Oncology Research, Vol. 21, pp. 129–136 Printed in the USA. All rights reserved. Copyright Ó 2014 Cognizant Comm. Corp.
0965-0407/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096504013X13832473329999 E-ISSN 1555-3906 www.cognizantcommunication.com
MicroRNA-200c Inhibits Apoptosis in Pituitary Adenoma Cells by Targeting the PTEN/Akt Signaling Pathway Chuangxin Liao,* Wenli Chen,* Xiang Fan,* Xiaobing Jiang,* Lubing Qiu,* Chunhua Chen,† Yonghong Zhu,† and Haijun Wang* *Department of Neurosurgery and Pituitary Tumor Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China †Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
MicroRNAs (miRNAs) are important regulators that are involved in the development of different types of tumors. MicroRNA-200c (miR-200c) has been characterized as a tumor suppressor or oncogene in different cancers. However, the role of miR-200c in pituitary tumorigenesis remains unknown. We observed that miR200c was overexpressed in pituitary adenoma cell lines. We transfected a miR-200c inhibitor into pituitary adenoma cells (MMQ cell line) to inhibit miR-200c expression and found that the percentage of apoptotic MMQ cells increased. Using bioinformatics analyses, we predicted that the tumor suppressor gene PTEN was targeted by miR-200c, and we confirmed the presence of a functional miR-200c binding site in the 3¢-UTR of PTEN using luciferase reporter assays. We determined that the inhibition of miR-200c expression can upregulate PTEN expression and decrease the expression of phosphorylated Akt (p-Akt). Furthermore, the siRNAmediated knockdown of PTEN abrogated the effect of inhibiting miR-200c expression. Taken together, these findings suggest that miR-200c regulates pituitary tumor formation through the PTEN/Akt signaling pathway. Therefore, we propose that the inhibition of miR-200c could have therapeutic potential in pituitary adenoma. Key words: MicroRNA; Pituitary adenoma; PTEN/Akt signaling pathway; Apoptosis
INTRODUCTION Pituitary adenomas account for approximately 15% of all brain tumors. Although most pituitary adenomas are benign tumors, some are aggressive and can spread to neighboring tissue (1). In addition to the symptoms caused by compression, such as vision impairment and headache, pituitary adenomas can induce symptoms related to hormone parasecretion, such as amenorrhea, lactation, sex disorders, gigantism, and acromegaly (2). The molecular biological mechanisms underlying the development of pituitary adenoma remain unclear. MicroRNAs (miRNAs) are small noncoding RNAs that consist of 19–25 nucleotides. MiRNAs can bind to complementary sequences within the 3¢ untranslated regions (3¢-UTRs) of their target genes, inducing mRNA degradation or posttranslational repression (3). miRNAs not only are involved in the development of normal cells but also play a role in the pathogenesis of many human cancers (4). Increasing evidence has shown that many miRNAs are deregulated in pituitary adenoma. Our research team found 35 miRNAs that were abnormally expressed in pituitary adenoma using TaqMan miRNA array assays. Among the upregulated miRNAs, miR-200c is upregulated 3.5-fold (5).
An increasing number of studies have revealed that miR200c is overexpressed in cervical cancer and esophageal cancer, acting as an oncogene (6,7). However, miR-200c is downregulated in non-small cell lung cancer (NSCLC), in which it acts as a tumor suppressor gene (8,9). Therefore, the biological function and mechanism of miR-200c in pituitary adenoma warrant further investigation. In this study, we validated our previous miRNA array assay results by real-time PCR (qRT-PCR). We downregulated miR-200c expression in pituitary adenoma cells through transfection with a miR-200c inhibitor and found that apoptosis increased. Luciferase reporter assays confirmed that PTEN is a target gene of miR-200c and that the suppression of miR-200c expression upregulates PTEN protein expression while downregulating p-Akt expression. These results suggest that miR-200c is involved in the tumorigenesis of pituitary adenomas through the PTEN/ Akt signaling pathway. MATERIALS AND METHODS Cell Culture The pituitary adenoma cell lines GH3 and MMQ, the normal rat pituitary cell line RPC, and the human
Address correspondence to Haijun Wang, M.D., Ph.D., Department of Neurosurgery and Pituitary Tumor Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China. Tel/Fax: +86-20-82393181; E-mail: [email protected]
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transformed embryonal epithelial cell HEK-293T originated from ATCC and were provided by the Institute of Biochemistry and Cell Biology of the Chinese Academy of Science (Shanghai, China). The three adenoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3.5% fetal bovine serum and penicillin in a 37°C humidified incubator with a 5% CO2 atmosphere. HEK-293T cells were cultured in DMEM containing 5% bovine serum and l-glutamine and incubated in a 37°C incubator with a 10% CO2 atmosphere. RNA Isolation Total RNA was isolated from cultured cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. miRNA qRT-PCR Analysis miR-200c expression levels were measured by qRTPCR. Sample RNA (10 ng) was reversely transcribed into cDNA along with miR-200c-specific primers (Applied Biosystems, Foster City, CA, USA) by using the ABI miRNA reverse transcription kit (Applied Biosystems), and qRT-PCR was then performed using the ABI 7700 Thermocycler (Applied Biosystems) according to the manufacturer’s protocol. The U6 gene was used as an endogenous control for all samples. mRNA qRT-PCR Analysis Synthesis of cDNA was performed on 1 μg of total RNA per sample with the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s manual. Primers for PTEN and 18S rRNA were designed according to the NCBI sequences using Invitrogen primer design Web-based software. PCR was performed using a Fast Start Master SYBR Green Kit (Roche, Switzerland) on a LightCycler (Roche). The expression levels of PTEN mRNA were analyzed using RealQuant software (Roche) and normalized to that of 18S rRNA mRNA. The oligonucleotide sequences of primers in this section are as follows: PTEN forward: 5¢-TATAGAGCGTGCGGATAATG-3¢, reverse: 5¢-AACTGCTAGCCTCTGGATTT-3¢ and 18S rRNA forward: 5¢-CCTGGATACCGCAGCTAGGA-3¢, reverse: 5¢-GCGGCGCAATACGAATGCCCC-3¢. miR-200c Inhibitor Transfection or Cotransfection With Short Interfering RNA (siRNA) The rat miR-200c inhibitor and negative control oligonucleotide inhibitor (NC inhibitor) were supplied by RiboBio (Guangzhou, China). The oligonucleotide sequence of miR-200c inhibitor was 5¢-CGUCUUACC CAGCAGUGUUUG-3¢. The oligonucleotide sequence of miR-200c NC inhibitor was 5¢-CAGUACUUUUGUGU AGUACAA-3¢. MMQ cells were transfected with 50 nM of miR200c inhibitor or NC inhibitor using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s instructions. Transfection efficiency was optimized using 6- carboxyfluorescein-labeled microRNA at approximately 60–70%. After transfection, the cells were subjected to further assays or for RNA/protein extraction. RNA was extracted 24 h after miR-200c inhibitor or NC inhibitor transfection; then qRT-PCR was performed. Total cell protein was extracted 48 h after transfection and used for Western blot assays. miR-200c inhibitor and PTEN siRNA (Ambion, Austin, TX, USA) or negative controls were delivered to MMQ cells using Lipofectamine 2000 as described by the manufacturer. MMQ cells were seeded in six-well plates at 50% confluence. On the next day, the appropriate amount of miR-200c inhibitor and PTEN siRNA or miR-200c inhibitor and negative control PTEN siRNA (NC siRNA) were diluted in 100 µl serum-free medium and incubated at room temperature for 15 to 20 min with 5 µl of Lipofectamine 2000 to allow the formation of transfection complexes, which were added to the cell cultures and gently swirled. Sequence specificities were confirmed by BLAST search. The culture medium was replaced with fresh medium after 5–6 h, and the cells were incubated for 48 h. The same protocol was followed for all subsequent experiments, unless otherwise specified. The oligonucleotide sequences of PTEN siRNA were sense: 5¢-AA CCCACCACAGCUAGAACTT-3¢; anti-sense: 5¢-AA GUUCUAGCUGUGGUGGGTT-3¢. The oligonucleotide sequences of NC PTEN siRNA were sense: 5¢-UUCUC CGAACGUGUCACGUTT-3¢; anti-sense: 5¢-A CGUGAC ACGUUCGGAGAATT-3¢. miRNA Target Prediction We used the database TargetScan 6.0 (http://www. targetscan.org/index.html) to search for putative miRNA target genes. We obtained the DNA sequence of the 3¢-UTR region of the putative target mRNA from GenBank (http:/ www.ncbi.nlm.nih.gov/). Apoptosis Assay Cells were trypsinized 48 h after miR-200c inhibitor transfection using the Annexin V-FITC/PI apoptosis detection kit (KeyGen Biotech) according to the manufacturer’s instructions. All samples were tested by flow cytometry. Nuclear Staining With 4,6-Diamidino-2-Phenylindole (DAPI) To examine the apoptosis in treated and untreated cells, DAPI nuclear staining assay was performed. Cells were harvested, washed with phosphate-buffered saline (PBS), and fixed with 3.7% paraformaldehyde (Sigma, St. Louis, MO, USA) in PBS for 10 min at room temperature. Fixed cells were washed twice with PBS, and then DAPI solution was spread over the plates followed by incubation for
miR-200c AND PITUITARY ADENOMA CELL APOPTOSIS
1 h at 4°C in the dark, and observed through a FluoView confocal laser microscope (Fluoview FV10i, Olympus Corporation, Tokyo, Japan). Caspase-3/7 Assay Caspase-3/7 activity was performed using the CaspaseGlo 3/7 assay (Promega, Madison, WI, USA). Cells cultured in a white-walled 96-well plate were added with 100 μl of Caspase-Glo® 3/7 Reagent. The contents of the plate were gently mixed with a plate shaker at 100 ´ g for 30 s. Cells were incubated at room temperature for 30 min in the dark. The luminescence of each sample was measured in a plate-reading luminometer of Tecan’s Infinite M200. Vector Construction We amplified the 3¢-UTR of the PTEN mRNA (containing the miR-200c binding site) using PCR and then cloned it into the XhoI/NotI site of the PSICHECK-2 vector (Promega) to construct the luc-PTEN plasmid. Seven nucleotides within the core binding site of the PTEN 3¢-UTR were mutated (CAGTATT to GCTTTGC) to generate the mutant luc-PTEN plasmid. The oligonucleotide primers used for PTEN were PTEN XhoI forward: 5¢-CCG CTCGAGTTTTTTCTTATCAAGAGGGAT-3¢ and PTEN NotI reverse: 5¢-ATAAGAATGCGGCCGCTGACAAGAA TGAGACTTTAATC-3¢ (RiboBio, Guangzhou, China). Luciferase Assay HEK-293T cells were plated in 24-well plates and transfected with the luc-PTEN plasmid (or mut-luc-PTEN plasmid) and miR-200c mimic (RiboBio, Guangzhou, China) or NC mimic (RiboBio) using Lipofectamine 2000. The luciferase activity was examined 48 h after cotransfection using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Relative luciferase activity was normalized to the Renilla luciferase activity from the same vector. Western Blot Cells were harvested and lysed in lysis buffer enriched with protease inhibitors. After centrifugation, samples were collected. The protein concentrations were determined using the BCA protein assay reagent kit (Boster, Wuhan, China). Proteins were resolved in a 12% SDSpolyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk. Blots were developed with anti-PTEN antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Akt antibody (Cell Signalling, Frankfurt, Germany), or anti-phospho-Akt-Ser473 antibody (Cell Signaling), and then incubated with a peroxidaseconjugated secondary antibody. The protein bands were visualized using enhanced chemiluminescence reagents (Amersham, UK). A housekeeping gene GAPDH in the
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same lane was used to ensure equal loading. Bands were quantified using Quantity One software (Bio-Rad). Statistical Analysis The data are presented as the mean ± SEM. Experiments were performed in triplicate separately. PRISM 5.0 (GraphPad Software Inc., San Diego, CA, USA) was used for all statistical tests. The statistical significance was calculated with Student’s t test, and values of p
0965-0407/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096504013X13832473329999 E-ISSN 1555-3906 www.cognizantcommunication.com
MicroRNA-200c Inhibits Apoptosis in Pituitary Adenoma Cells by Targeting the PTEN/Akt Signaling Pathway Chuangxin Liao,* Wenli Chen,* Xiang Fan,* Xiaobing Jiang,* Lubing Qiu,* Chunhua Chen,† Yonghong Zhu,† and Haijun Wang* *Department of Neurosurgery and Pituitary Tumor Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China †Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
MicroRNAs (miRNAs) are important regulators that are involved in the development of different types of tumors. MicroRNA-200c (miR-200c) has been characterized as a tumor suppressor or oncogene in different cancers. However, the role of miR-200c in pituitary tumorigenesis remains unknown. We observed that miR200c was overexpressed in pituitary adenoma cell lines. We transfected a miR-200c inhibitor into pituitary adenoma cells (MMQ cell line) to inhibit miR-200c expression and found that the percentage of apoptotic MMQ cells increased. Using bioinformatics analyses, we predicted that the tumor suppressor gene PTEN was targeted by miR-200c, and we confirmed the presence of a functional miR-200c binding site in the 3¢-UTR of PTEN using luciferase reporter assays. We determined that the inhibition of miR-200c expression can upregulate PTEN expression and decrease the expression of phosphorylated Akt (p-Akt). Furthermore, the siRNAmediated knockdown of PTEN abrogated the effect of inhibiting miR-200c expression. Taken together, these findings suggest that miR-200c regulates pituitary tumor formation through the PTEN/Akt signaling pathway. Therefore, we propose that the inhibition of miR-200c could have therapeutic potential in pituitary adenoma. Key words: MicroRNA; Pituitary adenoma; PTEN/Akt signaling pathway; Apoptosis
INTRODUCTION Pituitary adenomas account for approximately 15% of all brain tumors. Although most pituitary adenomas are benign tumors, some are aggressive and can spread to neighboring tissue (1). In addition to the symptoms caused by compression, such as vision impairment and headache, pituitary adenomas can induce symptoms related to hormone parasecretion, such as amenorrhea, lactation, sex disorders, gigantism, and acromegaly (2). The molecular biological mechanisms underlying the development of pituitary adenoma remain unclear. MicroRNAs (miRNAs) are small noncoding RNAs that consist of 19–25 nucleotides. MiRNAs can bind to complementary sequences within the 3¢ untranslated regions (3¢-UTRs) of their target genes, inducing mRNA degradation or posttranslational repression (3). miRNAs not only are involved in the development of normal cells but also play a role in the pathogenesis of many human cancers (4). Increasing evidence has shown that many miRNAs are deregulated in pituitary adenoma. Our research team found 35 miRNAs that were abnormally expressed in pituitary adenoma using TaqMan miRNA array assays. Among the upregulated miRNAs, miR-200c is upregulated 3.5-fold (5).
An increasing number of studies have revealed that miR200c is overexpressed in cervical cancer and esophageal cancer, acting as an oncogene (6,7). However, miR-200c is downregulated in non-small cell lung cancer (NSCLC), in which it acts as a tumor suppressor gene (8,9). Therefore, the biological function and mechanism of miR-200c in pituitary adenoma warrant further investigation. In this study, we validated our previous miRNA array assay results by real-time PCR (qRT-PCR). We downregulated miR-200c expression in pituitary adenoma cells through transfection with a miR-200c inhibitor and found that apoptosis increased. Luciferase reporter assays confirmed that PTEN is a target gene of miR-200c and that the suppression of miR-200c expression upregulates PTEN protein expression while downregulating p-Akt expression. These results suggest that miR-200c is involved in the tumorigenesis of pituitary adenomas through the PTEN/ Akt signaling pathway. MATERIALS AND METHODS Cell Culture The pituitary adenoma cell lines GH3 and MMQ, the normal rat pituitary cell line RPC, and the human
Address correspondence to Haijun Wang, M.D., Ph.D., Department of Neurosurgery and Pituitary Tumor Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China. Tel/Fax: +86-20-82393181; E-mail: [email protected]
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transformed embryonal epithelial cell HEK-293T originated from ATCC and were provided by the Institute of Biochemistry and Cell Biology of the Chinese Academy of Science (Shanghai, China). The three adenoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3.5% fetal bovine serum and penicillin in a 37°C humidified incubator with a 5% CO2 atmosphere. HEK-293T cells were cultured in DMEM containing 5% bovine serum and l-glutamine and incubated in a 37°C incubator with a 10% CO2 atmosphere. RNA Isolation Total RNA was isolated from cultured cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. miRNA qRT-PCR Analysis miR-200c expression levels were measured by qRTPCR. Sample RNA (10 ng) was reversely transcribed into cDNA along with miR-200c-specific primers (Applied Biosystems, Foster City, CA, USA) by using the ABI miRNA reverse transcription kit (Applied Biosystems), and qRT-PCR was then performed using the ABI 7700 Thermocycler (Applied Biosystems) according to the manufacturer’s protocol. The U6 gene was used as an endogenous control for all samples. mRNA qRT-PCR Analysis Synthesis of cDNA was performed on 1 μg of total RNA per sample with the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s manual. Primers for PTEN and 18S rRNA were designed according to the NCBI sequences using Invitrogen primer design Web-based software. PCR was performed using a Fast Start Master SYBR Green Kit (Roche, Switzerland) on a LightCycler (Roche). The expression levels of PTEN mRNA were analyzed using RealQuant software (Roche) and normalized to that of 18S rRNA mRNA. The oligonucleotide sequences of primers in this section are as follows: PTEN forward: 5¢-TATAGAGCGTGCGGATAATG-3¢, reverse: 5¢-AACTGCTAGCCTCTGGATTT-3¢ and 18S rRNA forward: 5¢-CCTGGATACCGCAGCTAGGA-3¢, reverse: 5¢-GCGGCGCAATACGAATGCCCC-3¢. miR-200c Inhibitor Transfection or Cotransfection With Short Interfering RNA (siRNA) The rat miR-200c inhibitor and negative control oligonucleotide inhibitor (NC inhibitor) were supplied by RiboBio (Guangzhou, China). The oligonucleotide sequence of miR-200c inhibitor was 5¢-CGUCUUACC CAGCAGUGUUUG-3¢. The oligonucleotide sequence of miR-200c NC inhibitor was 5¢-CAGUACUUUUGUGU AGUACAA-3¢. MMQ cells were transfected with 50 nM of miR200c inhibitor or NC inhibitor using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s instructions. Transfection efficiency was optimized using 6- carboxyfluorescein-labeled microRNA at approximately 60–70%. After transfection, the cells were subjected to further assays or for RNA/protein extraction. RNA was extracted 24 h after miR-200c inhibitor or NC inhibitor transfection; then qRT-PCR was performed. Total cell protein was extracted 48 h after transfection and used for Western blot assays. miR-200c inhibitor and PTEN siRNA (Ambion, Austin, TX, USA) or negative controls were delivered to MMQ cells using Lipofectamine 2000 as described by the manufacturer. MMQ cells were seeded in six-well plates at 50% confluence. On the next day, the appropriate amount of miR-200c inhibitor and PTEN siRNA or miR-200c inhibitor and negative control PTEN siRNA (NC siRNA) were diluted in 100 µl serum-free medium and incubated at room temperature for 15 to 20 min with 5 µl of Lipofectamine 2000 to allow the formation of transfection complexes, which were added to the cell cultures and gently swirled. Sequence specificities were confirmed by BLAST search. The culture medium was replaced with fresh medium after 5–6 h, and the cells were incubated for 48 h. The same protocol was followed for all subsequent experiments, unless otherwise specified. The oligonucleotide sequences of PTEN siRNA were sense: 5¢-AA CCCACCACAGCUAGAACTT-3¢; anti-sense: 5¢-AA GUUCUAGCUGUGGUGGGTT-3¢. The oligonucleotide sequences of NC PTEN siRNA were sense: 5¢-UUCUC CGAACGUGUCACGUTT-3¢; anti-sense: 5¢-A CGUGAC ACGUUCGGAGAATT-3¢. miRNA Target Prediction We used the database TargetScan 6.0 (http://www. targetscan.org/index.html) to search for putative miRNA target genes. We obtained the DNA sequence of the 3¢-UTR region of the putative target mRNA from GenBank (http:/ www.ncbi.nlm.nih.gov/). Apoptosis Assay Cells were trypsinized 48 h after miR-200c inhibitor transfection using the Annexin V-FITC/PI apoptosis detection kit (KeyGen Biotech) according to the manufacturer’s instructions. All samples were tested by flow cytometry. Nuclear Staining With 4,6-Diamidino-2-Phenylindole (DAPI) To examine the apoptosis in treated and untreated cells, DAPI nuclear staining assay was performed. Cells were harvested, washed with phosphate-buffered saline (PBS), and fixed with 3.7% paraformaldehyde (Sigma, St. Louis, MO, USA) in PBS for 10 min at room temperature. Fixed cells were washed twice with PBS, and then DAPI solution was spread over the plates followed by incubation for
miR-200c AND PITUITARY ADENOMA CELL APOPTOSIS
1 h at 4°C in the dark, and observed through a FluoView confocal laser microscope (Fluoview FV10i, Olympus Corporation, Tokyo, Japan). Caspase-3/7 Assay Caspase-3/7 activity was performed using the CaspaseGlo 3/7 assay (Promega, Madison, WI, USA). Cells cultured in a white-walled 96-well plate were added with 100 μl of Caspase-Glo® 3/7 Reagent. The contents of the plate were gently mixed with a plate shaker at 100 ´ g for 30 s. Cells were incubated at room temperature for 30 min in the dark. The luminescence of each sample was measured in a plate-reading luminometer of Tecan’s Infinite M200. Vector Construction We amplified the 3¢-UTR of the PTEN mRNA (containing the miR-200c binding site) using PCR and then cloned it into the XhoI/NotI site of the PSICHECK-2 vector (Promega) to construct the luc-PTEN plasmid. Seven nucleotides within the core binding site of the PTEN 3¢-UTR were mutated (CAGTATT to GCTTTGC) to generate the mutant luc-PTEN plasmid. The oligonucleotide primers used for PTEN were PTEN XhoI forward: 5¢-CCG CTCGAGTTTTTTCTTATCAAGAGGGAT-3¢ and PTEN NotI reverse: 5¢-ATAAGAATGCGGCCGCTGACAAGAA TGAGACTTTAATC-3¢ (RiboBio, Guangzhou, China). Luciferase Assay HEK-293T cells were plated in 24-well plates and transfected with the luc-PTEN plasmid (or mut-luc-PTEN plasmid) and miR-200c mimic (RiboBio, Guangzhou, China) or NC mimic (RiboBio) using Lipofectamine 2000. The luciferase activity was examined 48 h after cotransfection using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Relative luciferase activity was normalized to the Renilla luciferase activity from the same vector. Western Blot Cells were harvested and lysed in lysis buffer enriched with protease inhibitors. After centrifugation, samples were collected. The protein concentrations were determined using the BCA protein assay reagent kit (Boster, Wuhan, China). Proteins were resolved in a 12% SDSpolyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk. Blots were developed with anti-PTEN antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Akt antibody (Cell Signalling, Frankfurt, Germany), or anti-phospho-Akt-Ser473 antibody (Cell Signaling), and then incubated with a peroxidaseconjugated secondary antibody. The protein bands were visualized using enhanced chemiluminescence reagents (Amersham, UK). A housekeeping gene GAPDH in the
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same lane was used to ensure equal loading. Bands were quantified using Quantity One software (Bio-Rad). Statistical Analysis The data are presented as the mean ± SEM. Experiments were performed in triplicate separately. PRISM 5.0 (GraphPad Software Inc., San Diego, CA, USA) was used for all statistical tests. The statistical significance was calculated with Student’s t test, and values of p
