Email this article 
Hide
Show all
Knockdown by MSH2 and EPCAM siRNA suppress Wnt/β-catenin pathway in HCT116 cell line
Abstract
Small interfering RNA (siRNA) has the potential as a therapeutic approach against selective pathways in colorectal cancer (CRC). EPCAM, a transmembrane glycoprotein mediating cell adhesion, was known to be involved in suppressing Wnt/β-catenin pathway, an important pathway for tumour progression in colon cancer cells. EPCAM deletions caused a transcriptional read-through that may silence its neighbouring gene, MSH2. This study aimed to investigate the effect of co-siRNA targeted genes, MSH2 and EPCAM, in colon cancer cell line, HCT116, and their effect in regulating the Wnt/β-catenin pathway. Methods: Pre-designed siRNA of MSH2 and EPCAM were transfected into HCT116 cells. The cells were divided into six group of treatments: untreated cell group, cells treated with negative control siRNA, MSH2-siRNA treated cells, EPCAM-siRNA treated cells, cells treated with both EPCAM and MSH2-siRNA, and cells treated with transfection reagent (mock control). The mRNA and protein expression following the individual and combined siRNA treatments were assessed by quantitative polymerase chain reaction and Western blot. Results: The mRNA and protein expression levels of MSH2, EPCAM and β-catenin were reduced in the individual MSH2 and EPCAM-siRNA treated samples as compared to the untreated sample. Further reduction of mRNA and protein expressions for MSH2, EPCAM and β-catenin were identified in combined siRNA treatments. Conclusion: Reduction of β-catenin expression by simultaneous silencing of MSH2 and EPCAM suggested that these genes may play a role in supressing the Wnt/β-catenin pathway in cancer cells.
Keywords
Full Text:
PDFReferences
Haggar FA, Boushey RP. Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors. Clin Colon Rectal Surg. 2009;22(4):191-197.
Rawla P, Sunkara T, Barsouk A. Epidemiology of colorectal cancer: Incidence, mortality, survival, and risk factors. Prz Gastroenterol. 2019;14(2):89-103
Peltomäki P, De la Chapelle A. Mutations predisposing to hereditary nonpolyposis colorectal cancer. Adv Cancer Res. 1997;71:94-119.
Tutlewska K, Lubinski J, Kurzawski G. Germline deletions in the EPCAM gene as a cause of Lynch syndrome–literature review. Hered Cancer Clin Pract. 2013;11(1):1-9.
Ligtenberg MJ, Kuiper RP, van Kessel AG, Hoogerbrugge N. EPCAM deletion carriers constitute a unique subgroup of Lynch syndrome patients. Fam Cancer. 2013;12(2):169-74.
Guarinos C, Castillejo A, Barberá V-M, Pérez-Carbonell L, Sánchez-Heras A-B, Segura Á, et al. EPCAM germ line deletions as causes of Lynch syndrome in Spanish patients. J Mol Diagn. 2010;12(6):765-70.
Zhou F, Qi Y, Xu H, Wang Q, Gao X, Guo H. Expression of EpCAM and Wnt/beta-catenin in human colon cancer. Genet Mol Res. 2015;14(2):4485-94.
Ligtenberg MJ, Kuiper RP, Chan TL, Goossens M, Hebeda KM, Voorendt M, et al. Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3′ exons of TACSTD1. Nat Genet. 2009;41(1):112.
Miyaki M, Iijima T, Kimura J, Yasuno M, Mori T, Hayashi Y, et al. Frequent mutation of β-catenin and APC genes in primary colorectal tumors from patients with hereditary nonpolyposis colorectal cancer. Cancer Res. 1999;59(18):4506-9.
Polakis P. Wnt signaling in cancer. Cold Spring Harbor perspectives in biology. 2012; 4(5):a008052.
Liang W, Mason AJ, Lam JK. Western blot evaluation of siRNA delivery by pH-responsive peptides. Target Identification and Validation in Drug Discovery: Methods and Protocols. 2013:73-87.
Pirini F, Tedaldi G, Danesi R, Cangini I, Tumedei MM, Ferrari A, et al. Identification of a novel large EPCAM-MSH2 duplication, concurrently with LOHs in chromosome 20 and X, in a family with Lynch syndrome. Int J Colorectal Dis. 2019;34(11):1999-2002.
Doan CC, Le LT, Hoang SN, Do SM, Van Le D. Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells. Biol Res. 2014;47(1):70.
Xu C-f, Wang J. Delivery systems for siRNA drug development in cancer therapy. Asian J Pharm Sci. 2015;10(1):1-12.
Daka A, Peer D. RNAi-based nanomedicines for targeted personalized therapy. Adv Drug Deliv Rev. 2012;64(13):1508-21.
Shen K, Ye Y, Jiang K, Liang B, Yang X, Wang S. MSH2 is required for cell proliferation, cell cycle control and cell invasiveness in colorectal cancer cells. Chin Sci Bull. 2012;57(20):2580-5.
Bultman SJ. Interplay between diet, gut microbiota, epigenetic events, and colorectal cancer. Mol Nutr Food Res. 2017;61(1):1500902.
Nagasaka T, Rhees J, Kloor M, Gebert J, Naomoto Y, Boland CR, et al. Somatic hypermethylation of MSH2 is a frequent event in Lynch Syndrome colorectal cancers. Cancer Res. 2010;70(8):3098-108.
Yamashita T, Budhu A, Forgues M, Wang XW. Activation of hepatic stem cell marker EpCAM by Wnt–β-catenin signaling in hepatocellular carcinoma. Cancer Res. 2007;67(22):10831-9.
Novellasdemunt L, Antas P, Li VS. Targeting Wnt signaling in colorectal cancer. A review in the theme: cell signaling: proteins, pathways and mechanisms. Am J Physiol Cell Physiol. 2015;309(8):C511-21.
Liu C, Li Y, Semenov M, Han C, Baeg G-H, Tan Y, et al. Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell. 2002;108(6):837-47.
Castiglia D, Bernardini S, Alvino E, Pagani E, De Luca N, Falcinelli S, et al. Concomitant activation of Wnt pathway and loss of mismatch repair function in human melanoma. Genes Chromosomes Cancer. 2008;47(7):614-24.
Belcheva A, Irrazabal T, Robertson SJ, Streutker C, Maughan H, Rubino S, et al. Gut microbial metabolism drives transformation of MSH2-deficient colon epithelial cells. Cell. 2014;158(2):288-99.
Ji J, Yamashita T, Wang XW. Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma. Cell Biosci. 2011;1(1):1-8.
Network CGA. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407):330.
Liang G, Fang X, Yang Y, Song Y. Silencing of CEMIP suppresses Wnt/β-catenin/Snail signaling transduction and inhibits EMT program of colorectal cancer cells. Acta Histochem. 2018;120(1):56-63.
Li J, Zhang Z, Wang L, Zhang Y. The oncogenic role of Wnt10a in colorectal cancer through activation of canonical Wnt/β‑catenin signaling. Oncol Lett. 2019;17(4):3657-64.
Van de Water FM, Boerman OC, Wouterse AC, Peters JG, Russel FG, Masereeuw R. Intravenously administered short interfering RNA accumulates in the kidney and selectively suppresses gene function in renal proximal tubules. Drug Metab Dispose. 2006;34(8):1393-7.
Copyright (c) 2023 Journal of Biomedical and Clinical Sciences (JBCS)
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Copyright © 2016 AMDI Publisher, Universiti Sains Malaysia.
Disclaimer : This website has been updated to the best of our knowledge to be accurate. However, Universiti Sains Malaysia shall not be liable for any loss or damage caused by the usage of any information obtained from this web site.
Best viewed: Mozilla Firefox 4.0 & Google Chrome at 1024 × 768 resolution.