Introduction Odd-skipped related transcription factor 1 (OSR1) is usually a newly identified tumor suppressor in many tumor types. invasion and migration in COAD cells in vitro. Mechanistically, OSR1 exerted anticancer effects by inhibiting FAK-mediated activation of Akt and MAPK pathways. Conclusion Our findings suggest that OSR1 functions as a tumor suppressor in COAD by suppressing FAK-mediated activation of Akt and MAPK pathways. strong class=”kwd-title” Keywords: OSR1, colon adenocarcinoma, tumor suppressor, FAK, Akt, MAPK Introduction Colon adenocarcinoma (COAD) is one of the most common malignancies worldwide. The incidence of COAD ranks the third among malignancies, and the lethality of COAD ranks the second among malignancies.1 Despite the development of advanced diagnostic and therapeutic techniques, more than half of COAD patients die every year, mainly Actinomycin D supplier because they are diagnosed at an advanced stage.2 Therefore, it is urgent to further understand the mechanism of COAD and identify the key molecules involved in COAD progression. The odd-skipped related transcription factor 1 (OSR1) gene is located at human 2p24.1.3,4 OSR1 is a protein of 266 amino acids containing three highly conserved C2H2 zinc finger domains, a tyrosine kinase phosphorylation site (Tyr 203) and several hypothetical proline-XX-proline (PXXP) SH3 binding Actinomycin D supplier motifs. OSR1 is usually expressed in the human colon, small intestine, bladder, testicles, fetal lungs, mesenchymal stem cells and osteoblasts.5 OSR1 is an important regulator of embryo, heart and genitourinary development.6,7 In recent years, increasing studies have suggested that OSR1 exerts antitumor effect in multiple tumors, including gastric cancer,4 tongue squamous carcinoma,8 renal cell carcinoma,9 and lung adenocarcinoma.10,11 However, the role of OSR1 in COAD is not fully understood. Therefore, in our study, we focused on the role and mechanism of OSR1 in COAD. Materials and Methods Patient Samples and Immunohistochemistry (IHC) Total 21 fresh COAD and corresponding paracancerous colon tissue samples were collected from patients who underwent surgery at the First Affiliated Hospital of Chongqing Medical University for mRNA detection, and 91 formalin-fixed, paraffin-embedded COAD tissue samples were collected from patients who underwent surgery at the First Affiliated Hospital of Chongqing Medical University or college between 2012 and 2013 for IHC. The patients were enrolled based on the following inclusion criteria: (1) no radiotherapy or chemotherapy before surgery and (2) no other history of surgery. Our protocol was in accordance with the ethical guidelines of the Declaration of Helsinki and was Mouse monoclonal to CD54.CT12 reacts withCD54, the 90 kDa intercellular adhesion molecule-1 (ICAM-1). CD54 is expressed at high levels on activated endothelial cells and at moderate levels on activated T lymphocytes, activated B lymphocytes and monocytes. ATL, and some solid tumor cells, also express CD54 rather strongly. CD54 is inducible on epithelial, fibroblastic and endothelial cells and is enhanced by cytokines such as TNF, IL-1 and IFN-g. CD54 acts as a receptor for Rhinovirus or RBCs infected with malarial parasite. CD11a/CD18 or CD11b/CD18 bind to CD54, resulting in an immune reaction and subsequent inflammation approved by Ethical Review Committee of the First Affiliated Hospital of Chongqing Medical University or college. All patients signed written informed consent. IHC was conducted using IHC kit (ZSGB-BIO, China) according to the manufacturers protocols, and the results were evaluated based on staining intensity (0, no staining; 1, poor staining; 2, moderate staining; and 3, strong staining) and extent (1, 25%; 2, 25C50%; 3, 50C75%; and 4, 75%). Cell Culture and Transfection SW480, HT29, HCT116, HCT-8, SW620, and LoVo human COAD cells were purchased from your American Type Culture Collection (USA), and cultured in RPMI 1640 medium (HyClone, USA) made up of 10% fetal bovine serum at 37C with 5% CO2. COAD cells were divided into seven groups: the Vector group (cells transfected with blank lentivirus pEZ-Lv105-vector), the OSR1 group (cells transfected with recombinant lentivirus pEZ-Lv105-OSR1), the siCtrl group (cells transfected with a negative control siRNA), the siOSR1#1 group (cells transfected with the siRNA#1 targeting OSR1), the siOSR1#2 group (cells transfected with the siRNA#2 targeting OSR1), the PF573228 group (cells treated with the PF573228), the PF573228+siOSR1 group (cells transfected with the siRNA#1 or 2 targeting OSR1 and treated with the PF573228), and the PF573228+OSR1 group (cells transfected with recombinant lentivirus vector pEZ-Lv105-OSR1 Actinomycin D supplier and treated with the PF573228). The recombinant lentivirus vector pEZ-Lv105-OSR1 (OSR1 group) and blank pEZ-Lv105-vector (vector group) were purchased from Genecopoeia (USA). The recombinant lentivirus was transfected into COAD cells. Seventy-two hours after transfection, puromycin was used to select the stably transfected cell lines. Two small interfering RNAs (siRNAs) targeting OSR1 (siOSR1#1: 5-ACCGGTGAGACTGGTCCAATA-3 and siOSR1#2: 5-TTCTCCGAACGTGTCACGT-3), Actinomycin D supplier and a negative control (siCtrl) were provided by RiboBio (Guangzhou, China). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) RNA was extracted from tissue or cells using RNAiso Plus (Takara Biotechnology, Japan). RNA was reverse transcribed into cDNA using a PrimeScript? RT Master Mix cDNA Synthesis Kit (Takara Biotechnology,.