MicroRNA-221 and microRNA-222 regulate gastric carcinoma cell proliferation and radioresistance by targeting PTEN
- Zhang Chun-zhi†1, 2,
- Han Lei†1,
- Zhang An-ling1,
- Fu Yan-chao3,
- Yue Xiao1,
- Wang Guang-xiu1,
- Jia Zhi-fan1,
- Pu Pei-yu1,
- Zhang Qing-yu3Email author and
- Kang Chun-sheng1Email author
© Chun-zhi et al; licensee BioMed Central Ltd. 2010
Received: 6 January 2010
Accepted: 12 July 2010
Published: 12 July 2010
MicroRNAs (miRNAs) can function as either oncogenes or tumor suppressor genes via regulation of cell proliferation and/or apoptosis. MiR-221 and miR-222 were discovered to induce cell growth and cell cycle progression via direct targeting of p27 and p57 in various human malignancies. However, the roles of miR-221 and miR-222 have not been reported in human gastric cancer. In this study, we examined the impact of miR-221 and miR-222 on human gastric cancer cells, and identified target genes for miR-221 and miR-222 that might mediate their biology.
The human gastric cancer cell line SGC7901 was transfected with AS-miR-221/222 or transduced with pMSCV-miR-221/222 to knockdown or restore expression of miR-221 and miR-222, respectively. The effects of miR-221 and miR-222 were then assessed by cell viability, cell cycle analysis, apoptosis, transwell, and clonogenic assay. Potential target genes were identified by Western blot and luciferase reporter assay.
Upregulation of miR-221 and miR-222 induced the malignant phenotype of SGC7901 cells, whereas knockdown of miR-221 and miR-222 reversed this phenotype via induction of PTEN expression. In addition, knockdonwn of miR-221 and miR-222 inhibited cell growth and invasion and increased the radiosensitivity of SGC7901 cells. Notably, the seed sequence of miR-221 and miR-222 matched the 3'UTR of PTEN, and introducing a PTEN cDNA without the 3'UTR into SGC7901 cells abrogated the miR-221 and miR-222-induced malignant phenotype. PTEN-3'UTR luciferase reporter assay confirmed PTEN as a direct target of miR-221 and miR-222.
These results demonstrate that miR-221 and miR-222 regulate radiosensitivity, and cell growth and invasion of SGC7901 cells, possibly via direct modulation of PTEN expression. Our study suggests that inhibition of miR-221 and miR-222 might form a novel therapeutic strategy for human gastric cancer.
Gastric cancer, a highly invasive and aggressive malignancy that is characterized by resistance to apoptosis and radioresistance, is among the most common cancers and is the leading cause of cancer-related death in China [1–6]. Gastric cancer in China is often diagnosed at an advanced clinical stage, with evident lymphatic tumor dissemination . The 5-year survival rate is approximately 60% for patients with localized disease, but only 2% for those with metastatic disease. Although much has been learned about the genetic and biochemical bases of gastric cancer, few novel therapeutic targets have been identified, due to difficulties in target identification and validation.
MicroRNAs (miRNAs) are noncoding RNAs of approximate 22 nt in length that function as post-transcriptional regulators. By base-pairing with the complementary sites in the 3'untranslated region (3'UTR) of the mRNA, miRNAs control mRNA stability and translation efficiency [8–12]. Growing evidence indicates the important role of miRNA in the development of various cancers. Deregulation of some miRNAs, including miR-221 and miR-222, have been observed in lymphoma, colorectal, lung, and breast cancers, papillary thyroid and hepatocellular carcinoma, glioblastoma [13–21], and gastric cancer [22, 23].
The PTEN gene, located at 10q23.3, encodes a central domain with homology to the catalytic region of protein tyrosine phosphatases. This gene is an important regulator of protein phosphatases and 3'-phosphoinositol phosphatases. PTEN dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PIP3), the second messenger produced by phosphoinositide 3-kinase (PI3K), to negatively regulate the activity of the serine/threonine protein kinase, Akt [24, 25]. PTEN is inactivated in some malignant tumors, resulting in Akt hyper-activation, thereby promoting cell proliferation, inhibition of apoptosis, and enhanced cell invasion and radioresistance [26–28]. miRNA, specifically miR-21 and miR-214, have been established as regulators of PTEN expression [29–33].
In the current study, we predicted that PTEN would be a target gene of the miR-221 and miR-222 cluster by computer-aided algorithm. Moreover, we found binding sites for human miR-221 and miR-222 in the PTEN 3'-UTR. Based upon these findings, we confirmed PTEN as a target of miR-221 and miR-222, and demonstrated that co-suppression of the miR-221/222 cluster inhibits cell proliferation, induces cell apoptosis, inhibits cell invasion and enhances cell radiosensitivity by upregulating PTEN expression in SGC7901 gastric cancer cells.
Cells and cell culture
The human gastric cancer cell line SGC7901 was kindly provided by Dr. Daiming Fan (the Fourth Military Medical University, China). The human embryonic kidney cell line HEK293 was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were grown in Dulbecco's Modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum at 37°C in 5% CO2 atmosphere.
Identification of microRNA targets
The PicTar algorithm http://pictar.mdc-berlin.de was used to identify human microRNA binding sites in PTEN (GeneID 5728). Briefly, PicTar provides 3' UTR alignments with predicted sites and links to various public databases for prediction of microRNA binding sites.
Plasmids, oligonucleotides and cell transfection
Human full-length miR-221 and miR-222 in pMSCV vector were kindly provided by Reuven Agami (Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, Netherlands). The recombinant retroviruses pMSCV-miR-221 and pMSCV-miR-222 were produced as previously described , and transfected into PT67, the packaging cells, using Lipofectamine 2000. The titers of homogenous virus were calculated after infection of NIH3T3 cells. Wild-type PTEN lacking the 3'UTR region was constructed in the pcDNA vector (pcDNA-PTEN) by Genesil Biotechnology Co. Ltd. (Wuhan, China). 2'-OMe-oligonucleotides were chemically synthesized by GenePharma Co. Ltd. (Shanghai, China). All the bases were 2'-OMe modified and had the following sequences: 2'-OMe-anti-miR-221 (AS-miR-221), 5'-AGCUACAUUGUCUGCUGGGUUUC-3'; 2'-OMe-anti-miR-222 (AS-miR-222), 5'-AGCUACAUCUGGCUACUGGGU-3'; scrambled oligonucleotide (Scr), 5'-UCUA CUCUUUCUAGGAGGUUGUGA-3'.
SGC7901 cells were grown to 70-80% confluence and transfected with pcDNA- PTEN and 2'-OMe-oligonucleotides using Lipofectamine 2000 or infected with pMSCV-miR-221 and/or pMSCV-miR-222 at a multiplicity of infection (MOI) of 50 at 37°C. At 4 h after infection, the medium was replaced with fresh DMEM containing 10% fetal bovine serum, and the cells were incubated for an additional 72 h for further study.
Northern blot analysis
Total RNA was extracted using TRIzol reagent (Invitrogen). The protocol for Northern blotting of miRNA was adopted from Ramkissoon . Total RNA were separated on a 12% denaturing polyacrylamide gel and transferred to Hybond N+ nylon membrane (Ambion, USA). The membrane was dried, UV cross-linked, hybridized with digoxigenin (DIG)-labeled probes overnight at 37°C in a buffer containing 5× SSC, 20 mmol/L Na2HPO4 (pH = 7.2), 7% SDS, 1× Denhardt's solution and 0.2 mg/mL salmon sperm DNA. The specific probes, end-labeled with DIG, were miRNA-221, 5'-GAAACCCAGCAGACAATGTAGCT-3'; miRNA-222, 5'-GAGACC CAGTAGCCAGATGTAGCT-3'; and U6, 5'-ATTTGCGTGTCATCCTTGCG-3'. The probes were purchased from Proligo Primers & Probes (Sigma, USA). Membranes were washed with 1× SSC/1% SDS at 50°C. After equilibration in detection buffer, blots were detected with a DIG Luminescent Detection Kit (Roche, USA) and analyzed using GeneGenius.
Cell viability assay
Cells were seeded into 96 well plates at 4000 cells/well. After transfection, 20 μl MTT (5 mg/mL) was added into a corresponding test well, and incubated for 4 h. The supernatant was then discarded, and 200 μL of DMSO was added to each well to dissolve the precipitate. Optical density (OD) was measured at the wavelength of 570 nm. Each test was performed daily for six consecutive days and repeated in eight wells.
Cell cycle assay
For cell cycle analysis, parental and transfected cells in the log phase of growth were stained with propidium iodide and examined with a fluorescence-activated cell-sorting (FACS) flow cytometer (BD Biosciences, San Jose, CA), and DNA histograms were analyzed with modified software. Each test was repeated in triplicate.
Measurement of early apoptosis by Annexin V staining
Parental and transfected cells in the log phase of growth were harvested and collected by centrifugation and resuspended at a density of 1 × 106 cells/mL. For the apoptosis assay, an annexin V-FITC labeled Apoptosis Detection Kit (Abcam, USA) was used. The pre-labeled cells were detected and apoptosis was quantified using a FACSCalibur flow cytometer (Becton-Dickinson, USA). The data obtained were analyzed using CellQuest software. Each test was repeated in triplicate.
Using parental and transfected cells, the invasion potential of the cells were evaluated by measuring the number of cells invading Matrigel-coated Transwell chambers (Becton Dickinson). Transwell inserts with 8 μm pores were coated with Matrigel and reconstituted with fresh medium for 2 h before the experiment. Cells (2 × 104/mL) were seeded into the upper chambers in 250 μL serum free DMEM, while DMEM supplemented with 10% fetal bovine serum (750 μL) was placed in the lower chamber. Cells were incubated for 72 h. Cells that degraded the Matrigel and invaded the lower surface of the Matrigel-coated membrane were fixed with 70% ethanol, stained with hematoxylin and counted in five random fields at ×200 magnification under a light microscope. The results were expressed as the average number of invasive cells per field.
Radiation Exposure and Clonogenic assay
Irradiation was performed at room temperature in a linear accelerator (Varian600, Varian, USA) at a dose rate of 3.2 Gy/min. Cells were plated into six-well plates and exposed to the specified dose (0, 2, 4 and 6 Gy) of X-rays. At 24 h after irradiation, all cells were trypsinized and counted. Corresponding numbers of cells were seeded into 10 cm dishes containing DMEM supplemented with 10% fetal bovine serum in triplicate, incubated for 10-14 days to allow colony growth, and colonies were stained with crystal violet. Colonies containing 50 or more cells were counted. The plating efficiency was calculated by dividing the average number of colonies per dish by the number of cells plated. Survival fractions were calculated by normalization to the plating efficiency of appropriate control groups.
Luciferase reporter assay
The human 3'-UTR of the PTEN gene was amplified by PCR using the following primers: PTEN-3'UTR-Forward: 5'-CGATTCTAGAAATCATGTTCTGGTGG-3' and PTEN-3'UTR-Reverse: 5'-GCATTCTAGAATTCTGCACAGTAAGCATA-3'. The cDNA was cloned into the XbaI/XbaI site of the pGL3-control vector (Promega, USA), downstream of the luciferase gene, to generate the vector pGL3-PTEN. For the luciferase reporter assay, SGC7901 cells were cultured in 96-well plates, transfected with 0.2 μg of the pGL3-PTEN or pGL3-control plasmids and 5 pmol of AS-miRNAs (AS-miR-221 and/or AS-miR-222) using Lipofectamine 2000. At 48 h after transfection, luciferase activity was measured using the Luciferase Assay System (Promega).
Western blot analysis
Parental and transfected cells were washed with pre-chilled PBS and solubilized in 1% Nonidet P-40 lysis buffer. Homogenates were clarified by centrifugation at 20,000 ×g for 15 min at 4°C and the protein concentration was measured by bicinchoninic acid protein assay kit (Pierce Biotechnology). 40 μg of protein from each sample was subjected to SDS-PAGE on SDS-acrylamide gel. Separated proteins were transferred to PVDF membranes (Millipore) and incubated with primary antibody (1:1000 dilution; Santa Cruz) followed by incubation with an HRP-conjugated secondary antibody (1:1000 dilution; Zymed, San Diego, CA). The specific protein was detected using a SuperSignal protein detection kit (Pierce, USA). The membrane was stripped and reprobed with a primary antibody against β-actin (Santa Cruz; 1:1000 dilution) as a control.
Data are expressed as the mean ± standard error (S.E.). P < 0.01 was considered statistically significant using ANOVA and the STD t test or SNK Q test t test.
Modulation of miR-221 and miR-222 expression in SGC7901 cell lines
miR-221 and miR-222 co-modulate SGC7901 cell proliferation
Apoptosis is a genetically encoded cascade of cellular reaction that results in the disposal of unwanted cells. Disruption to this pathway has been implicated as a cause of cancer . Some miRNAs regulate proteins that are involved in apoptosis . Using Annexin V analysis, the number of apoptotic cells in early phase was found to be significantly increased in cells transfected with AS-miR-221/222 compared with that in other groups (p = 0.0012). In comparison with parental cells, the apoptotic rate was very low in pMSCV-miR-221/222 infected cells (Figure 2C). These data demonstrated that the proliferation and survival rates of SGC7901 cells might be co-modulated by miR-221 and miR-222.
miR-221 and miR-222 co-modulate SGC7901 cell invasion
We also assessed the role of miR-221 and miR-222 on cell invasion by Transwell assay. As shown in Figure 2D, as compared with blank and negative control cells, the invasion potential of SGC7901 cells transfected with AS-miR-221/222 was significantly decreased (0.3813-fold, p = 0.0067), while cells transduced with pMSCV-miR-221/222 displayed markedly increased invasive ability (1.3577-fold, P = 0.0099). These results suggested that miR-221 and miR-222 could co-modulate SGC7901 cell invasion.
miR-221 and miR-222 co-modulate SGC7901 cell radiosensitivity
Impact of miRNA221/222 expression on SGC7901 cell radiosensitivity.
control + irradiation
Scrambled + irradiation
AS-miRNA221/222 + irradiation
pMSCV-miR-221/222 + irradiation
miR-221 and miR-222 targeting of the PTEN gene
miR-221 and miR-222 affect the phenotype of SGC7901 cell in a PTEN-dependent pattern
Impact of PTEN on miRNA221/222-mediated SGC7901 cell radiosensitivity.
pMSCV-miR-221/222 and pcDNA-PTEN + irradiation
In this study, we demonstrated that miR-221 and miR-222 regulate gastric cancer cell viability, apoptosis, cell cycle progression and invasive ability. Our data suggests that downregulation of PTEN expression and enhanced Akt phosphorylation (p-Akt) are important mediators of these cellular processes. As pAkt impacts cell proliferation, cell transit from the G0/G1 to the S phase, apoptosis, cell invasive ability, and cell radiosensitivity, downregulation of miR-221 and miR-222 expression have important biologic effects on the malignant phenotype of SGC7901 cells. These results identify AS-miR-221/222 as a potential therapeutic approach for gastric cancer via upregulation of PTEN.
PTEN functions as a tumor suppressor gene, specifically by negatively regulating the Akt/PKB signaling pathway. Genetic inactivation of PTEN is a hallmark of many cancers, including glioblastoma, endometrial and prostate cancers, and reduced expression occurs in many other tumor types. Deficiency of PTEN in the intestine has been reported to induce precancerous polyps, via the induction of formation and fission of crypts, structures located at the base of the intestine containing a rapidly dividing pool of intestinal stem cells . Guo JM et al studied the microRNAs expression in primary gastric cancer tissues via microRNA microarray assay and were the first to demonstrate that PTEN was the target of miR-21 ; however, little is known regarding the impact of miR-221 and miR-222 on PTEN expression in gastric cancer.
miR-221 and miR-222 expression is abnormally increased in gastric cancer , however the mechanism by which miR-221 and miR-222 modulates tumor progression within the gut remains unknown. Here, we observed miR-221 and miR-222 upregulation in the human gastric cancer cell line SGC7901 compared with HEK293 epithelial cells, corroborating the findings of Young-kook et al . miR-221 and miR-222 modulate a variety of biological functions in the SGC7901 cell, including cell proliferation, apoptosis, invasion, and radioresistance. We identified binding sites for miR-221 and miR-222 in the PTEN 3'-UTR by bioinformatics analysis, suggesting that increased expression of the miR-221/222 cluster might impact on PTEN expression. Indeed, we demonstrated that PTEN is a target gene of miR-221 and miR-222 by luciferase reporter assay. As PTEN can antagonize PI3K activity by dephosphorylating PIP3 and thereby negatively regulates the activity of Akt pathway [24, 25]. Several studies suggest that the loss of the PTEN function might be the underlying factor in Akt pathway activation [43–45]; thus, our findings are consistent with an emerging body of literature.
Akt represents a subfamily of the serine/threonine kinase family . It modulates the function of numerous substrates related to the cell proliferation, apoptosis and invasion and is putatively involved in the development of some cancers, such as in colon , prostate , lung  and thyroid cancer . It has been shown that Akt activation in cancer cells can increase their invasive ability and resistance to radiotherapy [51–53]. In our study, we found that knockdown of miR-221 and miR-222 in SGC7901 cells resulted in downregulation of pAkt expression, affecting the expression of several Akt-regulated proteins including cyclin D1, Bcl-2, and MMP2/9. The malignant phenotype of the SGC7901 cells was reversed by knockdown miR-221 and miR-222, and cells were sensitized to radiation, corroborating the results of Garofalo et al . As PTEN is a target of miR-221 and miR-222, and has been described previously as an important regulator of radiation sensitivity [24, 55], these results suggest that increasing PTEN expression by silencing miR-221/222 could enhance the radiosensitivity of SGC7901 cells. Whether PTEN/Akt signaling is the sole target for miRNA-221/222 regulation of radiosensitivity remains unknown.
The PTEN gene is an important functional target of the miR-221/222 cluster in gastric cancer cells. Modulation of miR-221/222 expression by antisense or overexpression strategies directly affected PTEN expression. At present, anti-miRNA oligonucleotides have been shown to specifically inactivate endogenous target miRNAs, although rather inefficiently [59, 60]. We provide evidence that co-suppression of both miR-221 and miR-222 affects gastric cancer cell biology in vitro, and might represent a novel therapeutic strategy for gastric cancer through upregulation of PTEN expression.
This work was supported by the China National Natural Scientific Fund (30772231), the Tianjin Science and Technology Committee (10JCZDJC18500), the Program for New Century Excellent Talents in University (NCET-07-0615). The authors wish to thank Dr. R Agami, Division of Tumor Biology, the Netherlands Cancer Institute, Amsterdam, the Netherlands, for kindly providing the retroviral constructs of miR-221 and miR-222. The authors also wish to thank Dr. Daiming Fan, the Fourth Military Medical University, China, for kindly providing SGC7901 gastric cancer cells.
- Yu HG, Ai YW, Yu LL, Zhou XD, Li JH, Xu XM, Liu S, Chen J, Liu F, Qi YL, Deng QJ, Cao J, Liu SQ, Luo HS, Yu JP: Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death. Int J Cancer. 2008, 122: 433-443. 10.1002/ijc.23049.View ArticlePubMedGoogle Scholar
- Qiu H, Yashiro M, Shinto O, Matsuzaki T, Hirakawa K: DNA methyltransferase inhibitor 5-aza-CdR enhances the radiosensitivity of gastric cancer cells. Cancer Sci. 2009, 100: 181-188. 10.1111/j.1349-7006.2008.01004.x.View ArticlePubMedGoogle Scholar
- Cinti C, Vindigni C, Zamparelli A, La Sala D, Epistolato MC, Marrelli D, Cevenini G, Tosi P: Activated Akt as an indicator of prognosis in gastric cancer. Virchows Arch. 2008, 453: 449-455. 10.1007/s00428-008-0676-8.View ArticlePubMedGoogle Scholar
- Bandres E, Bitarte N, Arias F, Agorreta J, Fortes P, Agirre X, Zarate R, Diaz-Gonzalez JA, Ramirez N, Sola JJ, Jimenez P, Rodriguez J, Garcia-Foncillas J: microRNA-451 regulates macrophage migration inhibitory factor production and proliferation of gastrointestinal cancer cells. Clin Cancer Res. 2009, 15: 2281-2290. 10.1158/1078-0432.CCR-08-1818.View ArticlePubMedGoogle Scholar
- Lee BL, Lee HS, Jung J, Cho SJ, Chung HY, Kim WH, Jin YW, Kim CS, Nam SY: Nuclear factor-κB activation correlates with better prognosis and Akt activation in human gastric cancer. Clin Cancer Res. 2005, 11: 2518-2525. 10.1158/1078-0432.CCR-04-1282.View ArticlePubMedGoogle Scholar
- Cai SR, Wang Z, Chen CQ, Wu WH, He YL, Zhan WH, Zhang CH, Cui J, Wu H: Role of silencing phosphatase of regenerating liver-3 expression by microRNA interference in the growth of gastric cancer. Chin Med J. 2008, 121: 2534-2538.PubMedGoogle Scholar
- Rojo F, Tabernero J, Albanell J, Van Cutsem E, Ohtsu A, Doi T, Koizumi W, Shirao K, Takiuchi H, Ramon y Cajal S, Baselga J: Pharmacodynamic studies of gefitinib in tumor biopsy specimens from patients with advanced gastric carcinoma. J Clinical Oncology. 2006, 24: 4309-4315. 10.1200/JCO.2005.04.2424.View ArticleGoogle Scholar
- Liu T, Tang H, Lang Y, Liu M, Li X: MicroRNA-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin. Cancer Letters. 2008, 273: 233-242. 10.1016/j.canlet.2008.08.003.View ArticlePubMedGoogle Scholar
- Moriyama T, Ohuchida K, Mizumoto K, Yu J, Sato N, Nabae T, Takahata S, Toma H, Nagai E, Tanaka M: MicroRNA-21 modulates biological functions of pancreatic cancer cells including their proliferation, invasion and chemoresistance. Mol Cancer Ther. 2009, 8: 1067-1074. 10.1158/1535-7163.MCT-08-0592.View ArticlePubMedGoogle Scholar
- Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N: Widespread changes in protein synthesis induced by microRNAs. Nature. 2008, 455: 58-63. 10.1038/nature07228.View ArticlePubMedGoogle Scholar
- Tay Y, Zhang J, Thomson AM, Lim B, Rigoutsos I: MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature. 2008, 455: 1124-1128. 10.1038/nature07299.View ArticlePubMedGoogle Scholar
- Chi SW, Zang JB, Mele A, Darnell RB: Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature. 2009, 460: 479-486.PubMedPubMed CentralGoogle Scholar
- Calin GA, Croce CM: MicroRNA signatures in human cancers. Nature Reviews Cancer. 2006, 6: 857-866. 10.1038/nrc1997.View ArticlePubMedGoogle Scholar
- Metzler M, Wilda M, Busch K, Viehmann S, Borkhardt A: High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma. Genes Chromosomes Cancer. 2004, 39: 167-169. 10.1002/gcc.10316.View ArticlePubMedGoogle Scholar
- Michael MZ, O' Connor SM, van Holst Pellekaan NG, Young GP, James RJ: Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol Cancer Res. 2003, 1: 882-891.PubMedGoogle Scholar
- Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, Takahashi T: Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 2004, 64: 3753-3756. 10.1158/0008-5472.CAN-04-0637.View ArticlePubMedGoogle Scholar
- Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri E, Pedriali M, Fabbri M, Campiglio M, Ménard S, Palazzo JP, Rosenberg A, Musiani P, Volinia S, Nenci I, Calin GA, Querzoli P, Negrini M, Croce CM: MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 2005, 65: 7065-7070. 10.1158/0008-5472.CAN-05-1783.View ArticlePubMedGoogle Scholar
- He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, Calin GA, Liu CG, Franssila K, Suster S, Kloos RT, Croce CM, Chapelle A: The role of microRNA genes in papillary thyroid carcinoma. PNAS. 2005, 102: 19075-19080. 10.1073/pnas.0509603102.View ArticlePubMedPubMed CentralGoogle Scholar
- Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, Shimotohno K: Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene. 2006, 25: 2537-2545. 10.1038/sj.onc.1209283.View ArticlePubMedGoogle Scholar
- Chan JA, Krichevsky AM, Kosik KS: MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005, 65: 6029-6033. 10.1158/0008-5472.CAN-05-0137.View ArticlePubMedGoogle Scholar
- Ciafrè SA, Galardi S, Mangiola A, Ferracin M, Liu CG, Sabatino G, Negrini M, Maira G, Croce CM, Farace MG: Extensive modulation of a set of microRNAs in primary glioblastoma. Biochem Biophys Res Commun. 2005, 334: 1351-1358. 10.1016/j.bbrc.2005.07.030.View ArticlePubMedGoogle Scholar
- Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM: A microRNA expression signature of human solid tumors defines cancer gene targets. PNAS. 2006, 103: 2257-2261. 10.1073/pnas.0510565103.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim YK, Yu J, Tae SH, Park SY, Bumjin N, Dong HK, Keun H, Yoo MW, Lee HJ, Yang HK, Kim VN: Functional links between clustered microRNAs: suppression of cell-cycle inhibitors by microRNA clusters in gastric cancer. Nucleic Acids Research. 2009, 37: 1672-1681. 10.1093/nar/gkp002.View ArticlePubMedPubMed CentralGoogle Scholar
- Sun Y, St Clair DK, Fang F, Warren GW, Rangnekar VM, Crooks PA, St Clair WH: The radiosensitization effect of parthenolide in prostate cancer cells is mediated by nuclear factor-κB inhibition and enhanced by the presence of PTEN. Mol Cancer Ther. 2007, 6: 2477-2486. 10.1158/1535-7163.MCT-07-0186.View ArticlePubMedPubMed CentralGoogle Scholar
- Jiang Z, Pore N, Cerniglia GJ, Mick R, Georgescu MM, Bernhard EJ, Hahn SM, Gupta AK, Maity A: Phosphatase and tensin homologue deficiency in glioblastoma confers resistance to radiation and temozolomide that is reversed by the protease inhibitor nelfinavir. Cancer Res. 2007, 67: 4467-4473. 10.1158/0008-5472.CAN-06-3398.View ArticlePubMedGoogle Scholar
- Ge H, Cao YY, Chen LQ, Wang YM, Chen ZF, Wen DG, Zhang XF, Guo W, Wang N, Li Y, Zhang JH: PTEN polymorphisms and the risk of esophageal carcinoma and gastric cardiac carcinoma in a high incidence region of China. Diseases of the Esophagus. 2008, 21: 409-415. 10.1111/j.1442-2050.2007.00786.x.View ArticlePubMedGoogle Scholar
- Cinti C, Vindigni C, Zamparelli A, La Sala D, Epistolato MC, Marrelli D, Cevenini G, Tosi P: Activated Akt as an indicator of prognosis in gastric cancer. Virchows Arch. 2008, 453: 449-455. 10.1007/s00428-008-0676-8.View ArticlePubMedGoogle Scholar
- Pappas G, Zumstrin LA, Munshi A, Hobbs M, Meyn RE: Adenoviral-mediated PTEN expression radiosensitizes non-small cell lung cancer cells by suppressing DNA repair capacity. Cancer Gene Therapy. 2007, 14: 543-549. 10.1038/sj.cgt.7701050.View ArticlePubMedGoogle Scholar
- Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y, Klinge CM: Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic Acids Research. 2009, 37: 2584-2595. 10.1093/nar/gkp117.View ArticlePubMedPubMed CentralGoogle Scholar
- Qi L, Bart J, Tan LP, Platteel I, Sluis T, Huitema S, Harms G, Fu L, Hollema H, Berg A: Expression of miR-21 and its targets (PTEN, PDCD4, TM1) in flat epithelial atypia of the breast in relation to ductal carcinoma in situ and invasive carcinoma. BMC Cancer. 2009, 9: 163-10.1186/1471-2407-9-163.View ArticlePubMedPubMed CentralGoogle Scholar
- Talotta F, Cimmino A, Matarazzo MR, Casalino L, De Vita G, D'Esposito M, Di Lauro R, Verde P: An autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation. Oncogene. 2009, 28: 73-84. 10.1038/onc.2008.370.View ArticlePubMedGoogle Scholar
- Vinciguerra M, Sgroi A, Veyrat-Durebex C, Rubbia-Brandt L, Buhler LH, Foti M: Unsaturated fatty acids inhibit the expression of tumor suppressor phosphatase and tensin homolog (PTEN) via MicroRNA-21 up-regulation in hepatocytes. Hepatology. 2009, 49: 1176-1184. 10.1002/hep.22737.View ArticlePubMedGoogle Scholar
- Yang H, Kong W, He L, Zhao JJ, O'Donnell JD, Wang J, Wenham RM, Coppola D, Kruk PA, Nicosia SV, Cheng JQ: MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN. Cancer Res. 2008, 68: 425-433. 10.1158/0008-5472.CAN-07-2488.View ArticlePubMedGoogle Scholar
- Zhang JX, Han L, Ge YL, Zhou X, Zhang AL, Zhang CZ, Zhong Y, You YP, Pu PY, Kang CS: miR-221/222 promote malignant progression of glioma through activation of the Akt pathway. Int J Oncol. 2010, 36: 913-920.PubMedGoogle Scholar
- Ramkissoon SH, Mainwaring LA, Sloand EM, Young NS, Kajigaya S: Nonisotopic detection of microRNA using digoxigenin labeled RNA probes. Mol Cell Probes. 2006, 20: 1-4. 10.1016/j.mcp.2005.07.004.View ArticlePubMedGoogle Scholar
- Wang X, Tang S, Le SY, Lu R, Rader JS, Meyers C, Zheng ZM: Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One. 2008, 3: e2557-10.1371/journal.pone.0002557.View ArticlePubMedPubMed CentralGoogle Scholar
- Ito H, Kanzawa T, Miyoshi T, Hirohata S, Kyo S, Iwamaru A, Aoki H, Kondo Y, Kondo S: Therapeutic efficacy of PUMA for malignant glioma cells regardless of the p53 status. Hum Gene Ther. 2005, 16: 685-698. 10.1089/hum.2005.16.685.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang Y, Lee CG: MicroRNA and cancer-focus on apoptosis. J Cell Mol Med. 2009, 13: 12-23. 10.1111/j.1582-4934.2008.00510.x.View ArticlePubMedGoogle Scholar
- Kim RH, Mak TW: Tumours and tremors: PTEN regulation underlies both. Br J Cancer. 2006, 94: 620-624.PubMedPubMed CentralGoogle Scholar
- He XC, Yin T, Grindley JC, Tian Q, Sato T, Tao WA, Dirisina R, Porter-Westpfahl KS, Hembree M, Johnson T, Wiedemann LM, Barrett TA, Hood L, Wu H, Li L: PTEN-deficient intestinal stem cells initiate intestinal polyposis. Nat Genet. 2007, 39: 189-198. 10.1038/ng1928.View ArticlePubMedPubMed CentralGoogle Scholar
- Guo J, Miao Y, Xiao B, Huan R, Jiang Z, Meng D, Wang Y: Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues. J Gastroenterol Hepatol. 2009, 24: 652-657. 10.1111/j.1440-1746.2008.05666.x.View ArticlePubMedGoogle Scholar
- Kim YK, Yu J, Han TS, Park SY, Namkoong B, Kim DH, Hur K, Yoo MW, Lee HJ, Yang HK, Kim VN: Functional links between clustered microRNAs: suppression of cell-cycle inhibitors by microRNA clusters in gastric cancer. Nucleic Acids Res. 2009, 37: 1672-1681. 10.1093/nar/gkp002.View ArticlePubMedPubMed CentralGoogle Scholar
- Honjo S, Osaki M, Ardyanto TD, Hiramatsu T, Maeta N, Ito H: COX-2 inhibitor, NS398, enhances Fas-mediated apoptosis via modulation of the PTEN-Akt pathway in human gastric carcinoma cell lines. DNA Cell Biol. 2005, 24: 141-147. 10.1089/dna.2005.24.141.View ArticlePubMedGoogle Scholar
- Byun DS, Cho K, Ryu BK, Lee MG, Park JI, Chae KS, Kim HJ, Chi SG: Frequent monoallelic deletion of PTEN and its reciprocal association with PIK3CA amplification in gastric carcinoma. Int J Cancer. 2003, 104: 318-327. 10.1002/ijc.10962.View ArticlePubMedGoogle Scholar
- Oki E, Baba H, Tokunaga E, Nakamura T, Ueda N, Futatsugi M, Mashino K, Yamamoto M, Ikebe M, Kakeji Y, Maehara Y: Akt phosphorylation associates with LOH of PTEN and leads to chemoresistance for gastric cancer. Int J Cancer. 2005, 117: 376-380. 10.1002/ijc.21170.View ArticlePubMedGoogle Scholar
- Yu HG, Ai YW, Yu LL, Zhou XD, Liu J, Li JH, Xu XM, Liu S, Chen J, Liu F, Qi YL, Deng QJ, Cao J, Liu SQ, Luo HS, Yu JP: Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death. Int J Cancer. 2008, 122: 433-443. 10.1002/ijc.23049.View ArticlePubMedGoogle Scholar
- Itoh N, Semba S, Ito M, Takeda H, Kawata S, Yamakawa M: Phosphorylation of Akt/PKB is required for suppression of cancer cell apoptosis and tumor progression in human colorectal carcinoma. Cancer. 2002, 94: 3127-3134. 10.1002/cncr.10591.View ArticlePubMedGoogle Scholar
- Liao Y, Grobholz R, Abel U, Trojan L, Michel MS, Angel P, Mayer D: Increase of AKT/PKB expression correlates with Gleason pattern in human prostate cancer. In J Cancer. 2003, 107: 676-680.Google Scholar
- kudela K, Hayashi H, Ito T, Yazawa T, Suzuki T, Nakane Y, Sato H, Ishi H, Keqin X, Masuda A, Takahashi T, Kitamura H: K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation: possible contribution to non-invasive expansion of lung adenocarcinoma. Am J Pathol. 2004, 164: 91-100.View ArticleGoogle Scholar
- Vasko V, Saji M, Hardy E, Kruhlak M, Larin A, Savchenko V, Miyakawa M, Isozaki O, Murakami H, Tsushima T, Burman KD, De Micco C, Ringel MD: Akt activation and localisation correlate with tumour invasion and oncogene expression in thyroid cancer. J Med Genet. 2004, 41: 161-170. 10.1136/jmg.2003.015339.View ArticlePubMedPubMed CentralGoogle Scholar
- Grille SJ, Bellicosa A, Upson J, Klein-Szanto AJ, Van RF, Lee KW, Donowitz M, Tsichlis PN, Larue L: The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer Res. 2003, 63: 2172-2178.PubMedGoogle Scholar
- Brognard J, Clark AS, Ni Y, Dennis PA: Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 2001, 61: 3986-3997.PubMedGoogle Scholar
- Tanno S, Yanagawa N, Habiro A, Koizumi K, Nakano Y, Osanai M, Mizukami Y, Okumura T, Testa JR, Kohgo Y: Serine/threonine kinase AKT is frequently activated in human bile duct cancer and is associated with increased radioresistance. Cancer Res. 2004, 64: 3486-3490. 10.1158/0008-5472.CAN-03-1788.View ArticlePubMedGoogle Scholar
- Garofalo M, Di Leva G, Romano G, Nuovo G, Suh SS, Ngankeu A, Taccioli C, Pichiorri F, Alder H, Secchiero P, Gasparini P, Gonelli A, Costinean S, Acunzo M, Condorelli G, Croce CM: miR-221 & 222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer Cell. 2009, 16: 498-509. 10.1016/j.ccr.2009.10.014.View ArticlePubMedPubMed CentralGoogle Scholar
- Park JK, Jung HY, Park SH, Kang SY, Yi MR, Um HD, Hong SH: Combination of PTEN and gamma-ionizing radiation enhances cell death and G(2)/M arrest through regulation of AKT activity and p21 induction in non-small-cell lung cancer cells. Int J Radiat Oncol Biol Phys. 2008, 70: 1552-1560.View ArticlePubMedGoogle Scholar
- Milas L, Akimoto T, Hunter NR, Mason KA, Buchmiller L, Yamakawa M, Muramatsu H, Ang KK: Relationship between cyclin D1 expression and poor radioresponse of murine carcinomas. Int J Radiat Oncol Biol Phys. 2002, 2: 514-521.View ArticleGoogle Scholar
- Zhang C, Kang C, You Y, Pu P, Yang W, Zhao P, Wang G, Zhang A, Jia Z, Han L, Jiang H: Co-suppression of miR-221/222 cluster suppresses human glioma cell growth by targeting p27kip1 in vivo and in vivo. Int J Oncol. 2009, 34: 1653-1660. 10.3892/ijo_00000241.View ArticlePubMedGoogle Scholar
- Fornari F, Gramantieri L, Ferracin M, Veronese A, Sabbioni S, Calin GA, Grazi GL, Giovannini C, Croce CM, Bolondi L, Negrini M: MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma. Oncogene. 2008, 27: 5651-5661. 10.1038/onc.2008.178.View ArticlePubMedGoogle Scholar
- Lu Y, Xiao J, Lin H, Bai Y, Luo X, Wang Z, Yang B: A single anti-microRNA antisense oligodeoxyribonucleotide(AMO) targeting multiple microRNAs offers an improved approach for microRNA interference. Nucleic Acids Research. 2009, 37: e24-10.1093/nar/gkn1053.View ArticlePubMedPubMed CentralGoogle Scholar
- Esau CC: Inhibition of microRNA with antisense oligonucleotides. Methods. 2008, 44: 55-60. 10.1016/j.ymeth.2007.11.001.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/367/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.