Tumor Biol. DOI 10.1007/s13277-014-2698-y
RESEARCH ARTICLE
Expression of microRNA-96 and its potential functions by targeting FOXO3 in non-small cell lung cancer Juan Li & Ping Li & Tengfei Chen & Ge Gao & Xiaonan Chen & Yuwen Du & Ren Zhang & Rui Yang & Wei Zhao & Shaozhi Dun & Feng Gao & Guojun Zhang
Received: 11 September 2014 / Accepted: 30 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract MicroRNAs are implicated in the regulation of various cellular processes, including proliferation, differentiation, cell death, and cell mobility, and can function either as oncogenes or tumor suppressors in tumor progression. The effects of the expression of miR-96 in non-small cell lung cancer (NSCLC) remain unclear. In our study, qRT-PCR (quantitative reverse transcription PCR) was performed to identify the miR-96 expression level in 68 paired NSCLC and adjacent normal lung tissues. Trans-well, cell counting kit-8, and apoptosis assays were used to evaluate the effects of miR-96 expression on cell invasion, proliferation, and apoptosis. Dual-luciferase reporter assay and Western blotting were used to verify whether FOXO3 was a potential major target gene of miR-96. Finally, the effect of FOXO3 on miR96-induced cell survival was determined by transfection of the genes expressing FOXO3 lacking 3′UTR and miR-96. The expression level of miR-96 in NSCLC tissues was higher than that in adjacent normal lung tissues, and this increased expression was significantly associated with lymph node metastasis. In contrast to the cells in the blank and negative control groups, the number of cells migrating through the matrigel was significantly lower and the incidence of apoptosis was significantly higher in cells transfected with a miR-96 inhibitor. Western blotting and dual-luciferase reporter assays Juan Li and Ping Li contributed equally to this work. J. Li : P. Li : T. Chen : R. Yang : S. Dun : F. Gao : G. Zhang (*) Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China e-mail: [email protected] G. Gao : X. Chen : Y. Du : R. Zhang College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China W. Zhao Department of Pediatric Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China
demonstrated that miR-96 can bind to the putative seed region in FOXO3 mRNA 3′UTR, and can significantly lower the expression of FOXO3. The introduction of FOXO3 cDNA without 3′UTR restored miR-96 induced cell apoptosis and invasion. MiR-96 is up-regulated in NSCLC tissues. Downregulation of miR-96 inhibits invasion and promotes apoptosis in NSCLC cells A549 and SPC-A-1 by targeting FOXO3. Therefore, our study improves our understanding of the mechanisms underlying NSCLC pathogenesis and may promote the development of novel targeted therapies. Keywords miR-96 . FOXO3 . NSCLC . Proliferation . Apoptosis . Invasion
Introduction Lung cancer remains the leading cause of cancer-related mortality worldwide, with nearly 1.4 million deaths each year [1–4]. Due to the advances in diagnosis and appropriate systemic therapy, including surgery, radiation, and chemotherapy, the prognosis of lung cancer is encouraging. However, the number of cases and deaths related to lung cancer is still rising in many parts of the world. More than 80 % of all cases of lung cancer are non-small cell lung cancer (NSCLC), which includes adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and bronchioalveolar carcinoma [2]. Because the underlying mechanisms of lung cancer metastasis consist of multiple sequential steps that are not completely understood to date, further investigation of this mechanism is needed urgently. MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that negatively regulate gene expression at the post-transcriptional and/or translational level by usually imperfect base-pairing to the 3′ untranslated region (UTR) of one or more genes [5]. MiRNAs are implicated in the
Tumor Biol.
regulation of various cellular processes, including proliferation, differentiation, cell death, and cell mobility, through their interaction with one or more target genes. They can function either as oncogenes or tumor suppressors in tumor progression [6–8]. Therefore, miRNAs expression profiles constitute progress in cancer diagnosis, classification, clinical prognostic information, and therapy [9–13]. MiR-96, which maps to chromosomal region 17, is a member of the miR-183 family (miR-96, miR-182, and miR-183). Data from six NSCLC tissues and their matching normal controls from adjacent tissues defined that miR-96 was up-regulated significantly in NSCLC [14]. Upregulation of miR-96 results in the downregulation of the transcriptional factors FOXO3a and FOXO1, and thus induces cell proliferation in human breast cancer [15, 16]. The FOXO subfamily of forkhead transcription factors, including FOXO1, FOXO3a, FOXO4, and FOXO6 contains evolutionarily conserved transcriptional activators that are characterized by a highly conserved forkhead domain with a DNA-binding motif [17]. FOXO proteins play a pivotal role in biological processes, such as apoptosis, cell cycle control, differentiation, stress response, DNA damage repair, and glucose metabolism [18]. Inhibition of the transcriptional activity of FOXO3a protein in human breast cancer cells can promote cell transformation and tumor progression [19]. In the present study, we investigated miR-96 and FOXO3 expression levels in tumor and normal tissues from 68 NSCLC patients, and observed concomitant alterations in lung cancer cell proliferation, invasion, apoptosis, and metastasis.
Hyclone) supplemented with 10 % fetal bovine serum (FBS, Hyclone), as well as 100 units of penicillin/ml and 100 μg of streptomycin/ml (Enpromise, China), and incubated at 37 °C in a 5 % CO2 humidified atmosphere. Cell transfection MiR-96 inhibitor, miR-96 mimics, and a negative control (NC) were synthesized by Shanghai GenePharma Co. Ltd. A549 and SPC-A-1 cells were plated at a density of 1.5×105 cells/well in six-well plates. Once the cells reached ∼50 % confluence, transient transfection was conducted using Lipofectamine™2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Twenty-four to 48 h after initial transfection, the cells were harvested for further experiments. RNA extraction and quantitative real-time fluorescent Total RNA was extracted from tissue samples and cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. To verify mature miRNA expression levels, qRT-PCR was performed using a High-Specificity miR-96 qRT-PCR Detection Kit (Stratagene Corp., La Jolla, CA) in conjunction with an ABI 7500 thermal cycler, according to the manufacturer’s recommendations. U6 small nuclear RNA (U6 snRNA) was used as an endogenous control for normalization. The qRT-PCR results were expressed relative to miR-96 expression levels at the threshold cycle (Ct) and were converted to fold changes (2-ΔΔCt).
Materials and methods
Western blotting
Patient sample collection
Total proteins of cultured cells were extracted using RIPA buffer containing phenylmethanesulfonyl fluoride (PMSF). A BCA protein assay kit (Beyotime, Haimen, China) was used to determine the protein concentrations. Proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. The membranes were blocked in 5 % (v/v) skim milk for 1 h, washed four times with Tris-buffered saline containing Tween 20 (TBST) at room temperature and then incubated overnight at 4 °C with diluted primary antibodies (rabbit antihuman FOXO3 antibody, 1:500, Santa Cruz Biotechnology). Following extensive washing with TBST, secondary antibodies were incubated at room temperature for 1 h (HRP labeled goat anti-rabbit IgG, 1:1000, Santa Cruz Biotechnology) for 1 h. After four washes (15 min each) with TBST at room temperature, signals were determined using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). An antibody against β-actin (Santa Cruz Biotechnology) served as an endogenous reference.
Paired NSCLC and adjacent normal lung tissues (located more than 5 cm away from the tumors) were obtained from 68 patients who underwent primary surgical resection of NSCLC between March 2012 and January 2014 at the First Affiliated Hospital of Zhengzhou University. None of the patients had received preoperative adjuvant therapy. These samples were snap-frozen in liquid nitrogen after resection. Prior patient consent and approval from the ethics committees of Zhengzhou University were obtained for the use of these clinical materials for research purposes. Cell culture Human lung cancer cell lines A549 and SPC-A-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM,
Tumor Biol.
Trans-well migration assay Trans-well filters (Costar) were coated with matrigel (3.9 μg/μl, 60-80 μl) on the upper surface of a polycarbonate membrane (6.5 mm in diameter, 8 μm pore size). The concentration of cells in each group was adjusted to 2×105 cells/ml at 24 h post-transfection. The upper chamber was loaded with 200 μl of serum-free medium and the lower chamber was supplied with 500 μl of medium containing 10 % FBS. After incubation for 8 h at 37 °C in a humidified incubator with 5 % CO2, the medium was removed from the upper chamber and the cells in the upper chamber were scraped off with a cotton swab. Cells that had migrated to the basal side of the membrane were fixed with methanol, stained with hematoxylin, mounted and dried at 80 °C for 30 min. Five wells were used for each group. The number of cells invading the matrigel was counted in three randomly selected fields using an inverted microscope (200× magnification). Each test was performed in triplicate. Cell counting kit-8 assay The cell proliferation assay was performed using the Cell Counting Kit-8 (CCK8) solution (Dojindo, Gaithersburg, MD), in accordance with the manufacturer’s protocol. Briefly, cells were seeded in triplicate at a concentration of 2×103 cells/well, into 96-well plates and treated with 10 μl/well of Cell Counting Kit-8 solution during the last 4 h of culture, daily for 5 consecutive days. Optical density was measured at 450 nm using a microplate reader. Apoptosis assay Cells from each group were harvested at 48 h post-transfection by trypsinization. Cells were resuspended at a density of 1×106 cells/mL in 1× binding buffer. After double staining with Annexin V-FITC and propidium iodide (PI), using the Annexin V-FITC Apoptosis Detection Kit I (BestBio, Shanghai, China), the cells were analyzed using a FACScan® flow cytometer (BD Biosciences). Dual-luciferase assay The human FOXO3 fragments containing putative seed regions for miR-96 were amplified by PCR from human genomic DNA. The mutant FOXO3 3′UTRs were obtained by overlap extension PCR. The fragments were cloned into a pmirGLO reporter vector (Promega), downstream of the luciferase gene, to generate the recombinant vectors pmirGLO-FOXO3-wt, pmirGLO-FOXO3-mut. For the luciferase reporter assay, cells were seeded in 96-well plates and transfected with 50 nM of miR-96 inhibitor or NC and 100 ng of luciferase reporter
plasmid (pmirGLO-FOXO3-wt/pmirGLO-FOXO3-mut) using LipofectamineTM2000 (Invitrogen). At 24 h after transfection, cells were harvested, and luciferase activity was measured with a dual-luciferase assay kit (Promega) according to the manufacturer’s instructions. Statistical analysis Statistical testing was conducted with the assistance of SPSS 17.0 software. All data were expressed as means±standard deviation (SD). Student’s t test and one-way analysis of variance (ANOVA) were used to analyze data. For multiple comparisons, Tukey’s HSD was used in the comparison of the parental and control vector groups. Results were considered significant when P
RESEARCH ARTICLE
Expression of microRNA-96 and its potential functions by targeting FOXO3 in non-small cell lung cancer Juan Li & Ping Li & Tengfei Chen & Ge Gao & Xiaonan Chen & Yuwen Du & Ren Zhang & Rui Yang & Wei Zhao & Shaozhi Dun & Feng Gao & Guojun Zhang
Received: 11 September 2014 / Accepted: 30 September 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract MicroRNAs are implicated in the regulation of various cellular processes, including proliferation, differentiation, cell death, and cell mobility, and can function either as oncogenes or tumor suppressors in tumor progression. The effects of the expression of miR-96 in non-small cell lung cancer (NSCLC) remain unclear. In our study, qRT-PCR (quantitative reverse transcription PCR) was performed to identify the miR-96 expression level in 68 paired NSCLC and adjacent normal lung tissues. Trans-well, cell counting kit-8, and apoptosis assays were used to evaluate the effects of miR-96 expression on cell invasion, proliferation, and apoptosis. Dual-luciferase reporter assay and Western blotting were used to verify whether FOXO3 was a potential major target gene of miR-96. Finally, the effect of FOXO3 on miR96-induced cell survival was determined by transfection of the genes expressing FOXO3 lacking 3′UTR and miR-96. The expression level of miR-96 in NSCLC tissues was higher than that in adjacent normal lung tissues, and this increased expression was significantly associated with lymph node metastasis. In contrast to the cells in the blank and negative control groups, the number of cells migrating through the matrigel was significantly lower and the incidence of apoptosis was significantly higher in cells transfected with a miR-96 inhibitor. Western blotting and dual-luciferase reporter assays Juan Li and Ping Li contributed equally to this work. J. Li : P. Li : T. Chen : R. Yang : S. Dun : F. Gao : G. Zhang (*) Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China e-mail: [email protected] G. Gao : X. Chen : Y. Du : R. Zhang College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China W. Zhao Department of Pediatric Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China
demonstrated that miR-96 can bind to the putative seed region in FOXO3 mRNA 3′UTR, and can significantly lower the expression of FOXO3. The introduction of FOXO3 cDNA without 3′UTR restored miR-96 induced cell apoptosis and invasion. MiR-96 is up-regulated in NSCLC tissues. Downregulation of miR-96 inhibits invasion and promotes apoptosis in NSCLC cells A549 and SPC-A-1 by targeting FOXO3. Therefore, our study improves our understanding of the mechanisms underlying NSCLC pathogenesis and may promote the development of novel targeted therapies. Keywords miR-96 . FOXO3 . NSCLC . Proliferation . Apoptosis . Invasion
Introduction Lung cancer remains the leading cause of cancer-related mortality worldwide, with nearly 1.4 million deaths each year [1–4]. Due to the advances in diagnosis and appropriate systemic therapy, including surgery, radiation, and chemotherapy, the prognosis of lung cancer is encouraging. However, the number of cases and deaths related to lung cancer is still rising in many parts of the world. More than 80 % of all cases of lung cancer are non-small cell lung cancer (NSCLC), which includes adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and bronchioalveolar carcinoma [2]. Because the underlying mechanisms of lung cancer metastasis consist of multiple sequential steps that are not completely understood to date, further investigation of this mechanism is needed urgently. MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that negatively regulate gene expression at the post-transcriptional and/or translational level by usually imperfect base-pairing to the 3′ untranslated region (UTR) of one or more genes [5]. MiRNAs are implicated in the
Tumor Biol.
regulation of various cellular processes, including proliferation, differentiation, cell death, and cell mobility, through their interaction with one or more target genes. They can function either as oncogenes or tumor suppressors in tumor progression [6–8]. Therefore, miRNAs expression profiles constitute progress in cancer diagnosis, classification, clinical prognostic information, and therapy [9–13]. MiR-96, which maps to chromosomal region 17, is a member of the miR-183 family (miR-96, miR-182, and miR-183). Data from six NSCLC tissues and their matching normal controls from adjacent tissues defined that miR-96 was up-regulated significantly in NSCLC [14]. Upregulation of miR-96 results in the downregulation of the transcriptional factors FOXO3a and FOXO1, and thus induces cell proliferation in human breast cancer [15, 16]. The FOXO subfamily of forkhead transcription factors, including FOXO1, FOXO3a, FOXO4, and FOXO6 contains evolutionarily conserved transcriptional activators that are characterized by a highly conserved forkhead domain with a DNA-binding motif [17]. FOXO proteins play a pivotal role in biological processes, such as apoptosis, cell cycle control, differentiation, stress response, DNA damage repair, and glucose metabolism [18]. Inhibition of the transcriptional activity of FOXO3a protein in human breast cancer cells can promote cell transformation and tumor progression [19]. In the present study, we investigated miR-96 and FOXO3 expression levels in tumor and normal tissues from 68 NSCLC patients, and observed concomitant alterations in lung cancer cell proliferation, invasion, apoptosis, and metastasis.
Hyclone) supplemented with 10 % fetal bovine serum (FBS, Hyclone), as well as 100 units of penicillin/ml and 100 μg of streptomycin/ml (Enpromise, China), and incubated at 37 °C in a 5 % CO2 humidified atmosphere. Cell transfection MiR-96 inhibitor, miR-96 mimics, and a negative control (NC) were synthesized by Shanghai GenePharma Co. Ltd. A549 and SPC-A-1 cells were plated at a density of 1.5×105 cells/well in six-well plates. Once the cells reached ∼50 % confluence, transient transfection was conducted using Lipofectamine™2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Twenty-four to 48 h after initial transfection, the cells were harvested for further experiments. RNA extraction and quantitative real-time fluorescent Total RNA was extracted from tissue samples and cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. To verify mature miRNA expression levels, qRT-PCR was performed using a High-Specificity miR-96 qRT-PCR Detection Kit (Stratagene Corp., La Jolla, CA) in conjunction with an ABI 7500 thermal cycler, according to the manufacturer’s recommendations. U6 small nuclear RNA (U6 snRNA) was used as an endogenous control for normalization. The qRT-PCR results were expressed relative to miR-96 expression levels at the threshold cycle (Ct) and were converted to fold changes (2-ΔΔCt).
Materials and methods
Western blotting
Patient sample collection
Total proteins of cultured cells were extracted using RIPA buffer containing phenylmethanesulfonyl fluoride (PMSF). A BCA protein assay kit (Beyotime, Haimen, China) was used to determine the protein concentrations. Proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. The membranes were blocked in 5 % (v/v) skim milk for 1 h, washed four times with Tris-buffered saline containing Tween 20 (TBST) at room temperature and then incubated overnight at 4 °C with diluted primary antibodies (rabbit antihuman FOXO3 antibody, 1:500, Santa Cruz Biotechnology). Following extensive washing with TBST, secondary antibodies were incubated at room temperature for 1 h (HRP labeled goat anti-rabbit IgG, 1:1000, Santa Cruz Biotechnology) for 1 h. After four washes (15 min each) with TBST at room temperature, signals were determined using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). An antibody against β-actin (Santa Cruz Biotechnology) served as an endogenous reference.
Paired NSCLC and adjacent normal lung tissues (located more than 5 cm away from the tumors) were obtained from 68 patients who underwent primary surgical resection of NSCLC between March 2012 and January 2014 at the First Affiliated Hospital of Zhengzhou University. None of the patients had received preoperative adjuvant therapy. These samples were snap-frozen in liquid nitrogen after resection. Prior patient consent and approval from the ethics committees of Zhengzhou University were obtained for the use of these clinical materials for research purposes. Cell culture Human lung cancer cell lines A549 and SPC-A-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM,
Tumor Biol.
Trans-well migration assay Trans-well filters (Costar) were coated with matrigel (3.9 μg/μl, 60-80 μl) on the upper surface of a polycarbonate membrane (6.5 mm in diameter, 8 μm pore size). The concentration of cells in each group was adjusted to 2×105 cells/ml at 24 h post-transfection. The upper chamber was loaded with 200 μl of serum-free medium and the lower chamber was supplied with 500 μl of medium containing 10 % FBS. After incubation for 8 h at 37 °C in a humidified incubator with 5 % CO2, the medium was removed from the upper chamber and the cells in the upper chamber were scraped off with a cotton swab. Cells that had migrated to the basal side of the membrane were fixed with methanol, stained with hematoxylin, mounted and dried at 80 °C for 30 min. Five wells were used for each group. The number of cells invading the matrigel was counted in three randomly selected fields using an inverted microscope (200× magnification). Each test was performed in triplicate. Cell counting kit-8 assay The cell proliferation assay was performed using the Cell Counting Kit-8 (CCK8) solution (Dojindo, Gaithersburg, MD), in accordance with the manufacturer’s protocol. Briefly, cells were seeded in triplicate at a concentration of 2×103 cells/well, into 96-well plates and treated with 10 μl/well of Cell Counting Kit-8 solution during the last 4 h of culture, daily for 5 consecutive days. Optical density was measured at 450 nm using a microplate reader. Apoptosis assay Cells from each group were harvested at 48 h post-transfection by trypsinization. Cells were resuspended at a density of 1×106 cells/mL in 1× binding buffer. After double staining with Annexin V-FITC and propidium iodide (PI), using the Annexin V-FITC Apoptosis Detection Kit I (BestBio, Shanghai, China), the cells were analyzed using a FACScan® flow cytometer (BD Biosciences). Dual-luciferase assay The human FOXO3 fragments containing putative seed regions for miR-96 were amplified by PCR from human genomic DNA. The mutant FOXO3 3′UTRs were obtained by overlap extension PCR. The fragments were cloned into a pmirGLO reporter vector (Promega), downstream of the luciferase gene, to generate the recombinant vectors pmirGLO-FOXO3-wt, pmirGLO-FOXO3-mut. For the luciferase reporter assay, cells were seeded in 96-well plates and transfected with 50 nM of miR-96 inhibitor or NC and 100 ng of luciferase reporter
plasmid (pmirGLO-FOXO3-wt/pmirGLO-FOXO3-mut) using LipofectamineTM2000 (Invitrogen). At 24 h after transfection, cells were harvested, and luciferase activity was measured with a dual-luciferase assay kit (Promega) according to the manufacturer’s instructions. Statistical analysis Statistical testing was conducted with the assistance of SPSS 17.0 software. All data were expressed as means±standard deviation (SD). Student’s t test and one-way analysis of variance (ANOVA) were used to analyze data. For multiple comparisons, Tukey’s HSD was used in the comparison of the parental and control vector groups. Results were considered significant when P
