Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
MicroRNA-26a/b Regulate DNA Replication Licensing, Tumorigenesis and Prognosis by Targeting CDC6 in Lung Cancer Xin Zhang, Dakai Xiao, Ziyi Wang, et al. Mol Cancer Res Published OnlineFirst August 6, 2014.
Updated version Supplementary Material Author Manuscript
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MicroRNA-26a/b
Regulate
DNA
Replication
Licensing,
Tumorigenesis and Prognosis by Targeting CDC6 in Lung Cancer
Xin Zhang1*, Dakai Xiao1*, Ziyi Wang2,3*, Yongxin Zou2,3, Liyan Huang1, Weixuan Lin1, Qiuhua Deng1, Hui Pan1, Jiangfen Zhou1, Chun Liang2,3# and Jianxing He1#
1
Department of Cardiothoracic Surgery, The First Affiliated Hospital of Guangzhou Medical
University, State Key Laboratory of Respiratory Disease, Guangzhou, China. 2
Guangzhou HKUST Fok Ying Tung Research Institute, Guangzhou, China
3
Division of Life Science and Center for Cancer Research, Hong Kong University of Science and
Technology, Hong Kong, China
* These authors contributed equally to this work. #
Corresponding authors; CL: [email protected]; JH: [email protected]
Running title: MiR-26a/b target CDC6 in Lung Cancer and Its Prognosis Conflict of interests: None
1 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
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Abstract Cancer is characterized by mutations, genome rearrangements, epigenetic changes and altered gene expression that enhance cell proliferation, invasion and metastasis. To accommodate deregulated cellular proliferation, many DNA replication-initiation proteins are over-expressed in human cancers. However, the mechanism that represses the expression of these proteins in normal cells and the cellular changes that result in their over-expression is largely unknown. One possible mechanism is through microRNA (miR) expression differences. Here it is demonstrated that miR-26a and miR-26b inhibit replication licensing and the proliferation, migration and invasion of lung cancer cells by targeting CDC6. Importantly, miR-26a/b expression is significantly decreased in human lung cancer tissue specimens compared to the paired adjacent normal tissues, and that miR-26a/b down-regulation and the consequential up-regulation of CDC6 are associated with poorer prognosis of lung cancer patients. These results indicate that miR-26a/b repress replication licensing and tumorigenesis by targeting CDC6.
Implications: The current study suggests that miR-26a, miR-26b and CDC6 and factors regulating their expression represent potential cancer diagnostic and prognostic markers as well as anticancer targets.
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Introduction As a prerequisite for cell proliferation, genome duplication must be carried out under strict cell cycle control in normal cells. On the other hand, deregulation of DNA replication in cancer cells may be needed to accommodate enhanced and uncontrolled cell proliferation. One of the most prominent abnormalities in the regulation of DNA replication in cancer cells is the over-expression of several DNA replication-initiation proteins such as CDC6 and MCM proteins (1-7).
CDC6 is a key factor for loading the helicase MCM (minichromosome maintenance) proteins onto replication origins for the assembly of pre-replicative complex (pre-RCs) at the M-to-G1 phase transition to establish replication licensing (reviewed in (8-11). These and some other related replication-initiation proteins are required for DNA replication, and their subsequent removal or inactivation before and/or during DNA replication helps to prevent re-replication within the same cell cycle. Importantly, these proteins are expressed at lower levels in proliferating normal cells compared to cancer cells and are not even expressed in non-proliferating normal cells (2-7). Therefore, regulation of these proteins is not only important for ensuring faithful genetic inheritance in normal cell cycle progression, but also critical for preventing cancer. However, although it is known that CDC6 is positively regulated by the E2F transcriptional activators (12-14), the mechanisms that repress the expression of CDC6 in normal cells and the cellular changes that lead to the over-expression of CDC6 in cancer cells are not clear.
3 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
As one of the important cellular mechanisms that regulate gene expression, microRNAs (miRNAs) bind to the partially complementary sequences at the 3’-untranslated regions (3’-UTR) of the mRNAs of target genes, resulting in the degradation and/or blocking of translation of the mRNAs (15). However, no miRNA has been reported to target CDC6. In this study, we identified a target sequence of microRNA-26a (miR-26a) and -26b in the 3’-UTR of CDC6 and found that miR-26a and –26b indeed can suppress CDC6 gene expression.
Growing evidence indicates that miR-26 is down-regulated in human cancers, including nasopharyngeal carcinoma (16), breast cancer (17, 18), hepatocellular carcinoma (19), parathyroid tumors (20), rhabdomyosarcoma (21), and non-small cell lung cancer (22). In addition, ectopic expression miR-26b leads to apoptosis in cancer cells by targeting pRb5 (23) and PTGS2 (24). However, the roles of miR-26 in repressing DNA replication proteins and in lung cancer Tumorigenesis and prognosis are not clear.
In this report, we show that miR-26a/b directly suppress CDC6 expression and thus inhibiting the proliferation, migration and invasion of lung cancer cells. More importantly, we show that miR-26a/b expression is significantly decreased in human lung cancer tissues compared to the paired adjacent normal tissues, and that miR-26a/b down-regulation and the consequential up-regulation of CDC6 are associated with poorer prognosis of lung cancer patients. These results indicate that miR-26a/b control replication licensing by targeting CDC6 in normal cells, 4 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
and their down-regulation may be involved in the initiation and development of human lung cancer.
Materials and methods Cell culture, plasmids and chemicals Human lung cancer cells A549, H1299, H460 were cultured in DMEM medium supplemented with 10% FBS and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). All cells were maintained at 37°C in a humidified incubator with 5% CO2. CDC6 expression constructs were cloned in pEGFP-C3 vector (Clontech). Synthetic miR-26a, -26b and CDC6 siRNA were from Genepharm (Shanghai, China).
To construct the sense reporter plasmid, the 3’-UTR of CDC6 containing the predicted miR-26a/b binding site was amplified by PCR in a total volume of 25 l. The primers used were 5’-CCGCTCGAGATTCTTCTCTTACACCCCACCCG-3’
(sense)
5’-ATAAGAATGCGGCCGCCCATTTAATGCCACCAAACCA-3’
and
(antisense).
The
PCR
product was digested by Xho I and Not I and then inserted into psi-CHECK2 (Promega) which was digested by the same restriction enzymes. The mutant 3’-UTR construct was generated by site-directed
mutagenesis
with
the
following
mutagenic
primers:
AAAACAAATATGACCTTTTTATGAACTTGCCAATGAATTTTAATCTAT-3' mutagenic
sites
were
denoted
as
bold),
(sense;
5'-
the and
5'-ATAGATTAAAATTCATTGGCAAGTTCATAAAAAGGTCATATTTGTTTT-3' (anti-sense).
5 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Transient transfection Cells were plated in 6-well plates (1.5×105 cells/well) or 96-well plates (3x103 cells/well) one day before transfection. Cells were transfected with miRNA or siRNA in Opti-MEM medium using Lipofetamine RNAiMax transfection reagent (Invitrogen) according to the manufacturer’s protocol. After 6 hrs, the medium was replaced by fresh growth medium. The next day, the cells were transfected for the second time. Transfected cells were then analyzed after 48 hrs. Cells were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Cell synchronization and release Transfected or untreated cells were arrested at the G1/S phase boundary by treatment with 0.5 mM mimosine (Sigma) for 20 hrs. To release the cell into the cell cycle, the cells were washed three times with the growth medium and then cultured in fresh growth medium. Cell synchronization was confirmed by flow cytometry.
RNA extraction and quantitative reverse transcription real-time PCR (qRT-PCR) Total RNAs of lung cancer cells and lung cancer specimens were extracted using TRIzol reagent (Invitrogen). The quality and quantity of RNA were determined by agarose gel electrophoresis and photospectrometry. One μg of RNA was used for cDNA synthesis using the Reverse Transcription System (Promega) according to the manufacturer’s instructions. Real time-PCR 6 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
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was performed using GoTaq qPCR Master Mix (Promega). Thermal cycling conditions were as follow: 1 step for 10 min at 95°C followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. The quality of the PCR products was monitored using post-PCR melting curve analysis. β-Actin was used as the internal control. Relative mRNA levels were calculated using 2-ΔΔCt (ΔCt = CT-CA), where ΔCt represents the mean threshold cycle (Ct) differences between the test gene (CT) and that of the β-actin (CA) values. The primer sequences used were as follows: CDC6, 5’-AACCACTGTCTGAATGTAAATC-3’ (sense) and 5’-CTGTTACCATCAACTTCTGAG-3’ (anti-sense); β-Actin, 5’-AGAAGAGCTACGAGCTGCCTGACG-3’ (sense) and 5’-GGACTCCATGCCCAGGAAGGAA-3’ (anti-sense).
Immunoblotting Cells were harvested, and total cellular proteins were extracted by radioimmuno-precipitation assay (RIPA) lysis buffer containing protease inhibitors. Protein concentrations were measured using Bradford assay (Bio-Rad), with bovine serum albumin as the standard. Equal amounts of protein extracts from all samples were applied to SDS-PAGE and then transferred to PVDF membrane (Millipore). The membrane was blocked in TBST buffer containing 5% non-fat milk and 0.1% Tween-20 for 1 hr at room temperature followed by incubation with the primary antibody at 4°C overnight. GAPDH (Anti-GAPDH from Sigma) was used as the loading control. After being washed, membranes were incubated with the corresponding second antibodies and washed again. The signals were visualized by the enhanced chemiluminescence system (Bio-Rad) according to the manufacturer’s instructions. 7 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
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Luciferase sense reporter assay 293T cells (40% confluent) were co-transfected with psi-CHECK2/CDC6 3’-UTR or psiCHECK-2/CDC6 3’-UTR mutant reporter plasmid (200 ng) together with miR-26a, -26b mimics or control RNA (40 nM) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. A day later, the cells were transfected again with miR-26a, -26b mimics or control RNA (40 nM). After 48 hrs, cells were lysed, and the reporter activity was evaluated using the Dual Glo-Luciferase Assay System (Promega) according to the manufacturer’s instructions. The Renilla luciferase activity was normalised by the firefly luciferase activity.
EdU incorporation assay EdU (5-ethynyl-2′-deoxyuridine) incorporation was determined by using the Cell-Light™ EdU DNA Cell Proliferation Kit (Ribobio) according to the manufacturer’s instructions. In briefly, after G1 phase synchronization with mimosine, H1299 cells were released into fresh medium and cultured for 4 hrs with the presence of 50 μM EdU in the final 30 min. Cells were then fixed, permeabilized and stained for EdU. The nuclei were stained with Hoechst 33342 for 15 min. The proportion of the cells incorporated EdU was determined by fluorescence microscopy
Chromatin binding assay Cells synchronized with mimosine were harvested by trypsinization and washed twice with cold 8 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
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PBS. A volume (20 l/106 cells) of Extraction Buffer (EB; 100 mM KCl, 50 mM HEPES-KOH, pH7.5, 2.5 mM MgCl2, 50 mM NaF, 5 mM Na4P2O7, 0.1 mM NaVO3, 0.5% Triton X-100, 1 mM PMSF, 2 μg/ml Pepstatin A, 20 μg/ml Leupeptin, 20 μg/ml Aprotinin, 0.2 mM Pefabloc, 2 mM Benzamidine HCl and 0.2 mg/ml Bacitracin) was added to resuspend and lyse the cells with pippetting. The lysate was set on ice for 10 min, with mixing every 2-3 min during the incubation. An equal volume of 30% ice-cold sucrose containing the same protease inhibitors as in EB was added to the bottom of tube to underlay the cell lysate. The tube was then spun in a microcentrifuge at top speed for 10 min at 4oC to separate the chromatin and free proteins. The supernatant was transferred to a new tube and kept on ice. The pellet was washed with an equal volume of EB and spun again at top speed for 5 min. The two supernatants were combined as the soluble proteins. The pellet (chromatin fraction) was resuspended in EB of one half volume of the supernatant.
Flow cytometry Cells were collected by trypsinization and washed once with PBS. Cells were fixed in 70% ethanol for 1 hr to overnight at -20°C, washed thoroughly with ice-cold PBS, and then stained in propidium iodide solution (50 μg/ml RNase A, 0.1% Triton X-100, 0.1 mM EDTA and 50 μg/ml propidium iodide) for at least 30 min at 4°C. Samples were analyzed with a FACSort instrument (Becton Dickinson).
Cell proliferation assay 9 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
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Cell proliferation was determined by CCK-8 assay. Cells were seeded in 96-well plates (3×103 cells/well). After transfected with miRNA or siRNA, cells were incubated with CCK-8 (0.5 mg/ml) for 1 hr, and the absorbance was determined at 450 nm with Multiskan FC microtitter plate reader (Thermo scientific).
In vitro clonogenic assay Some 48 hrs post-transfection with miR-26a, -26b or CDC6 siRNA, cells were trypsinized and seeded in 6-well plates. After 10 days, the cells were fixed with methanol/acetic acid (3:1) for 15 min and stained with 0.5% crystal violet in methanol for 15 min. After staining, colonies were visually counted.
In vitro invasion and wound healing (migration) assays For in vitro invasion assay, 5×104 cells transfected with miR-26a, -26b or CDC6 siRNA were plated into the upper membrane of the Transwell inserts (8 μm) coated with Matrigel (BD Biosciences). After 24 hrs, the cells on the upper membrane were swiped off with cotton swabs, and the cells invaded into the lower membrane were stained with Wright and Giemsa Stain. The cells were photographed under a microscope (Nikon DS-Ril) and the cell numbers were counted using the ImageJ software.
For wound healing assay, cells were transfected with miR-26a, -26b or siRNA and cultured until confluent. The monolayer of the cells was then wounded by a tip for the 200-μl pipette, and the 10 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
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cells were washed with PBS three times and replaced with fresh growth medium. The wounded area was monitored every 24 hrs under a microscope (Leica DFC450) and the degree of wound healing by cell migration was quantified using Image-Pro Plus software (Media Cybernetics).
Lentivirus-based vector construct and transduction To construct the lentivirus-based CDC overexpression plasmid, total RNA extracted from lung tissue was used for cDNA synthesis. The full length of CDC6 with Myc-tag was amplified and cloned into the NheI and NotI sites of pCDH-CMV-MCS-EF1-puro (System Biosciences, USA) . The sequences were verified by Sanger sequencing. To generate lentiviral particles, CDC6 expression plasmid was co-transfected with psPAX2 and pMD2.G into 293T cells using calcium phosphate. The lentiviral particles were then used to transduce A549 cells in the growth medium containing 8 μg/ml polybrene. The expression of was confirmed by immunoblotting with the anti-myc tag antibody.
Patients and clinical specimens The tumor specimens and the adjacent normal tissues were obtained from 128 patients with NSCLC who underwent surgical resection in Department of Thoracic Surgery, The First Affiliated Hospital of Guangzhou Medical University, China, between 2007 and 2009. None of the patients received any anticancer therapies before surgery. Following resection, the tumor specimens were evaluated by pathologists to determine the tumor cellularity and TNM stage according to the Union International Contre le Cancer (UICC-7) staging system for lung cancer. 11 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
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The tumor tissues containing at least 50% of tumor cells were included in this study. The tumor specimens and the adjacent normal tissues were frozen in liquid nitrogen in tissue bank of our hospital. This study was approved by the Institutional Review Board of the First Affiliated Hospital, Guangzhou Medical University.
Statistical analysis The correlations of miR-26a/b and CDC6 mRNA expression with clinicopathological characteristics were determined by Pearson’s chi-square test. Student’s t test was used to compare the expression of miR-26a, -26b and CDC6 mRNA in normal and tumor specimens. The correlation of the expression of miR-26a/b and CDC6 mRNA was determined by Spearman’s correlation. The overall survival of the patients was analyzed by Kaplan-meier curve with log-rank test. The prognostic significance of the expression of miR-26a/b and CDC6 was evaluated by Cox regression model with forward LR method. p
MicroRNA-26a/b Regulate DNA Replication Licensing, Tumorigenesis and Prognosis by Targeting CDC6 in Lung Cancer Xin Zhang, Dakai Xiao, Ziyi Wang, et al. Mol Cancer Res Published OnlineFirst August 6, 2014.
Updated version Supplementary Material Author Manuscript
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MicroRNA-26a/b
Regulate
DNA
Replication
Licensing,
Tumorigenesis and Prognosis by Targeting CDC6 in Lung Cancer
Xin Zhang1*, Dakai Xiao1*, Ziyi Wang2,3*, Yongxin Zou2,3, Liyan Huang1, Weixuan Lin1, Qiuhua Deng1, Hui Pan1, Jiangfen Zhou1, Chun Liang2,3# and Jianxing He1#
1
Department of Cardiothoracic Surgery, The First Affiliated Hospital of Guangzhou Medical
University, State Key Laboratory of Respiratory Disease, Guangzhou, China. 2
Guangzhou HKUST Fok Ying Tung Research Institute, Guangzhou, China
3
Division of Life Science and Center for Cancer Research, Hong Kong University of Science and
Technology, Hong Kong, China
* These authors contributed equally to this work. #
Corresponding authors; CL: [email protected]; JH: [email protected]
Running title: MiR-26a/b target CDC6 in Lung Cancer and Its Prognosis Conflict of interests: None
1 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Abstract Cancer is characterized by mutations, genome rearrangements, epigenetic changes and altered gene expression that enhance cell proliferation, invasion and metastasis. To accommodate deregulated cellular proliferation, many DNA replication-initiation proteins are over-expressed in human cancers. However, the mechanism that represses the expression of these proteins in normal cells and the cellular changes that result in their over-expression is largely unknown. One possible mechanism is through microRNA (miR) expression differences. Here it is demonstrated that miR-26a and miR-26b inhibit replication licensing and the proliferation, migration and invasion of lung cancer cells by targeting CDC6. Importantly, miR-26a/b expression is significantly decreased in human lung cancer tissue specimens compared to the paired adjacent normal tissues, and that miR-26a/b down-regulation and the consequential up-regulation of CDC6 are associated with poorer prognosis of lung cancer patients. These results indicate that miR-26a/b repress replication licensing and tumorigenesis by targeting CDC6.
Implications: The current study suggests that miR-26a, miR-26b and CDC6 and factors regulating their expression represent potential cancer diagnostic and prognostic markers as well as anticancer targets.
2 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Introduction As a prerequisite for cell proliferation, genome duplication must be carried out under strict cell cycle control in normal cells. On the other hand, deregulation of DNA replication in cancer cells may be needed to accommodate enhanced and uncontrolled cell proliferation. One of the most prominent abnormalities in the regulation of DNA replication in cancer cells is the over-expression of several DNA replication-initiation proteins such as CDC6 and MCM proteins (1-7).
CDC6 is a key factor for loading the helicase MCM (minichromosome maintenance) proteins onto replication origins for the assembly of pre-replicative complex (pre-RCs) at the M-to-G1 phase transition to establish replication licensing (reviewed in (8-11). These and some other related replication-initiation proteins are required for DNA replication, and their subsequent removal or inactivation before and/or during DNA replication helps to prevent re-replication within the same cell cycle. Importantly, these proteins are expressed at lower levels in proliferating normal cells compared to cancer cells and are not even expressed in non-proliferating normal cells (2-7). Therefore, regulation of these proteins is not only important for ensuring faithful genetic inheritance in normal cell cycle progression, but also critical for preventing cancer. However, although it is known that CDC6 is positively regulated by the E2F transcriptional activators (12-14), the mechanisms that repress the expression of CDC6 in normal cells and the cellular changes that lead to the over-expression of CDC6 in cancer cells are not clear.
3 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
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As one of the important cellular mechanisms that regulate gene expression, microRNAs (miRNAs) bind to the partially complementary sequences at the 3’-untranslated regions (3’-UTR) of the mRNAs of target genes, resulting in the degradation and/or blocking of translation of the mRNAs (15). However, no miRNA has been reported to target CDC6. In this study, we identified a target sequence of microRNA-26a (miR-26a) and -26b in the 3’-UTR of CDC6 and found that miR-26a and –26b indeed can suppress CDC6 gene expression.
Growing evidence indicates that miR-26 is down-regulated in human cancers, including nasopharyngeal carcinoma (16), breast cancer (17, 18), hepatocellular carcinoma (19), parathyroid tumors (20), rhabdomyosarcoma (21), and non-small cell lung cancer (22). In addition, ectopic expression miR-26b leads to apoptosis in cancer cells by targeting pRb5 (23) and PTGS2 (24). However, the roles of miR-26 in repressing DNA replication proteins and in lung cancer Tumorigenesis and prognosis are not clear.
In this report, we show that miR-26a/b directly suppress CDC6 expression and thus inhibiting the proliferation, migration and invasion of lung cancer cells. More importantly, we show that miR-26a/b expression is significantly decreased in human lung cancer tissues compared to the paired adjacent normal tissues, and that miR-26a/b down-regulation and the consequential up-regulation of CDC6 are associated with poorer prognosis of lung cancer patients. These results indicate that miR-26a/b control replication licensing by targeting CDC6 in normal cells, 4 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
and their down-regulation may be involved in the initiation and development of human lung cancer.
Materials and methods Cell culture, plasmids and chemicals Human lung cancer cells A549, H1299, H460 were cultured in DMEM medium supplemented with 10% FBS and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). All cells were maintained at 37°C in a humidified incubator with 5% CO2. CDC6 expression constructs were cloned in pEGFP-C3 vector (Clontech). Synthetic miR-26a, -26b and CDC6 siRNA were from Genepharm (Shanghai, China).
To construct the sense reporter plasmid, the 3’-UTR of CDC6 containing the predicted miR-26a/b binding site was amplified by PCR in a total volume of 25 l. The primers used were 5’-CCGCTCGAGATTCTTCTCTTACACCCCACCCG-3’
(sense)
5’-ATAAGAATGCGGCCGCCCATTTAATGCCACCAAACCA-3’
and
(antisense).
The
PCR
product was digested by Xho I and Not I and then inserted into psi-CHECK2 (Promega) which was digested by the same restriction enzymes. The mutant 3’-UTR construct was generated by site-directed
mutagenesis
with
the
following
mutagenic
primers:
AAAACAAATATGACCTTTTTATGAACTTGCCAATGAATTTTAATCTAT-3' mutagenic
sites
were
denoted
as
bold),
(sense;
5'-
the and
5'-ATAGATTAAAATTCATTGGCAAGTTCATAAAAAGGTCATATTTGTTTT-3' (anti-sense).
5 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Transient transfection Cells were plated in 6-well plates (1.5×105 cells/well) or 96-well plates (3x103 cells/well) one day before transfection. Cells were transfected with miRNA or siRNA in Opti-MEM medium using Lipofetamine RNAiMax transfection reagent (Invitrogen) according to the manufacturer’s protocol. After 6 hrs, the medium was replaced by fresh growth medium. The next day, the cells were transfected for the second time. Transfected cells were then analyzed after 48 hrs. Cells were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Cell synchronization and release Transfected or untreated cells were arrested at the G1/S phase boundary by treatment with 0.5 mM mimosine (Sigma) for 20 hrs. To release the cell into the cell cycle, the cells were washed three times with the growth medium and then cultured in fresh growth medium. Cell synchronization was confirmed by flow cytometry.
RNA extraction and quantitative reverse transcription real-time PCR (qRT-PCR) Total RNAs of lung cancer cells and lung cancer specimens were extracted using TRIzol reagent (Invitrogen). The quality and quantity of RNA were determined by agarose gel electrophoresis and photospectrometry. One μg of RNA was used for cDNA synthesis using the Reverse Transcription System (Promega) according to the manufacturer’s instructions. Real time-PCR 6 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
was performed using GoTaq qPCR Master Mix (Promega). Thermal cycling conditions were as follow: 1 step for 10 min at 95°C followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. The quality of the PCR products was monitored using post-PCR melting curve analysis. β-Actin was used as the internal control. Relative mRNA levels were calculated using 2-ΔΔCt (ΔCt = CT-CA), where ΔCt represents the mean threshold cycle (Ct) differences between the test gene (CT) and that of the β-actin (CA) values. The primer sequences used were as follows: CDC6, 5’-AACCACTGTCTGAATGTAAATC-3’ (sense) and 5’-CTGTTACCATCAACTTCTGAG-3’ (anti-sense); β-Actin, 5’-AGAAGAGCTACGAGCTGCCTGACG-3’ (sense) and 5’-GGACTCCATGCCCAGGAAGGAA-3’ (anti-sense).
Immunoblotting Cells were harvested, and total cellular proteins were extracted by radioimmuno-precipitation assay (RIPA) lysis buffer containing protease inhibitors. Protein concentrations were measured using Bradford assay (Bio-Rad), with bovine serum albumin as the standard. Equal amounts of protein extracts from all samples were applied to SDS-PAGE and then transferred to PVDF membrane (Millipore). The membrane was blocked in TBST buffer containing 5% non-fat milk and 0.1% Tween-20 for 1 hr at room temperature followed by incubation with the primary antibody at 4°C overnight. GAPDH (Anti-GAPDH from Sigma) was used as the loading control. After being washed, membranes were incubated with the corresponding second antibodies and washed again. The signals were visualized by the enhanced chemiluminescence system (Bio-Rad) according to the manufacturer’s instructions. 7 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Luciferase sense reporter assay 293T cells (40% confluent) were co-transfected with psi-CHECK2/CDC6 3’-UTR or psiCHECK-2/CDC6 3’-UTR mutant reporter plasmid (200 ng) together with miR-26a, -26b mimics or control RNA (40 nM) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. A day later, the cells were transfected again with miR-26a, -26b mimics or control RNA (40 nM). After 48 hrs, cells were lysed, and the reporter activity was evaluated using the Dual Glo-Luciferase Assay System (Promega) according to the manufacturer’s instructions. The Renilla luciferase activity was normalised by the firefly luciferase activity.
EdU incorporation assay EdU (5-ethynyl-2′-deoxyuridine) incorporation was determined by using the Cell-Light™ EdU DNA Cell Proliferation Kit (Ribobio) according to the manufacturer’s instructions. In briefly, after G1 phase synchronization with mimosine, H1299 cells were released into fresh medium and cultured for 4 hrs with the presence of 50 μM EdU in the final 30 min. Cells were then fixed, permeabilized and stained for EdU. The nuclei were stained with Hoechst 33342 for 15 min. The proportion of the cells incorporated EdU was determined by fluorescence microscopy
Chromatin binding assay Cells synchronized with mimosine were harvested by trypsinization and washed twice with cold 8 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
PBS. A volume (20 l/106 cells) of Extraction Buffer (EB; 100 mM KCl, 50 mM HEPES-KOH, pH7.5, 2.5 mM MgCl2, 50 mM NaF, 5 mM Na4P2O7, 0.1 mM NaVO3, 0.5% Triton X-100, 1 mM PMSF, 2 μg/ml Pepstatin A, 20 μg/ml Leupeptin, 20 μg/ml Aprotinin, 0.2 mM Pefabloc, 2 mM Benzamidine HCl and 0.2 mg/ml Bacitracin) was added to resuspend and lyse the cells with pippetting. The lysate was set on ice for 10 min, with mixing every 2-3 min during the incubation. An equal volume of 30% ice-cold sucrose containing the same protease inhibitors as in EB was added to the bottom of tube to underlay the cell lysate. The tube was then spun in a microcentrifuge at top speed for 10 min at 4oC to separate the chromatin and free proteins. The supernatant was transferred to a new tube and kept on ice. The pellet was washed with an equal volume of EB and spun again at top speed for 5 min. The two supernatants were combined as the soluble proteins. The pellet (chromatin fraction) was resuspended in EB of one half volume of the supernatant.
Flow cytometry Cells were collected by trypsinization and washed once with PBS. Cells were fixed in 70% ethanol for 1 hr to overnight at -20°C, washed thoroughly with ice-cold PBS, and then stained in propidium iodide solution (50 μg/ml RNase A, 0.1% Triton X-100, 0.1 mM EDTA and 50 μg/ml propidium iodide) for at least 30 min at 4°C. Samples were analyzed with a FACSort instrument (Becton Dickinson).
Cell proliferation assay 9 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Cell proliferation was determined by CCK-8 assay. Cells were seeded in 96-well plates (3×103 cells/well). After transfected with miRNA or siRNA, cells were incubated with CCK-8 (0.5 mg/ml) for 1 hr, and the absorbance was determined at 450 nm with Multiskan FC microtitter plate reader (Thermo scientific).
In vitro clonogenic assay Some 48 hrs post-transfection with miR-26a, -26b or CDC6 siRNA, cells were trypsinized and seeded in 6-well plates. After 10 days, the cells were fixed with methanol/acetic acid (3:1) for 15 min and stained with 0.5% crystal violet in methanol for 15 min. After staining, colonies were visually counted.
In vitro invasion and wound healing (migration) assays For in vitro invasion assay, 5×104 cells transfected with miR-26a, -26b or CDC6 siRNA were plated into the upper membrane of the Transwell inserts (8 μm) coated with Matrigel (BD Biosciences). After 24 hrs, the cells on the upper membrane were swiped off with cotton swabs, and the cells invaded into the lower membrane were stained with Wright and Giemsa Stain. The cells were photographed under a microscope (Nikon DS-Ril) and the cell numbers were counted using the ImageJ software.
For wound healing assay, cells were transfected with miR-26a, -26b or siRNA and cultured until confluent. The monolayer of the cells was then wounded by a tip for the 200-μl pipette, and the 10 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
cells were washed with PBS three times and replaced with fresh growth medium. The wounded area was monitored every 24 hrs under a microscope (Leica DFC450) and the degree of wound healing by cell migration was quantified using Image-Pro Plus software (Media Cybernetics).
Lentivirus-based vector construct and transduction To construct the lentivirus-based CDC overexpression plasmid, total RNA extracted from lung tissue was used for cDNA synthesis. The full length of CDC6 with Myc-tag was amplified and cloned into the NheI and NotI sites of pCDH-CMV-MCS-EF1-puro (System Biosciences, USA) . The sequences were verified by Sanger sequencing. To generate lentiviral particles, CDC6 expression plasmid was co-transfected with psPAX2 and pMD2.G into 293T cells using calcium phosphate. The lentiviral particles were then used to transduce A549 cells in the growth medium containing 8 μg/ml polybrene. The expression of was confirmed by immunoblotting with the anti-myc tag antibody.
Patients and clinical specimens The tumor specimens and the adjacent normal tissues were obtained from 128 patients with NSCLC who underwent surgical resection in Department of Thoracic Surgery, The First Affiliated Hospital of Guangzhou Medical University, China, between 2007 and 2009. None of the patients received any anticancer therapies before surgery. Following resection, the tumor specimens were evaluated by pathologists to determine the tumor cellularity and TNM stage according to the Union International Contre le Cancer (UICC-7) staging system for lung cancer. 11 Downloaded from mcr.aacrjournals.org on September 4, 2014. © 2014 American Association for Cancer Research.
Author Manuscript Published OnlineFirst on August 6, 2014; DOI: 10.1158/1541-7786.MCR-13-0641 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
The tumor tissues containing at least 50% of tumor cells were included in this study. The tumor specimens and the adjacent normal tissues were frozen in liquid nitrogen in tissue bank of our hospital. This study was approved by the Institutional Review Board of the First Affiliated Hospital, Guangzhou Medical University.
Statistical analysis The correlations of miR-26a/b and CDC6 mRNA expression with clinicopathological characteristics were determined by Pearson’s chi-square test. Student’s t test was used to compare the expression of miR-26a, -26b and CDC6 mRNA in normal and tumor specimens. The correlation of the expression of miR-26a/b and CDC6 mRNA was determined by Spearman’s correlation. The overall survival of the patients was analyzed by Kaplan-meier curve with log-rank test. The prognostic significance of the expression of miR-26a/b and CDC6 was evaluated by Cox regression model with forward LR method. p
