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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.
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