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Rnai-Mediated HOXD3 Knockdown Inhibits Growth in Human RKO Cells

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Rnai-Mediated HOXD3 Knockdown Inhibits Growth in Human RKO Cells ONCOLOGY REPORTS 36: 1793-1798, 2016 RNAi-mediated HOXD3 knockdown inhibits growth in human RKO cells FANGJUN CHEN1,2, GUOPING SUN1 and JUN PENG3 1Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022; 2Department of Clinical Medicine, Anqing Medical College, Anqing, Anhui 246052; 3Department of Pathology, The Affiliated Anqing Municipal Hospital of Anhui Medical University, Anqing, Anhui 246003, P.R. China Received January 25, 2016; Accepted March 2, 2016 DOI: 10.3892/or.2016.4993 Abstract. Numerous studies have shown that the multifunc- regulators of cell differentiation, morphogenesis, and tional Homeobox-containing (HOX) D3 gene is involved in organogenesis during development (5,6). In humans and other various physiological and pathological processes. To elucidate mammals, 39 HOX genes are clustered in four complexes the role and mechanism of HOXD3 in colorectal cancer called HOXA, B, C, and D. These are further subdivided into (CRC), we measured its expression in five CRC cell lines. 13 paralogous groups based on their sequence similarities and After determining that HOXD3 was highly expressed in the relative positions along the clusters (7-11). Mutational analyses human RKO cancer cell line, we used lentiviral-mediated demonstrated that HOXD genes play a pivotal role in deter- small interfering RNAs (siRNAs) to knock down HOXD3 mining the regional specificity of cells (12,13). For example, expression and assessed proliferation, cell cycle distribution, the HOXD13 gene is involved in the normal morphogenesis of apoptosis and colony formation using cell proliferation, flow limbs (12) and of the anal sphincter (13). HOXD3 is a member cytometric, and colony formation assays. The expression of of the third paralogous group of the HOXD gene family and is HOXD3 was strongly suppressed in the RKO cells infected involved in embryonic development (14,15). with the lentiviruse expressing an HOXD3 short hairpin RNA Studies have shown that the human HOXD3 gene plays (shRNA). The downregulation of HOXD3 expression in RKO a multifunctional role in cancer. For example, HOXD3 cells significantly decreased proliferation and colony forma- overexpression was found to regulate cell adhesion in tion, and increased apoptosis in vitro, compared to the cells human erythroleukemia HEL cells (16), induce coordinate infected with the mock control (p<0.01). Moreover, specific metastasis-related gene expression, and enhance the motility downregulation of HOXD3 led to the accumulation of cells at and invasiveness of human lung cancer A549 (17-19) and the G2 phase of the cell cycle. Our findings revealed that the melanoma cells (20). Additionally, studies have shown that HOXD3 gene promotes CRC cell growth and plays a pivotal HOXD3 induces angiogenesis by increasing pro-angiogenic role in the development and survival of malignant human molecules (21-25). While these studies clearly demonstrated colorectal cancer cells. that HOXD3 is involved in the development and growth of various types of cancers, the functional role of HOXD3 in Introduction human CRC has not yet been determined. In the present study, we demonstrated that HOXD3 is highly Worldwide, colorectal carcinoma (CRC) is one of the most expressed in the human CRC RKO cell line. Consequently, common malignant cancers and is the second most frequent we used a lentiviral vector to deliver small interfering RNA cause of cancer-related deaths (1-3). Despite marked advances (siRNA) to knock down HOXD3 expression in the RKO cells. in diagnostic and therapeutic approaches, the prognosis for Finally, we assessed the effects of HOXD3 knockdown on CRC patients remains poor (4). Consequently, there is an human CRC cell growth and survival in vitro. urgent need to develop new CRC treatment methods. Since homeobox-containing (HOX) genes encode Materials and methods DNA-binding proteins, they are master transcriptional Cell lines. DLD-1, HCT-116, SW620, HT-29, and RKO human colon carcinoma cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, Correspondence to: Professor Guoping Sun, Department of China). Cell lines were maintained in Dulbecco's modi- Oncology, The First Affiliated Hospital of Anhui Medical University, fied Eagle's medium (DMEM; Corning, Shanghai, China) 218 Jixi Road, Hefei, Anhui 230022, P.R. China supplemented with 10% fetal bovine serum (FBS; Ausbian, E-mail: [email protected] Australia) at 37˚C in a 5% CO2 incubator. Key words: colorectal cancer, HOXD3, RKO cells, proliferation, colony formation, apoptosis Quantitative RT-PCR. Total RNA was extracted from the cells in each group (DLD-1, HCT-116, SW620, HT-29, and RKO) 1794 CHEN et al: HOXD3 knockdown inhibits RKO cell growth using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), post-infection, the cells were incubated with 3-(4,5-dimethyl- according to the manufacturer's protocol. Two micrograms of thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ total RNA from each sample was reverse transcribed to single- ml; Promega, Shanghai, China) at a final concentration of stranded cDNA. One microgram of cDNA was used as a 0.5 mg/ml for 4 h. After discarding the supernatants, 150 µl template for the quantitative real-time PCR. The primers used of dimethyl sulfoxide (DMSO; Sigma-Aldrich Co., LLC, St. were as follows: HOXD3 forward, 5'-CGG CAA CTT CGT Louis, MO, USA) was added to each well. The plates were read CGA GTC C-3' and reverse, 5'-ATG AGG GTC GCA AGG at 490 nm using an ELISA reader (Tecan Infinite, Männedorf, TCC A-3'; and GAPDH forward, 5'-TGA CTT CAA CAG Switzerland). All experiments were performed in triplicate. CGA CAC CCA-3' and reverse, 5'-CAC CCT GTT GCT GTA GCC AAA-3'. Cycling conditions for quantitative RT-PCR Cell cycle distribution analysis. Flow cytometry (FCM) were as follows: 95˚C for 30 sec, then 45 cycles of 95˚C for was used to determine cell cycle distribution as previously 5 sec and 60˚C for 30 sec. The PCR products of HOXD3 and described (27). Briefly, RKO cells were infected with shCtrl GAPDH were 145 and 121 bp, respectively. The data were or shHOXD3 vector and incubated at 37˚C for 1, 2, 3, 4, or quantified using the 2 -ΔΔCt method. All analyses were performed 5 days. At the indicated time-point, the cells were collected in triplicate. and washed with ice-cold phosphate-buffered saline (PBS). Cells were centrifuged at 1,000 x g for 10 min and then fixed Recombinant lentiviral vector production and cell infection. in ice-cold 70% ethanol for 30 min at 4˚C. The cells were To create the RNAi target site, the complementary DNA washed with PBS and then resuspended and incubated in PBS sequence (CCA AAT CAC AGC CCA ATA T) of HOXD3 was containing 50 µg/ml propidium iodide (PI; Sigma-Aldrich) designed by Shanghai GeneChem Co., Ltd. (Shanghai, China) and 100 µg/ml RNase A (Fermentas, Shanghai, China) in the using the full-length human sequence (GenBank no. dark at 4˚C for 30 min. Cell cycle phase was analyzed using a NM_006898). The HOXD3 hairpin oligonucleotides were BD FACSCalibur flow cytometer (BD Biosciences, San Diego, synthesized and inserted into the pGV115-GFP (GeneChem CA, USA). All studies were performed in triplicate. Co. Ltd.) lentiviral vector. Lentivirus particles were prepared as previously described (26). Analysis of apoptosis. FCM was used to measure apoptosis For lentiviral infection, RKO cells were cultured in 6-well and was performed as previously described (28). At 48 h plates. The HOXD3-siRNA lentivirus (shHOXD3) or negative prior to transfection, the RKO cells were seeded in 6-well control lentivirus (shCtrl) was added according to a multiplicity plates. At 72 h post-transfection, the cells were collected and of infection (MOI 1:5). At 3 days post-infection, the cells were washed twice with ice-cold PBS. Cell densities were adjusted observed for presence of the GFP marker with a fluorescence to 1x106/ml using 1X staining buffer. Cell suspension (100 µl) microscope (MicroPublisher 3.3RTV; Olympus, Tokyo, and 5 µl Annexin V-APC (eBioscience, San Diego, CA, USA) Japan). At 5 days post-infection, the cells were harvested and were thoroughly mixed and incubated in darkness at room knockdown efficiency was analyzed by quantitative RT-PCR temperature for 15 min. All rates of apoptosis were measured and western blot analysis. by FCM within 1 h. Each experiment was performed in triplicate. Western blot analysis. While on ice, cell lysates were incu- bated for 10-15 min in ice-cold lysis buffer (100 mM Tris, Cell colony formation assay. At 72 h post-transfection, the pH 6.8, 2% β-mercaptoethanol, 20% glycerol, 4% SDS). The RKO cells were reseeded at 600 cells/well in 6-well plates lysates were centrifuged at 12,000 x g for 15 min at 4˚C, and and cultured at 37˚C for 10 days. The cells were collected and the supernatants were collected. Protein concentration was washed with ice-cold PBS. When a majority of single colonies determined using a BCA protein assay kit (Beyotime, Beijing, contained more than 50 cells, the samples were fixed using China). An equal amount of total protein from each sample 1 ml/well of 4% paraformaldehyde (Sinopharm, Shanghai, was partially separated in a 10% SDS-PAGE gel and blotted China) for 30 min at 37˚C. According to instructions, the onto PVDF membranes. Membranes were incubated with GFP samples were stained with 500 µl of Giemsa stain (Dingguo or GAPDH primary antibodies (Santa Cruz Biotechnology, Bio, Shanghai, China) at room temperature for 10 min and Santa Cruz, CA, USA) at 4˚C overnight, followed by horse- images were acquired. All experiments were performed in radish peroxidase (HRP)-conjugated goat anti-mouse IgG triplicate.
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