Acta Biochimica et Biophysica Sinica Advance Access published May 21, 2015 Acta Biochim Biophys Sin, 2015, 1–8 doi: 10.1093/abbs/gmv039 Original Article
Original Article
miR-125a-3p targets MTA1 to suppress NSCLC cell proliferation, migration, and invasion Hong Zhang1,†, Xiaoxia Zhu1,†, *, Na Li1, Dianhe Li1, Zhou Sha1, Xiaokang Zheng1,*, and Haofei Wang2,* Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China, and Department of Cardiothoracic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
2 †
These authors contributed equally to this work. *Correspondence address. Tel: +86-20-62787484; Fax: +86-20-61641034; E-mail: [email protected] (X.X.Z.)/zkn1268@fimmu. com (X.K.Z.)/[email protected] (H.F.W.)
Received 18 January 2015; Accepted 13 March 2015
Abstract Metastasis-associated gene 1 (MTA1) is associated with cell growth, metastasis, and survival in nonsmall-cell lung cancer (NSCLC). Several previous reports have demonstrated that microRNAs affect gene expression through interaction between their seed region and the 3′-untranslated region of the target mRNA, resulting in post-transcriptional regulation. The aim of this study was to identify miRNAs that suppress malignancy in NSCLC cells by targeting MTA1. Two human NSCLC cell lines were analyzed for the expression of MTA1 by quantitative RT-PCR and western blotting after transfection with MTA1 mimics. A luciferase reporter assay was established to test the direct connection between MTA1 and its upstream miRNAs. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay, 5-ethynyl-2′-deoxyuridine analysis, and colony formation assay. Cell migration and invasive capacity were evaluated by wound-healing assay and transwell assay. The miRNA/MTA1 axis was also probed by quantitative RT-PCR and western blotting in samples from eight NSCLC patients. Among the candidate miRNAs, miR-125a-3p was shown to posttranscriptionally regulate MTA1 in NSCLC cells. These data were reinforced by the luciferase reporter assay, in addition to the demonstration that MTA1 is inversely correlated with miR-125a-3p in NSCLC tissues. Furthermore, miR-125a-3p was found to inhibit NSCLC cell proliferation, migration, and invasion, through the same mechanisms of down-regulated MTA1. Our report demonstrates that miR-125a-3p inhibits the proliferation, migration, and invasion of NSCLC cells through down-regulation of MTA1, indicating the role of the miR-125a-3p/MTA1 axis in NSCLC, and may provide novel insight into the molecular mechanisms underpinning the disease and potential therapeutic targets. Key words: miR-125a-3p, metastasis-associated gene 1 (MTA1), non-small-cell lung cancer, proliferation, invasion
Introduction Lung cancer is the leading cause of cancer mortality worldwide [1], and 80%–85% of lung cancers are non-small-cell lung cancer (NSCLC) [2]. Approximately 56% of lung cancers are diagnosed at a distant stage, while only 15% of patients are diagnosed at a local stage [3]. The overall 5-year survival rate for NSCLC is as low as 17.1% [3]. Metastatic disease seriously impacts the treatment
outcome and overall survival rate in NSCLC patients. Further insight into the molecular mechanisms underpinning metastasis is significantly important and may facilitate the identification of new molecular targets for the treatment of the disease. Metastasis-associated gene 1 (MTA1) was discovered by screening a cDNA library from rat metastatic breast tumors [4]. It is one of the metastasis-related genes which is over-expressed in numerous
© The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 1
Downloaded from http://abbs.oxfordjournals.org/ at Florida International University on May 28, 2015
1
2
miRNA mimics and siRNAs miRNA mimics, small interfering RNA (siRNA) against MTA1 (si-MTA1), and their negative controls were synthesized by GenePharma (Shanghai, China). The sequences were as follows: miR-199a-5p mimic: 5′-CCCAGUGUUCAGACUACCUGUUC-3′; miR-199b-5p mimic: 5′-CCCAGUGUUUAGACUAUCUGUUC-3′; miR-125a-3p mimic: 5′-ACAGGUGAGGUUCUUGGGAGCC-3′; scrambled mimic: 5′-UUCUCCGAACGUGUCACGUTT-3′ (negative control for miRNA mimics); si-MTA1: 5′-CCAGCAUCAUUGAGUACUATT-3′; and scrambled siRNA: 5′-GCGACGAUCUGCCUAAGAUdTdT-3′ (negative control for si-MTA1).
Transfection Cells were seeded in 6-well plates and cultured for 18–24 h. Then, cells were transfected with miRNA mimics or siRNAs (100 μM) using Lipofectamine 3000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Co-transfection of miR-125a-3p mimics and si-MTA1 in SPC-A-1 and 95D cells were performed according to the similar procedures. At 48 h post-transfection, the cells were harvested for further analysis.
Western blot analysis Proteins were extracted from whole cell lysates and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride membranes (Merck Millpore, Milford, USA). The following primary antibodies were used: mouse anti-MTA1 (1:1500; Abcam, Cambridge, USA) and mouse anti-β-actin (1:5000; Sigma, St Louis, USA). Membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz, Santa Cruz, USA). Luminata™ Chemiluminescent Detection Substrates (Merck Millpore) were used to visualize the immunoreactive bands, and the density of each band was analyzed using ImageJ software.
RNA preparation and quantitative real-time PCR analysis
Eight pairs of primary NSCLC tissues and their corresponding normal lung tissue samples were obtained from three squamous cell lung carcinoma patients (one highly differentiated and two moderately differentiated) and five lung adenocarcinoma patients (one highly differentiated, two moderately differentiated, and two poorly differentiated). Consent forms were signed by all patients. The procedures were approved by the Clinical Research Ethics Committee of Nanfang Hospital. None of the patients had been treated with radiotherapy or chemotherapy prior to surgery. All samples were immediately placed in liquid nitrogen and stored at −80°C. Both normal and tumor tissues were verified by histological analysis.
Total RNAs were isolated from cells or tissues using Trizol reagent (Invitrogen). The TaqMan Reverse Transcription Kit (Takara, Dalian, China) was used to obtain cDNA for mRNA detection, whereas TaqMan MicroRNA Reverse Transcription Kit (Takara) was applied to reverse transcribe RNA for miRNA detection. For miRNA and mRNA, real-time PCR was performed using miRscript SYBR Green PCR Kit and SYBR Green PCR Kit (Takara), respectively, on a Roche LightCycler 480 platform (Roche, Basel, Switzerland). The PCR steps included enzyme activation for 10 min at 95°C followed by 40 cycles of 95°C for 15 s and annealing at 60°C for 60 s, according to manufacturer’s protocol. U6 and GAPDH were used as internal controls for miRNA and mRNA analysis, respectively. The primers for miRNAs and mRNAs are listed in Supplementary Table S2. Data were expressed as fold changes relative to GAPDH or U6 and calculated based on the following formula: RQ = 2−ΔΔCt.
Cell culture
Luciferase reporter assay
HEK293T, SPC-A-1, and 95D cell lines were purchased from Shanghai Cell Bank of Chinese Academy of Science (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Gaithersburg, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.
The pmirGLO vectors containing wild type or mutant putative miR-125a-3p binding site in human MTA1 3′-UTR were synthesized by GenePharma (Shanghai, China). HEK293T or SPC-A-1 cells were seeded into 24-well plates the day before transfection and then cotransfected with 50 ng of wild-type or mutant-type luciferase vector and 20 μM miR-125a-3p mimic or negative control. After 48 h,
Materials and Methods Tissue sample
Downloaded from http://abbs.oxfordjournals.org/ at Florida International University on May 28, 2015
carcinomas, including NSCLC, colorectal carcinoma, hepatocellular carcinoma (HCC), and breast cancer [5–9]. In the past decades, MTA1 has been confirmed to be a critical component of the nucleosome remodeling and histone deacetylase (NuRD) complex. It is a key regulator of the DNA damage response, and is involved in epithelial-mesenchymal transition (EMT) and inflammation in both transcription-dependent and -independent manners [10]. However, the mechanism of MTA1 deregulation remains to be fully elucidated in order to achieve MTA1-targeted therapies. MicroRNAs (miRNAs) are a category of small non-coding RNA molecules with 18–23 nucleotides. miRNAs affect gene expression through interaction between their seed region and the 3′- or 5′-untranslated region (UTR) of the target mRNA [11], which leads to mRNA cleavage, translational repression or activation, and the formation of heterochromatin [12]. Numerous studies have shown that different miRNAs in cancer might function either as activators or suppressors of tumors through regulation of different targets. For instance, the miR-200 family was shown to abrogate the ability of cancer cells to undergo EMT [13]. Through modulation of the PTEN signaling pathway, up-regulation of miR-21 in NSCLC might promote the invasion and migration ability of cancer cells [14]. However, the relationship between MTA1 signaling and miRNA remains largely undetermined. Here, the regulation of MTA1 expression by tumor suppressive miRNAs was characterized by using bioinformatics analysis. Based on two commonly used algorithms, Targetscan and microrna.org, complete complementary sequences to three miRNAs (miR-199a/b-5p and miR-125a-3p) were found in the 3′-UTR of MTA1. These three candidate miRNAs have been reported to be anti-oncogenically involved in carcinogenesis, invasion, and metastasis [9,15–19]. In this study, miR-125a-3p was found to be a regulator of MTA1 expression through interaction with its 3′-UTR. The miR-125a-3pinduced suppression of the motility and proliferation of NSCLC cells was completely or partially mediated by the silencing of MTA1 expression. Furthermore, MTA1 protein level was found to be inversely correlated with miR-125a-3p expression in NSCLC tissues.
miR-125a-3p targets MTA1 in NSCLC
3
miR-125a-3p targets MTA1 in NSCLC luciferase activity was assayed by using the Dual-luciferase Reporter Assay System (Promega, Madison, USA).
Cell proliferation assay
Statistical analysis The data were expressed as the mean ± SD of three independent experiments. All statistical analyses were conducted using SPSS 14.0 statistics software package (SPSS Inc., Chicago, USA). A P-value of
Original Article
miR-125a-3p targets MTA1 to suppress NSCLC cell proliferation, migration, and invasion Hong Zhang1,†, Xiaoxia Zhu1,†, *, Na Li1, Dianhe Li1, Zhou Sha1, Xiaokang Zheng1,*, and Haofei Wang2,* Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China, and Department of Cardiothoracic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
2 †
These authors contributed equally to this work. *Correspondence address. Tel: +86-20-62787484; Fax: +86-20-61641034; E-mail: [email protected] (X.X.Z.)/zkn1268@fimmu. com (X.K.Z.)/[email protected] (H.F.W.)
Received 18 January 2015; Accepted 13 March 2015
Abstract Metastasis-associated gene 1 (MTA1) is associated with cell growth, metastasis, and survival in nonsmall-cell lung cancer (NSCLC). Several previous reports have demonstrated that microRNAs affect gene expression through interaction between their seed region and the 3′-untranslated region of the target mRNA, resulting in post-transcriptional regulation. The aim of this study was to identify miRNAs that suppress malignancy in NSCLC cells by targeting MTA1. Two human NSCLC cell lines were analyzed for the expression of MTA1 by quantitative RT-PCR and western blotting after transfection with MTA1 mimics. A luciferase reporter assay was established to test the direct connection between MTA1 and its upstream miRNAs. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay, 5-ethynyl-2′-deoxyuridine analysis, and colony formation assay. Cell migration and invasive capacity were evaluated by wound-healing assay and transwell assay. The miRNA/MTA1 axis was also probed by quantitative RT-PCR and western blotting in samples from eight NSCLC patients. Among the candidate miRNAs, miR-125a-3p was shown to posttranscriptionally regulate MTA1 in NSCLC cells. These data were reinforced by the luciferase reporter assay, in addition to the demonstration that MTA1 is inversely correlated with miR-125a-3p in NSCLC tissues. Furthermore, miR-125a-3p was found to inhibit NSCLC cell proliferation, migration, and invasion, through the same mechanisms of down-regulated MTA1. Our report demonstrates that miR-125a-3p inhibits the proliferation, migration, and invasion of NSCLC cells through down-regulation of MTA1, indicating the role of the miR-125a-3p/MTA1 axis in NSCLC, and may provide novel insight into the molecular mechanisms underpinning the disease and potential therapeutic targets. Key words: miR-125a-3p, metastasis-associated gene 1 (MTA1), non-small-cell lung cancer, proliferation, invasion
Introduction Lung cancer is the leading cause of cancer mortality worldwide [1], and 80%–85% of lung cancers are non-small-cell lung cancer (NSCLC) [2]. Approximately 56% of lung cancers are diagnosed at a distant stage, while only 15% of patients are diagnosed at a local stage [3]. The overall 5-year survival rate for NSCLC is as low as 17.1% [3]. Metastatic disease seriously impacts the treatment
outcome and overall survival rate in NSCLC patients. Further insight into the molecular mechanisms underpinning metastasis is significantly important and may facilitate the identification of new molecular targets for the treatment of the disease. Metastasis-associated gene 1 (MTA1) was discovered by screening a cDNA library from rat metastatic breast tumors [4]. It is one of the metastasis-related genes which is over-expressed in numerous
© The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 1
Downloaded from http://abbs.oxfordjournals.org/ at Florida International University on May 28, 2015
1
2
miRNA mimics and siRNAs miRNA mimics, small interfering RNA (siRNA) against MTA1 (si-MTA1), and their negative controls were synthesized by GenePharma (Shanghai, China). The sequences were as follows: miR-199a-5p mimic: 5′-CCCAGUGUUCAGACUACCUGUUC-3′; miR-199b-5p mimic: 5′-CCCAGUGUUUAGACUAUCUGUUC-3′; miR-125a-3p mimic: 5′-ACAGGUGAGGUUCUUGGGAGCC-3′; scrambled mimic: 5′-UUCUCCGAACGUGUCACGUTT-3′ (negative control for miRNA mimics); si-MTA1: 5′-CCAGCAUCAUUGAGUACUATT-3′; and scrambled siRNA: 5′-GCGACGAUCUGCCUAAGAUdTdT-3′ (negative control for si-MTA1).
Transfection Cells were seeded in 6-well plates and cultured for 18–24 h. Then, cells were transfected with miRNA mimics or siRNAs (100 μM) using Lipofectamine 3000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Co-transfection of miR-125a-3p mimics and si-MTA1 in SPC-A-1 and 95D cells were performed according to the similar procedures. At 48 h post-transfection, the cells were harvested for further analysis.
Western blot analysis Proteins were extracted from whole cell lysates and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride membranes (Merck Millpore, Milford, USA). The following primary antibodies were used: mouse anti-MTA1 (1:1500; Abcam, Cambridge, USA) and mouse anti-β-actin (1:5000; Sigma, St Louis, USA). Membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz, Santa Cruz, USA). Luminata™ Chemiluminescent Detection Substrates (Merck Millpore) were used to visualize the immunoreactive bands, and the density of each band was analyzed using ImageJ software.
RNA preparation and quantitative real-time PCR analysis
Eight pairs of primary NSCLC tissues and their corresponding normal lung tissue samples were obtained from three squamous cell lung carcinoma patients (one highly differentiated and two moderately differentiated) and five lung adenocarcinoma patients (one highly differentiated, two moderately differentiated, and two poorly differentiated). Consent forms were signed by all patients. The procedures were approved by the Clinical Research Ethics Committee of Nanfang Hospital. None of the patients had been treated with radiotherapy or chemotherapy prior to surgery. All samples were immediately placed in liquid nitrogen and stored at −80°C. Both normal and tumor tissues were verified by histological analysis.
Total RNAs were isolated from cells or tissues using Trizol reagent (Invitrogen). The TaqMan Reverse Transcription Kit (Takara, Dalian, China) was used to obtain cDNA for mRNA detection, whereas TaqMan MicroRNA Reverse Transcription Kit (Takara) was applied to reverse transcribe RNA for miRNA detection. For miRNA and mRNA, real-time PCR was performed using miRscript SYBR Green PCR Kit and SYBR Green PCR Kit (Takara), respectively, on a Roche LightCycler 480 platform (Roche, Basel, Switzerland). The PCR steps included enzyme activation for 10 min at 95°C followed by 40 cycles of 95°C for 15 s and annealing at 60°C for 60 s, according to manufacturer’s protocol. U6 and GAPDH were used as internal controls for miRNA and mRNA analysis, respectively. The primers for miRNAs and mRNAs are listed in Supplementary Table S2. Data were expressed as fold changes relative to GAPDH or U6 and calculated based on the following formula: RQ = 2−ΔΔCt.
Cell culture
Luciferase reporter assay
HEK293T, SPC-A-1, and 95D cell lines were purchased from Shanghai Cell Bank of Chinese Academy of Science (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Gaithersburg, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.
The pmirGLO vectors containing wild type or mutant putative miR-125a-3p binding site in human MTA1 3′-UTR were synthesized by GenePharma (Shanghai, China). HEK293T or SPC-A-1 cells were seeded into 24-well plates the day before transfection and then cotransfected with 50 ng of wild-type or mutant-type luciferase vector and 20 μM miR-125a-3p mimic or negative control. After 48 h,
Materials and Methods Tissue sample
Downloaded from http://abbs.oxfordjournals.org/ at Florida International University on May 28, 2015
carcinomas, including NSCLC, colorectal carcinoma, hepatocellular carcinoma (HCC), and breast cancer [5–9]. In the past decades, MTA1 has been confirmed to be a critical component of the nucleosome remodeling and histone deacetylase (NuRD) complex. It is a key regulator of the DNA damage response, and is involved in epithelial-mesenchymal transition (EMT) and inflammation in both transcription-dependent and -independent manners [10]. However, the mechanism of MTA1 deregulation remains to be fully elucidated in order to achieve MTA1-targeted therapies. MicroRNAs (miRNAs) are a category of small non-coding RNA molecules with 18–23 nucleotides. miRNAs affect gene expression through interaction between their seed region and the 3′- or 5′-untranslated region (UTR) of the target mRNA [11], which leads to mRNA cleavage, translational repression or activation, and the formation of heterochromatin [12]. Numerous studies have shown that different miRNAs in cancer might function either as activators or suppressors of tumors through regulation of different targets. For instance, the miR-200 family was shown to abrogate the ability of cancer cells to undergo EMT [13]. Through modulation of the PTEN signaling pathway, up-regulation of miR-21 in NSCLC might promote the invasion and migration ability of cancer cells [14]. However, the relationship between MTA1 signaling and miRNA remains largely undetermined. Here, the regulation of MTA1 expression by tumor suppressive miRNAs was characterized by using bioinformatics analysis. Based on two commonly used algorithms, Targetscan and microrna.org, complete complementary sequences to three miRNAs (miR-199a/b-5p and miR-125a-3p) were found in the 3′-UTR of MTA1. These three candidate miRNAs have been reported to be anti-oncogenically involved in carcinogenesis, invasion, and metastasis [9,15–19]. In this study, miR-125a-3p was found to be a regulator of MTA1 expression through interaction with its 3′-UTR. The miR-125a-3pinduced suppression of the motility and proliferation of NSCLC cells was completely or partially mediated by the silencing of MTA1 expression. Furthermore, MTA1 protein level was found to be inversely correlated with miR-125a-3p expression in NSCLC tissues.
miR-125a-3p targets MTA1 in NSCLC
3
miR-125a-3p targets MTA1 in NSCLC luciferase activity was assayed by using the Dual-luciferase Reporter Assay System (Promega, Madison, USA).
Cell proliferation assay
Statistical analysis The data were expressed as the mean ± SD of three independent experiments. All statistical analyses were conducted using SPSS 14.0 statistics software package (SPSS Inc., Chicago, USA). A P-value of
