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Effect of HOXA6 on the Proliferation, Apoptosis, Migration and Invasion of Colorectal Cancer Cells

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Effect of HOXA6 on the Proliferation, Apoptosis, Migration and Invasion of Colorectal Cancer Cells INTERNATIONAL JOURNAL OF ONCOLOGY 52: 2093-2100, 2018 Effect of HOXA6 on the proliferation, apoptosis, migration and invasion of colorectal cancer cells SHASHA WU1*, FEIXIANG WU2* and ZHENG JIANG1 Departments of 1Gastroenterology and 2Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China Received December 1, 2017; Accepted March 27, 2018 DOI: 10.3892/ijo.2018.4352 Abstract. Colorectal cancer (CRC) is one of the most common B-cell lymphoma-2 (Bcl-2)-associated X protein, caspase-3, types of tumor worldwide. The morbidity and mortality rates poly(ADP-ribose) polymerase and E-cadherin, but promoted of CRC have increased significantly in adults <50 years of age. the expression of Bcl-2, N-cadherin and Vimentin, whereas In the present study, the effects of homeobox A6 (HOXA6) the opposite effect was observed in cells with downregulated on the proliferation, apoptosis, migration and invasion of HOXA6. These results indicated that HOXA6 regulated CRC cells were investigated. The results of reverse tran- apoptosis through the Bcl-2 signaling pathway, and regulated scription-quantitative polymerase chain reaction (RT-qPCR) migration and invasion through the EMT process. In conclu- analysis demonstrated that the expression of HOXA6 in sion, the present study confirmed that HOXA6 was involved in CRC tumor tissue was higher than that in adjacent normal the regulation of CRC, which may inform the development of tissue. Appropriate cell lines and plasmids were selected by strategies for the diagnosis and treatment of CRC. RT-PCR and western blot analyses, and recombinant plasmids were transfected into Caco2 or HT-29 cells. The results of Introduction RT-qPCR and western blot analyses demonstrated that the expression of HOXA6 was effectively enhanced, or inhibited, Colorectal cancer (CRC) is one of the leading causes of following transfection. The rate of cell proliferation was cancer-associated mortality worldwide. Although the overall measured with cell counting kit-8, colony formation assay morbidity and mortality rates for CRC have been declining for and 5-ethynyl-2'-deoxyuridine assay, apoptosis was detected decades, they have increased considerably in adults <50 years using terminal deoxynucleotidyl transferase dUTP nick end of age. In addition, the 5-year relative survival rate for patients labeling and flow cytometry assays, and migration and inva- with CRC diagnosed at an advanced stage is just 14%, as not sion were evaluated using Transwell and wound-healing all of the tumor can be surgically removed (1-3). In addition to assays. The results demonstrated that the upregulation of old age and lifestyle factors, genetic changes are also a cause HOXA6 promoted cell proliferation, migration and invasion, of CRC (4). In these cases of CRC, targeted therapy may be but inhibited apoptosis, whereas the downregulated expres- effective. Therefore, the identification of novel tumor markers sion of HOXA6 produced the opposite effects. In addition, the may contribute to the diagnosis and treatment of CRC, such expression levels of apoptosis- and epithelial-mesenchymal as the effect of the clinical application of carcinoembryonic transition (EMT)-related proteins were examined. The antigen and carbohydrate antigen 19-9 (5). results of the western blot analysis revealed that the upregu- The homeobox (HOX) family includes a series of devel- lated expression of HOXA6 suppressed the expression of opmental genes. The encoded homeodomain proteins are important transcription factors that bind to specific DNA sequences as monomers or multimers to regulate cell differ- entiation, proliferation and apoptosis. At present, 39 human Correspondence to: Dr Zheng Jiang, Department of Gastroenterology, HOX genes have been identified, which are divided into four The First Affiliated Hospital of Chongqing Medical University, 1 Youyi clusters: A, B, C and D. Previous studies have reported that Road, Chongqing 400016, P.R. China HOX family genes are involved in the development of human E-mail: [email protected] malignancies and are associated with prognosis (6,7). It was previously observed that HOXA9, HOXA13 and HOXB7 *Contributed equally were overexpressed in esophageal cancer, and promoted the Abbreviations: CRC, colorectal cancer; CCK-8, cell counting proliferation of esophageal cancer cells (8-10). The expres- kit-8; RT-qPCR, reverse transcription-quantitative polymerase chain sion levels of HOXA1, HOXA10, HOXA13, HOXB7 and reaction; EMT, epithelial-mesenchymal transition HOXC6 were also found to be increased in gastric cancer tissue, and may promote cell proliferation and metastasis, in Key words: homeobox A6, colorectal cancer, cell proliferation, addition to being associated with prognosis (11-17). In CRC, apoptosis, invasion HOXB7 was also reported to be expressed at a high level, and interacted with the mitogen-activated protein kinase and 2094 WU et al: HOXA6 AFFECTS CRC CELL PROLIFERATION, APOPTOSIS, MIGRATION AND INVASION phosphoinositide 3-kinase/AKT pathways to promote tumor Table Ⅰ. Characteristics of the patients with colorectal cancer. cell proliferation (18). HOXA6 is a member of the HOX family and encodes a Age Sample collection date DNA-binding transcription factor, which may regulate gene (years) Sex Diagnosis (all in the year 2017) expression, morphogenesis and differentiation. Previous studies have reported that the HOXA6 gene is expressed at 74 Male Colon carcinoma September 28 a high level in several types of malignant tumor, including 72 Male Rectal carcinoma September 30 cerebral glioma and acute myeloid leukemia, and that it is 67 Male Rectal carcinoma October 9 associated with cell invasion, proliferation, colony formation 63 Female Rectal carcinoma October 11 and chemosensitivity (19,20). HOX family members have been 63 Male Colon carcinoma October 13 confirmed to be involved in tumor regulation, including in 68 Male Rectal carcinoma October 13 the occurrence and development of CRC. As HOXA6 is also 65 Female Rectal carcinoma October 14 involved in the regulation of certain types of malignant tumor, 62 Female Colon carcinoma October 17 it is reasonable to suggest that HOXA6 may have a similar 66 Female Colon carcinoma October 20 effect on tumor regulation in CRC. Based on this, the aim of 74 Male Colon carcinoma October 21 the present study was to investigate the expression of HOXA6 63 Male Colon carcinoma October 25 in CRC, and the effect of the expression of HOXA6 on the proliferation, apoptosis, migration and invasion of CRC cells. 80 Female Colon carcinoma October 25 69 Male Colon carcinoma October 31 Materials and methods 65 Female Colon carcinoma November 6 49 Male Colon carcinoma November 9 Patient specimens. A total of 16 CRC tumor and paired adjacent 63 Male Colon carcinoma November 10 normal colorectal tissue samples were collected from the First Affiliated Hospital of Chongqing Medical University (Chongqing, China) between September 28, 2017 and November 11, 2017 (Table I). Informed consent was signed by all patients, and 5'-TGGAACGGTGAAGGTGACAG-3' and reverse, 5'-AAC ethics approval was obtained from the Ethics Committee of the AACGCATCTCATATTTGGAA-3' (product length, 125 bp). First Affiliated Hospital of Chongqing Medical University. RT-qPCR was performed with GoTaq qPCR Master mix (Promega Corporation) (nuclease-free water: 3.6 µl; upstream Cell culture and transfection. The Caco2, HCT116, SW480 and primer: 0.2 µl; downstream primer: 0.2 µl; 2X GoTaq® qPCR HT-29 human CRC cell lines were purchased from the Master Mix: 5 µl; cDNA: 1 µl. Thermocycling steps: American Type Culture Collection (Manassas, VA, USA) and 95˚C-10 min; 95˚C-15 sec; 60˚C-1 min, 40 cycles) and the data cultured in RPMI-1640 medium (HyClone/GE Healthcare Life were analyzed using the 2-∆∆Cq method (22). RT-PCR was Sciences, Logan, UT, USA) supplemented with 10% fetal performed with GoTaq polymerase (Promega Corporation) bovine serum (PAN-Biotech GmbH, Aidenbach, Germany) at according to the manufacturer's protocol (2X GoTaq Green 37˚C in 5% CO2. Plasmids expressing HOXA6, short hairpin Master Mix: 5 µl; upstream primer: 0.6 µl; downstream primer: interfering RNA against HOXA6 (shHOXA6; sh1, 5'-CCTTGT 0.6 µl; cDNA: 2 µl; nuclease-free water: 1.8 µl. Thermocycling TTCTACCAACAGTCC-3'; sh2, 5'-CCTCGTGTTTCTATT steps: 95˚C-2 min; 95˚C-30 sec, 55˚C-30 sec, 72˚C-30 sec; CTGATA-3'; sh3, 5'-GGCGCGCAAATGAGTTCCTAT-3'; 72˚C-3 min. HOXA6: 32 cycles, β-actin: 23 cycles.). The PCR sh4, 5'-GACAAGACGTACACCTCACCT-3') (21) or a negative products were analyzed with 2% agarose gel electrophoresis,and control (NC) sequence (non-targeting shRNA) were purchased subjected to densitometric analysis with a gel imaging system from GeneCopeia, Inc. (Rockville, MD, USA). The CRC cells (Bio-Rad Laboratories, Inc., Hercules, CA, USA). were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according Western blot analysis. Proteins were extracted from the cells to the manufacturer's protocol. For the selection of stable cell at 72 h post-transfection using RIPA lysis buffer (Beyotime lines, G418 and Puromycin (Sigma-Aldrich; Merck Millipore, Institute of Biotechnology, Haimen, China). The protein Darmstadt, Germany) were added to the medium at 48 h concentration was determined using a BCA kit (Beyotime following transfection and the cells were maintained under the Institute of Biotechnology). Subsequent to 10% SDS-PAGE aforementioned conditions. separation (40 µg), the proteins were
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