked to pathways associated with cell differentiation, proliferation, survival, and apoptosis, and their abnormal expression has been shown to be involved in anticancer drug resistance [5]. Recently, a number of studies showed that miR-143 was downregulated in a range of human cancers, such as gastric cancer [6], colorectal cancer [7], lung cancer [8], cervical cancer [9], breast carcinoma [10], B-cell malignancies [11], and bladder cancer [12], suggesting that it might have potential roles as tumor-suppressor gene in these cancers mentioned above. In this study, we reported that miR-143 was significantly downregulated in both gastric cancer tissues and various gastric cancer cell lines. Moreover, it was downregulated in cisplatin-resistant gastric cancer cell line SGC7901/ cisplatin (DDP). We demonstrated that miR-143 might modulate cisplatin resistance of human gastric cancer cell line at least in part by targeting IGF1R and BCL2.
cisplatin (DDP, with final concentration of 1 μg/ml) was added to the culture media for SGC7901/DDP cells. Quantitative real-time PCR analysis for miRNA Both biopsy specimens and cells were isolated with Trizol reagent (Invitrogen, Carlsbad, CA) and miRNA fraction was further purified using a mirVanaTM miRNA isolation kit (Ambion, Austin, TX). The concentration and purity of the RNA samples were determined spectroscopically. Expression of mature miRNA was assayed using stem-loop RT followed by real-time PCR analysis. The primers of reverse transcription and polymerase chain reaction were purchased from RiboBio Co., Ltd., (Guangzhou, China) named BulgeLoopTM miRNA qRT-PCR Primer Set as previously described [13]. qRT-PCR was performed according to the protocol of the primer set. PCR product amplification was detected by the level of fluorescence emitted by SYBR Green (SYBR®Premix Ex Taq™ II, TaKaRa) which intercalated into double stranded DNA [13]. The U6 gene was used for normalizing each sample. The ΔCt method was used for miRNA expression analysis of biopsy specimens. First, the cycle number at the threshold level of fluorescence (Ct) for each sample was determined. Next, the ΔCt value was calculated. The ΔCt value was the difference between the Ct value of miR-143 and U6 (ΔCt=Ct (miR-143) −Ct (U6)). The fold-change for miRNA from cells relative to each control cells was calculated using the 2-ΔΔCt method [13], where ΔΔCt=ΔCt MKN45, MGC803, BGC823, SGC7901−ΔCt GES-1 or ΔΔCt=ΔCt SGC7901/DDP−ΔCt SGC7901. PCR was performed in triplicate.
Material and methods In vitro drug sensitivity assay Clinical samples Endoscopic biopsy specimens including 24 cases of gastric cancer and 30 cases of non-tumor gastric mucosa collected from the First Affiliated Hospital of Nanjing Medical University between March 2012 and December 2012 were included. All the samples were confirmed by pathological examination and stored in liquid nitrogen for miRNA analysis. Cell culture Human gastric epithelium cell line GES-1 and gastric adenocarcinoma cell lines MKN45, MGC803, BGC823, and SGC7901 were purchased from the National Institute of Cells (Shanghai, China). Cisplatin-resistant variant SGC7901/DDP was obtained from KeyGEN Biotechnology Company (Nanjing, China). All the cells were cultured in RPMI-1640 medium supplemented with 10 % fetal calf serum (Gibco BRL, Grand Island, NY) in a humidified atmosphere containing 5 % CO2 at 37 °C. To maintain the cisplatin-resistant phenotype,
SGC7901/DDP and SGC7901 cells were plated in 6-well plates (6×105 cells/well). One hundred nanometer of the miR-143 mimic or 100 nM of the miRNA mimic control were transfected in SGC7901/DDP cells, while 100 nM of the miR143 inhibitor or 100 nM of the miRNA inhibitor control were transfected in SGC7901 cells, using lipofectamine 2000 (Invitrogen, Long Island, NY, USA) according to the manufacturer’s protocol, respectively. The miR-143 mimic, miRNA mimic control, 2′-O-methyl (2′-O-Me)-modified miR-143 inhibitor, and miRNA inhibitor control were chemically synthesized by Shanghai GenePharma Company (Shanghai, China). The sequence of each was shown in Supplementary data 1. Twenty-four hours after transfection, cells were seeded into 96-well plates (5×103 cells/well) for next step experiment. After cellular adhesion, freshly prepared cisplatin (DDP) was added with the final concentration being 0.01, 0.1, 1, and 10 times of the human peak plasma concentration for cisplatin as previously described [14]. The peak serum concentration of cisplatin was 2.0 μg/ml [14]. Forty-eight hours after the
Tumor Biol.
addition of drugs, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The absorbance at 490 nm (A490) of each well was read on a spectrophotometer. The concentration, at which cisplatin produced 50 % inhibition of growth (IC50), was estimated by the relative survival curve. Three independent experiments were performed in quadruplicate.
0.1 for 1–9 %, 0.5 for 10–49 %, and 1.0 for 50 % or more. We multiplied the staining intensity by the proportion score of the percentage of positive cancer cells. Thus, we separated the patients into positive ones (the product >1) and negative ones (the product ≤1).
Dual-luciferase activity assay
GES-1, MKN45, MGC803, BGC823, SGC7901, and SGC7901/DDP cells were grown on glass coverslips and fixed with 4 % paraformaldehyde at 4 °C for 15 min and were further permeabilized and blocked with 0.5 % Triton X-100 and 5 % bovine serum albumin in phosphate buffered saline (PBS) for 30 min. The coverslips were then exposed to primary antibodies as mentioned above at 4 °C overnight, followed by the appropriate secondary antibodies. The preparations were visualized using an Olympus IX70 fluorescence microscope.
The 3′UTR of human IGF1R and BCL2 cDNA containing the putative target sites for the miR-143 (sequence shown in Supplementary data 2) was chemically synthesized and inserted at the XbaI site, immediately downstream of the luciferase gene in the pGL3-control vector (Promega, Madison, WI) by Integrated Biotech Solutions Co., Ltd (Shanghai, China), respectively. Twenty-four hours before transfection, cells were plated at 1.5×105 cells/well in 24-well plates. Two hundered nanogram of pGL3-IGF1R-3′-UTR or pGL3BCL2-3′-UTRplus 80 ng pRL-TK (Promega) were transfected in combination with 60 pmol of the miR-143 mimic or miRNA mimic control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol as described, respectively [14]. Luciferase activity was measured 24 h after transfection using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Three independent experiments were performed in triplicate. Immunohistochemistry Twenty cases of tissue samples were obtained from surgical specimens diagnosed with gastric cancer, from May 2011 to December 2012, at the First Affiliated Hospital of Nanjing Medical University. Tissue samples were formalin-fixed and paraffin-embedded, 4-μm-thick sections were cut and stained by using the avidin-biotin complex method. After that, the slides were pretreated with microwaves for antigen retrieval in 10 mM citrate buffer (pH 6.0) and incubated in the primary antibody at 4 °C overnight. The antibody of IGF1Rα (Catalog sc-271606) and BCL2 (Catalog BS1511) were purchased from Santa Cruz Biotechnology and Bioworld Technology, respectively. If the staining was uncertain, we repeated to confirm it. For the scoring of the above proteins, the slides were scored by two separate observers blinded to the clinical data. They evaluated the immunostainings of the slides under an optical microscope of a magnification of 400×. If there were any intra-observer differences, the slides were reevaluated to reach consensus. The staining intensity of the above protein expressions was scored on a scale of 1–3 as follows: 0 score for no staining, 1 for weak staining, 2 for moderate staining, and 3 for strong staining. The percentage of positive cancer cells was scored as follows: 0 score for 0 %,
Immunofluorescence staining
Western blot analysis SGC7901/DDP cells were plated in 6-well plates (6×105 cells/well); 72 h after the transfection of miR-143 mimic or miRNA mimic control, cells were harvested and homogenized with lysis buffer. Total protein was separated by denaturing 10 % SDS—polyacrylamide gel electrophoresis. Total protein of GES-1, MKN45, MGC803, BGC823, SGC7901, and SGC7901/DDP was also extracted and separated. Western blot analysis was performed as described [14]. The primary antibodies for IGF1Rα (Catalog sc-271606), IGF1R (Catalog BS1183), BCL2 (Catalog BS1511), and GAPDH (Catalog BS6945) were purchased from Santa Cruz Biotechnology and Bioworld Technology, respectively. Protein levels were normalized to GAPDH. Fold changes were determined. Cell proliferation assay Six hours after the transfection of miR-143 mimic or miRNA mimic control, SGC7901/DDP cells were trypsinized and seeded into 96-well culture plates at a density of 5×103 cells/well in growth medium supplemented with 10 % serum. The MTT assay was performed 24, 48, and 72 h post-transfection, and absorbance was measured using a spectrophotometer at 490 nm. Each assay was performed in triplicate with three independent replicates. Clonogenic assay SGC7901/DDP cells were transfected with miR-143 mimic or miRNA mimic control as previously described and plated into 6-well plates at a density of 300 cells per well, incubated at 37 °C for 2 weeks, and fixed and stained with crystal violet. The mean±SEM, number of colonies containing>50 cells,
Tumor Biol.
were counted under a microscope from three independent replicates. Apoptosis assay SGC7901/DDP cells were plated in 6-well plates (6×105 cells/well). Twenty-four hours after the transfection of miR143 mimic or miRNA mimic control as previously described, cells were treated by DDP, with final concentration of 10 μg/ml, respectively. Forty-eight hours after the treatment of DDP, flow cytometry was used to detect apoptosis of the transfected SGC7901/DDP cells by determining the relative amount of annexin V-FITC-positive-PI-negative cells as previously described [14]. Statistical analysis Each experiment was repeated at least 3 times. Numerical data were presented as mean±SD. The difference between means was analyzed with Student’s t test. All statistical analyses were performed using SPSS11.0 software (Chicago, IL). Differences were considered significant when p
