Down-regulation of miR-9 promotes epithelial mesenchymal transition via regulating anoctamin-1 (ANO1) in CRC cells

  • Author Footnotes
    1 Contributed equally.
    Young Ran Park
    Footnotes
    1 Contributed equally.
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    1 Contributed equally.
    Soo Teik Lee
    Footnotes
    1 Contributed equally.
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • Se Lim Kim
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • Shi Mao Zhu
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • Min Ro Lee
    Affiliations
    Department of Surgery, Chonbuk National University Hospital, Chonbuk National University Medical School, Jeonju, Republic of Korea
    Search for articles by this author
  • Seong Hun Kim
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • In Hee Kim
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • Seung Ok Lee
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • Seung Young Seo
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • Sang Wook Kim
    Correspondence
    Corresponding author at: Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea.
    Affiliations
    Department of Internal Medicine, Chonbuk National University Hospital, Chonbuk National University Medical School, 20 Geonji-ro, Deokjin-gu, Jeonju, Jeonbuk 54907, Republic of Korea

    Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    1 Contributed equally.

      Highlights

      • ANO1 is significantly up-regulated in CRC tumor with lymph node metastasis and late stage.
      • ANO1 is a direct target of miR-9.
      • Overexpression of miR-9 suppressed cell proliferation, migration, and invasion.
      • miR-9 suppressed p-AKT, cyclin-D1, and p-ERK expression by targeting ANO1 in CRC cells.

      Abstract

      MicroRNA-9 (miR-9) has been reported to play a suppressive or promoting role according to cancer type. In this study, we investigated the effects of anoctamin-1 (ANO1) and miR-9 on colorectal cancer (CRC) cell proliferation, migration, and invasion and determined the underlying molecular mechanisms.
      Thirty-two paired CRC tissues and adjacent normal tissues were analyzed for ANO1 expression using quantitative real-time PCR (qRT-PCR). HCT116 cells were transiently transfected with miR-9 mimic, miR-9 inhibitor, or si-ANO1. Cell proliferation was determined by MTT, and flow cytometric analysis, while cell migration and invasion were assayed by trans-well migration and invasion assay in HCT116 cells. ANO1 was validated as a target of miR-9 using luciferase reporter assay and bioinformatics algorithms.
      We found that ANO1 expression was up-regulated in CRC tissues compared with adjacent normal tissues. ANO1 expression was associated with advanced tumor stage and lymph node metastasis, and there was an inverse relationship between miR-9 and ANO1 mRNA expression in CRC specimens, but no significant difference was found between miR-9 and ANO1 expression. ANO1 is a direct target of miR-9, and overexpression of miR-9 suppressed both mRNA and protein expression of ANO1 and inhibited cell proliferation, migration, and invasion of HCT116 cells. We also showed that overexpression of miR-9 suppressed expression of p-AKT, cyclin D1, and p-ERK in HCT116 cells.
      We conclude that miR-9 inhibits CRC cell proliferation, migration, and invasion by directly targeting ANO1, and miR-9/ANO1 could be a potential therapeutic target for CRC.

      Keywords

      To read this article in full you will need to make a payment

      Subscribe:

      Subscribe to Cancer Genetics
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Jemal A.
        • Bray F.
        • Center M.M.
        • et al.
        Global cancer statistics.
        CA Cancer J Clin. 2011; 61: 69-90
        • Torre L.A.
        • Bray F.
        • Siegel R.L.
        • et al.
        Global cancer statistics, 2012.
        CA Cancer J Clin. 2015; 65: 87-108
        • Kopetz S.
        • Chang G.J.
        • Overman M.J.
        • et al.
        Improved survival in metastatic colorectal cancer is associated with adoption of hepatic resection and improved chemotherapy.
        J Clin Oncol. 2009; 27: 3677-3683
        • Lee R.C.
        • Feinbaum R.L.
        • Ambros V.
        The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.
        Cell. 1993; 75: 843-854
        • Bartel D.P.
        MicroRNAs: genomics, biogenesis, mechanism, and function.
        Cell. 2004; 116: 281-297
        • Wang Y.
        • Liu L.
        • Liu X.
        • et al.
        Shugoshin1 enhances multidrug resistance of gastric cancer cells by regulating MRP1, Bcl-2, and Bax genes.
        Tumour Biol. 2013; 34: 2205-2214
        • Feng H.
        • Wang Y.
        • Su J.
        • et al.
        MicroRNA-148a Suppresses the proliferation and migration of pancreatic cancer cells by down-regulating ErbB3.
        Pancreas. 2016; 45: 1263-1271
        • Chen L.
        • Luo L.
        • Chen W.
        • et al.
        MicroRNA-24 increases hepatocellular carcinoma cell metastasis and invasion by targeting p53: miR-24 targeted p53.
        Biomed Pharmacother. 2016; 84: 1113-1118
        • Wang L.
        • Yu P.
        miR-300 promotes proliferation and EMT-mediated colorectal cancer migration and invasion by targeting p53.
        Oncol Rep. 2016; 36: 3225-3232
        • Liu M.
        • Xiusheng H.
        • Xiao X.
        • et al.
        Overexpression of miR-422a inhibits cell proliferation and invasion, and enhances chemosensitivity in osteosarcoma cells.
        Oncol Rep. 2016; 36: 3371-3378
        • Chi Y.
        • Zhou D.
        MicroRNAs in colorectal carcinoma–from pathogenesis to therapy.
        J Exp Clin Cancer Res. 2016; 35: 43
        • Fiaschetti G.
        • Abela L.
        • Nonoguchi N.
        • et al.
        Epigenetic silencing of miRNA-9 is associated with HES1 oncogenic activity and poor prognosis of medulloblastoma.
        Br J Cancer. 2014; 110: 636-647
        • Jeon H.M.
        • Sohn Y.W.
        • Oh S.Y.
        • et al.
        ID4 imparts chemoresistance and cancer stemness to glioma cells by derepressing miR-9*-mediated suppression of SOX2.
        Cancer Res. 2011; 71: 3410-3421
        • Zhou X.
        • Marian C.
        • Makambi K.H.
        • et al.
        MicroRNA-9 as potential biomarker for breast cancer local recurrence and tumor estrogen receptor status.
        PLoS One. 2012; 7: e39011
      1. Sonneville F, Ruffin M, Coraux C, et al. MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology. 2017;8:710.

        • Selcuklu S.D.
        • Donoghue M.T.
        • Rehmet K.
        • et al.
        MicroRNA-9 inhibition of cell proliferation and identification of novel miR-9 targets by transcriptome profiling in breast cancer cells.
        J Biol Chem. 2012; 287: 29516-29528
        • Yu T.
        • Liu K.
        • Wu Y.
        • et al.
        MicroRNA-9 inhibits the proliferation of oral squamous cell carcinoma cells by suppressing expression of CXCR4 via the Wnt/beta-catenin signaling pathway.
        Oncogene. 2014; 33: 5017-5027
        • Zawistowski J.S.
        • Nakamura K.
        • Parker J.S.
        • et al.
        MicroRNA 9-3p targets beta1 integrin to sensitize claudin-low breast cancer cells to MEK inhibition.
        Mol Cell Biol. 2013; 33: 2260-2274
        • Zheng L.
        • Qi T.
        • Yang D.
        • et al.
        microRNA-9 suppresses the proliferation, invasion and metastasis of gastric cancer cells through targeting cyclin D1 and Ets1.
        PLoS One. 2013; 8: e55719
        • Xu D.
        • Chen X.
        • He Q.
        • et al.
        MicroRNA-9 suppresses the growth, migration, and invasion of malignant melanoma cells via targeting NRP1.
        Onco Targets Ther. 2016; 9: 7047-7057
        • Liu S.
        • Kumar S.M.
        • Lu H.
        • Liu A.
        • Yang R.
        • Pushparajan A.
        • Guo W.
        • et al.
        MicroRNA-9 up-regulates E-cadherin through inhibition of NF-kappaB1-Snail1 pathway in melanoma.
        J Pathol. 2012; 226: 61-72
        • Tsang D.P.
        • Wu W.K.
        • Kang W.
        • et al.
        Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-kappaB in hepatocellular carcinoma.
        J Pathol. 2016; 238: 651-664
        • Zhu M.
        • Xu Y.
        • Ge M.
        • et al.
        Regulation of UHRF1 by microRNA-9 modulates colorectal cancer cell proliferation and apoptosis.
        Cancer Sci. 2015; 106: 833-839
        • Park Y.R.
        • Lee S.T.
        • Kim S.L.
        • et al.
        MicroRNA-9 suppresses cell migration and invasion through downregulation of TM4SF1 in colorectal cancer.
        Int J Oncol. 2016; 48: 2135-2143
        • Huang X.
        • Gollin S.M.
        • Raja S.
        • et al.
        High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells.
        Proc Natl Acad Sci USA. 2002; 99: 11369-11374
        • Katoh M.
        • Katoh M..
        FLJ10261 gene, located within the CCND1-EMS1 locus on human chromosome 11q13, encodes the eight-transmembrane protein homologous to C12orf3, C11orf25 and FLJ34272 gene products.
        Int J Oncol. 2003; 22: 1375-1381
        • Huang X.
        • Godfrey T.E.
        • Gooding W.E.
        • et al.
        Comprehensive genome and transcriptome analysis of the 11q13 amplicon in human oral cancer and synteny to the 7F5 amplicon in murine oral carcinoma.
        Genes Chromosomes Cancer. 2006; 45: 1058-1069
        • Caputo A.
        • Caci E.
        • Ferrera L.
        • et al.
        TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity.
        Science. 2008; 322: 590-594
        • Duvvuri U.
        • Shiwarski D.J.
        • Xiao D.
        • et al.
        TMEM16A induces MAPK and contributes directly to tumorigenesis and cancer progression.
        Cancer Res. 2012; 72: 3270-3281
        • Ubby I.
        • Bussani E.
        • Colonna A.
        • et al.
        TMEM16A alternative splicing coordination in breast cancer.
        Mol Cancer. 2013; 12: 75
        • Atala A.
        Re: inhibition of Ca2+ -activated Cl-channel ANO1/TMEM16A expression suppresses tumor growth and invasiveness in human prostate carcinoma.
        J Urol. 2013; 189: 2393
        • Dixit R.
        • Kemp C.
        • Kulich S.
        • et al.
        TMEM16A/ANO1 is differentially expressed in HPV-negative versus HPV-positive head and neck squamous cell carcinoma through promoter methylation.
        Sci Rep. 2015; 5: 16657
        • Carneiro A.
        • Isinger A.
        • Karlsson A.
        • et al.
        Prognostic impact of array-based genomic profiles in esophageal squamous cell cancer.
        BMC Cancer. 2008; 8: 98
        • Sui Y.
        • Sun M.
        • Wu F.
        • et al.
        Inhibition of TMEM16A expression suppresses growth and invasion in human colorectal cancer cells.
        PLoS One. 2014; 9e115443
        • Mokutani Y.
        • Uemura M.
        • Munakata K.
        • et al.
        Down-regulation of microRNA-132 is associated with poor prognosis of colorectal cancer.
        Ann Surg Oncol. 2016; 23: 599-608
        • Guan L.
        • Song Y.
        • Gao J.
        • et al.
        Inhibition of calcium-activated chloride channel ANO1 suppresses proliferation and induces apoptosis of epithelium originated cancer cells.
        Oncotarget. 2016; 7: 78619-78630
        • Liu J.
        • Liu Y.
        • Ren Y.
        • et al.
        Transmembrane protein with unknown function 16A overexpression promotes glioma formation through the nuclear factor-kappaB signaling pathway.
        Mol Med Rep. 2014; 9: 1068-1074
        • Bae J.S.
        • Park J.Y.
        • Park S.H.
        • et al.
        Expression of ANO1/DOG1 is associated with shorter survival and progression of breast carcinomas.
        Oncotarget. 2018; 9: 607-621
        • Zhao B.
        • Jin Y.
        • Hu Y.
        • et al.
        Down-regulation of TMEM16A inhibited invasion and migration in rectal carcinoma.
        Int J Clin Exp Med. 2016; 9: 21224-21233
        • Ferretti E.
        • De Smaele E.
        • Po A.
        • et al.
        MicroRNA profiling in human medulloblastoma.
        Int J Cancer. 2009; 124: 568-577
        • Deng J.
        • Lei W.
        • Xiang X.
        • et al.
        Cullin 4A (CUL4A), a direct target of miR-9 and miR-137, promotes gastric cancer proliferation and invasion by regulating the Hippo signaling pathway.
        Oncotarget. 2016; 7: 10037-10050
        • Qu Z.
        • Yao W.
        • Yao R.
        • et al.
        The Ca(2+) -activated Cl(-) channel, ANO1 (TMEM16A), is a double-edged sword in cell proliferation and tumorigenesis.
        Cancer Med. 2014; 3: 453-461
        • Shiwarski D.J.
        • Shao C.
        • Bill A.
        • et al.
        To "grow" or "go": TMEM16A expression as a switch between tumor growth and metastasis in SCCHN.
        Clin Cancer Res. 2014; 20: 4673-4688
        • Ji X.
        • Peng Q.
        • Wang M.
        Anti-colon-cancer effects of polysaccharides: a mini-review of the mechanisms.
        Int J Biol Macromol. 2018; 114: 1127-1133
        • Atmaca H.
        • Ozkan A.N.
        • Zora M.
        Novel ferrocenyl pyrazoles inhibit breast cancer cell viability via induction of apoptosis and inhibition of PI3K/Akt and ERK1/2 signaling.
        Chem Biol Interact. 2017; 263: 28-35
        • Britschgi A.
        • Bill A.
        • Brinkhaus H.
        • et al.
        Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling.
        Proc Natl Acad Sci USA. 2013; 110: E1026-E1034
        • Cao Q.
        • Liu F.
        • Ji K.
        • et al.
        MicroRNA-381 inhibits the metastasis of gastric cancer by targeting TMEM16A expression.
        J Exp Clin Cancer Res. 2017; 36: 29