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The Fate of Krüppel-Like Factor 9-Positive Hepatic Carcinoma Cells May Be Determined by the Programmed Cell Death Protein 5

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The Fate of Krüppel-Like Factor 9-Positive Hepatic Carcinoma Cells May Be Determined by the Programmed Cell Death Protein 5 INTERNATIONAL JOURNAL OF ONCOLOGY 44: 153-160, 2014 The fate of Krüppel-like factor 9-positive hepatic carcinoma cells may be determined by the programmed cell death protein 5 DA-ZHI FU, YING CHENG, HUI HE, HAI-YANG LIU and YONG-FENG LIU Department of General Surgery, First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning, P.R. China Received August 10, 2013; Accepted October 7, 2013 DOI: 10.3892/ijo.2013.2147 Abstract. Liver cancer in men is the fifth most frequently In the study of Kang et al (8), downregulation of KLF9 is diagnosed cancer worldwide. Human Krüppel-like factor confirmed in human colorectal cancer. However, the roles (KLF9) gene, localized on human chromosome 9q13, has of KLF9 in hepatocellular carcinoma (HCC) were not clear. been implicated in mediating a diverse range of biological KLF9 is an evolutionary well-conserved member of the KLF processes including stem cell maintenance and differentiation family of transcriptional regulators (9). KLF9 is first identified of T- and B-lymphocytes. In this study, we confirmed that the as a transcriptional repressor of the rat Cyp1a1 (previously levels of KLF9 mRNA and protein were lower in hepatocel- P-4501A1) gene and originally named basic transcription lular carcinoma (HCC) tissue compared to normal tissue. In element-binding protein 1 (Bteb1) (10). Human KLF9 gene, addition, we confirmed that upregulation of KLF9 inhibited localized on human chromosome 9q13 (11), has been impli- cell proliferation and mobility and induce apoptosis in HepG2 cated in mediating a diverse range of biological processes cells depending on programmed cell death protein 5 (PDCD5) including stem cell maintenance (12) and differentiation of expression. However, no interaction was found between KLF9 T- and B-lymphocytes (13,14). and PDCD5 using fluorescence resonance energy transfer In our previous study, we confirmed that the levels of (FRET) and co-immunoprecipitation (co-IP). We confirmed PDCD5 mRNA and protein were lower in HCC tissue than that PDCD5 overexpression stimulated the promoter activities normal tissue (15). In this study, we investigated the poten- of KLF9 by luciferase reporter assays. tial role of KLF9 in HCC. We first demonstrated that KLF9 expression is correlated with clinicopathological features and Introduction patient survival, and it may be a useful predictor of prognosis in patients with HCC. Interesting, our results also showed that Liver cancer in men is the fifth most frequently diagnosed exogenous KLF9 expression played its role in HepG2 cells cancer worldwide but the second most frequent cause of depending on PDCD5 expression. cancer death (1). Half of these cases and deaths were estimated to occur in China (2). Materials and methods Krüppel-like factor (KLF) family members share a three C2H2 zinc-finger DNA binding domain, and are involved Cell culture and tumor specimens. Human liver cancer cell line, in cell proliferation and differentiation control in normal as HepG2, was cultured in DMEM (Hyclone, Logan, UT, USA) well as in pathological situations (3). An emerging body of containing 10% fetal bovine serum (Invitrogen Gibco, Carlsbad, evidence indicates that KLF is associated with various types CA, USA) and incubated in a 5% CO2 incubator at 37˚C. Liver of cancers. Downregulation of KLF4 expression is reported specimens were derived from 56 patients undergoing surgical in esophageal cancer (4) and colorectal cancer (5). Significant resection of primary hepatocellular carcinoma without prior genetic alterations of KLF6 expression are observed in pros- chemotherapeutic treatment or radiotherapy. Basic information tate cancer (6). LOH of KLF6 locus at chromosome 10p15 of these patients was described in our previous study (15). contributes to the development of colorectal carcinoma (7). Quantitative real-time RT-PCR (QRT-PCR). Total cellular RNA (1 µg) from each cell line was reverse-transcribed using Takara Reverse Transcription kit (Takara, Dalian, China) and Correspondence to: Dr Yong-Feng Liu, Department of General oligo(dT) 15 primers (Takara). The KLF9 primers were: Surgery, First Affiliated Hospital of China Medical University, 5'-TGGCTGTGGGAAAGTCTATGG-3' (sense) and 5'-CTC 155 Nanjing Road, Heping District, Shenyang 110001, Liaoning, GTCTGAGCGGGAGAACT-3' (antisense). SYBR Green P.R. China real-time PCR was performed with an ABI PRISM 7500 E-mail: [email protected] Sequence Detector. Thermal cycling conditions included pre- incubation at 50˚C for 2 min, 95˚C for 10 min followed by Key words: hepatocellular carcinoma, PDCD5, KLF9, apoptosis, 40 PCR cycles at 95˚C for 15 sec and 60˚C for 1 min. Relative fluorescence resonance energy transfer transcript levels were calculated using the relative standard curve method and results were normalized to GAPDH. The 154 FU et al: COMBINED EFFECTS OF KLF9 AND PDCD5 ON HCC CELLS GAPDH primers were: 5'-AGAAGGCTGGGGCTCATTTG-3' using the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetra- (sense) and 5'-AGGGGCCATCCACAGTCTTC-3' (antisense). ethylbenzimidazolycarbocyanine iodide (JC-1) following the manufacturer's protocol (KeyGen). Briefly, cells were plated Immunohistochemical staining (IHC). As described in our in a 6-well culture plate. After 24 h, cells were washed twice previous study (15), 4-µm sections of paraffin-embedded speci- with PBS, harvested and loaded with 20 nM JC-1 for 30 min in mens were performed using the KLF9 polyclonal antibody the dark. Afterwards, MMP was analyzed by a FACSCalibur (sc-12996, Santa Cruz Biotechnology, Santa Cruz, CA, USA). machine as described above. Briefly, after deparaffinization and hydration, the endogenous peroxidase activity was quenched by a 30-min incubation in a In vitro wound healing assay. Cells were grown in a 6-well mixture of 0.3% hydrogen peroxide solution in 100% methanol. dish. A confluent monolayer of cells was scratched with a The sections were blocked for 2 h at room temperature with 200-µl pipette tip to simulate a wound. Cells were washed 1.5% blocking serum in PBS and incubated with KLF9 antibody twice with PBS and then supplemented with medium and (1:1,000 dilution) at 4˚C in a moist chamber overnight, followed incubated for 4 h at 37˚C. Cell migration into the wounded by incubation with Envision reagent (Dako, Carpinteria, CA, area was monitored microscopically. Images were captured at USA) and color development in 3,3'-diaminobenzidine tetra- the interface of the unwounded and wounded areas. hydrochloride (DAB, Sigma-Aldrich, Carlsbad, CA, USA). The slides were then lightly counterstained with hematoxylin, Cell invasion assay. For invasive assay, cells were resuspended dehydrated with ethanol, cleaned with xylene, and mounted. in serum-free DMEM, and seeded in the control-membrane Adjacent non-cancer tissues were used as controls. Sections insert on the top portion of the Matrigel-coated chamber treated without primary antibodies were used as negative (BD Bioscience). The lower compartment of the chamber controls. The positive percentage of counting cells was graded contained 10% FBS as a chemo-attractant. After incubation semi-quantitatively according to a four-tier scoring system: for 24 h, cells on the membrane were scrubbed, washed with negative (-), 0-5%; weakly positive (+), 6-25%; moderately posi- PBS and fixed in 100% methanol and stained with Giemsa dye. tive (++), 26-50%; and strongly positive (+++), 51-100%. Imaging with confocal FRET microscopy. Confocal images Plasmid construction and transfection. The recombinant were acquired with a Leica TCS-SP2 confocal microscope plasmid, pEGFP-C1-KLF9 was kindly gifted by Mr. Lin Niu (Leica Microsystems, Heidelberg GmbH, Germany). To (China Medical University) and verified by DNA sequencing correct for spectral bleed-through (SBT) and for uncontrolled (Sunbiotech, Beijing, China). pEGFP-C1-KLF9 transfections variations in donor-acceptor concentrations, a combination of were performed using Lipofectamine 2000 (Invitrogen) donor, FRET and acceptor filter sets was used to isolate and according to the manufacturer's instructions. maximize three specific signals: donor fluorescence, acceptor fluorescence resulting from FRET and the directly excited Colony formation assay. Cells were seeded at 200 cells per acceptor fluorescence, respectively. Filter sets used were as well in 24-well tissue culture plates. Plates were incubated follows: the red channel (donor excitation/donor emission for one week in a humidified incubator at 37˚C. One week = 543/575 nm), the green/blue channel (acceptor excitation/ after seeding, colonies were stained with 0.05% crystal violet acceptor emission = 633/680 nm) and the FRET channel containing 50% methanol and counted. The colonies were (donor excitation/acceptor emission = 543/680 nm: FRET). counted in 4-5 random fields for each of the duplicate samples by using a microscope at x100 magnification. Co-immunoprecipitation. Transfected cells were washed once with PBS, lysed for 30 min in lysis buffer (50 mM Transmission electron microscopy. Specimens were immersed Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40) containing in 2% cacodylate-buffered glutaraldehyde, rinsed in caco- protease inhibitors (Cocktail, Roche, Basel, Switzerland) and dylate buffer supplemented with 15% sucrose, post-fixed phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4), and with 1% phosphate-buffered OsO4, dehydrated with alcohol, centrifuged at 15,000 x g at 4˚C for 15 min. Supernatants were clarified in propylene oxide, and embedded in Epon using flat pre-cleared with EZ View Red protein G-Sepharose (Sigma) molds. Ultrathin sections were made with an ultramicrotome, for 1 h at 4˚C. Then 5 µg of antibody specific for each target stained with uranyl acetate, followed by a saturated solution protein were added in each sample. Immune complexes were of bismuth subnitrate, and observed under a JEOL JSM 6400 precipitated by EZ View red protein G-Sepharose overnight scanning electron microscope (JEOL, Tokyo, Japan). at 4˚C and washed 3 times with lysis buffer. The immune complexes were boiled (100˚C) for 10 min in SDS sample Cell apoptosis assay. Cells (5x105) were collected without buffer (100 mM Tris-HCl, pH 8.8, 0.01% bromophenol blue, EDTA and washed with PBS.
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