Wang et al. Experimental & Molecular Medicine (2018) 50:57 DOI 10.1038/s12276-018-0082-5 Experimental & Molecular Medicine ARTICLE Open Access Long noncoding RNA DANCR promotes colorectal cancer proliferation and metastasis via miR-577 sponging Yong Wang1,ZhiLu2, Ningnin Wang3,JianzhouFeng1, Junjie Zhang4, Lan Luan4,WeiZhao1 and Xiandong Zeng 5 Abstract Long non-coding RNAs (lncRNAs) play key roles in various malignant tumors, including colorectal cancer (CRC). Long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) is overexpressed in CRC patients, but whether it affects CRC proliferation and metastasis via regulation of heat shock protein 27 (HSP27) remains unclear. In the present study, we found that DANCR was highly expressed and correlated with proliferation and metastasis in CRC. In addition, we demonstrated that DANCR and HSP27 were both targets of microRNA-577 (miR-577) and shared the same binding site. Furthermore, we revealed that DANCR promoted HSP27 expression and its mediation of proliferation/metastasis via miR-577 sponging. Finally, using an in vivo study, we confirmed that overexpression of DANCR promoted CRC tumor growth and liver metastasis. The present study demonstrated the function of DANCR in CRC and might provide a new target in the treatment of CRC. 1234567890():,; 1234567890():,; Introduction chromosome 4q12, has been reported to act as an onco- As the third most common cancer, colorectal cancer gene in various malignant tumors. Ma et al.7 reported that (CRC) ranks as one of the leading causes of cancer-related elevation of DANCR promoted tumor growth and metastasis death worldwide1. It has been reported that approximately in hepatocellular carcinoma. Jiang et al.8 revealed that more than a half of CRC patients die from distant DANCR promoted tumor progression and cancer stemness – metastasis, especially liver metastasis2 4. Most CRC features in osteosarcoma via inhibition of miR-33a-5p. Liu9 patients with already existing liver metastasis are not found that over-expression of DANCR was associated with suitable for surgical treatment and therefore have a 5-year advanced tumor progression and a poor prognosis in col- survival rate of <10%5, 6. Thus, it is critical to identify new orectal cancer patients. However, the detailed mechanism molecules and the underlying mechanisms associated concerning how DANCR functions remains unclear. with metastasis development. Among the multiple working mechanisms of lncRNAs, Long non-coding RNAs (lncRNAs) are a group of >200- the competitive endogenous RNA (ceRNA) theory was nucleotide non-coding RNAs involved in numerous dis- first proposed by Salmena and became prevalent and eases, including CRC. Differentiation antagonizing non- widely accepted10. The ceRNA hypothesis describes protein coding RNA (DANCR), located on human crosstalk between lncRNA and RNA transcript that occurs when they share the same miRNA response ele- ments (MREs)11. In the present study, we revealed that Correspondence: Xiandong Zeng ([email protected]) DANCR and heat shock protein 27 (HSP27), a down- 1The 4th Department of Orthopedic Surgery, Central Hospital Affiliated to Shenyang Medical College, Shenyang 110024, China stream target of microRNA-577 (miR-577) in CRC, 2Department of Nuclear Medicine, The First Affiliated Hospital of Dalian shared binding sites with miR-577. In addition, we Medical University, Dalian 116011, China found that overexpression of DANCR promoted CRC Full list of author information is available at the end of the article These authors contributed equally: Yong Wang, Zhi Lu. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.Toviewacopyofthislicense,http:// creativecommons.org/licenses/by-nc-nd/4.0/. Official journal of the Korean Society for Biochemistry and Molecular Biology Wang et al. Experimental & Molecular Medicine (2018) 50:57 Page 2 of 17 Fig. 1 Elevated DANCR is expressed in colorectal cancer tissues and cell lines. a, b DANCR endogenous expression levels were elevated in 91.49% (43/47) of CRC tissue specimens compared with those in matched non-CRC tissue specimens, as determined by qRT-PCR using GAPDH as the normalization control. **P < 0.01 vs. the non-CRC group. c The mean optical densities of DANCR were gradually elevated according to the grading of different clinical stages of CRC tissue specimens, as detected by in situ hybridization, normalized to the clinical stage I group. **P < 0.01, ***P < 0.001 vs. clinical stage I. d Endogenous expression of DANCR was up-regulated in the CRC cell lines HT29, HCT116, SW480, and LOVO compared with that in the normal human colon epithelial cell line NCM460, as measured by qRT-PCR using GAPDH as the normalization control. **P < 0.01, ***P < 0.001 vs. NCM460 group. The data are shown as the means ± SD from three independent experiments proliferation and metastasis, which were regulated by diagnosis, and the clinical stage of these patients was HSP27 acting as a ceRNA of miR-577. determined according to the tumor, node, metastasis (TNM) classification of the International Union Against Materials and methods Cancer (UICC). Written informed consent was obtained Patient and tissue samples from all participants. The Institute Research Medical In total 47 cases of CRC tissues and matched non-CRC Ethics Committee of Central Hospital Affiliated to She- tissues were used in this study and were collected after nyang Medical College granted approval for this study. receiving permission from patients during tumorectomy at the Central Hospital Affiliated to Shenyang Medical Cell culture College from February 2016 to February 2017. All 47 The human colon cancer cell lines HT29, HCT116, cases were diagnosed according to a definite pathological SW480, and LOVO and normal human colon epithelial Official journal of the Korean Society for Biochemistry and Molecular Biology Wang et al. Experimental & Molecular Medicine (2018) 50:57 Page 3 of 17 cell line NCM460 were purchased from the American Plasmid construction Type Culture Collection and were cultured in RPMI1640 DANCR fragments containing miR-577 binding sites medium (Gibco, El Paso, TX, USA) supplemented with 10 were amplified and cloned into pmirGLO vectors (Pro- % (v/v) fetal bovine serum (FBS, Sigma, St. Louis, MO, mega, Madison, WI, USA) to synthesize the wild-type USA), 100 IU/ml penicillin and 100 mg/ml streptomycin DANCR reporter plasmid pmirGLO-DANCR-wt. The (Baomanbio, Shanghai, China) at 37 °C in a humidified putative binding sites of miR-577 in DANCR were atmosphere containing 5% CO2. mutated using a QuikChange Site-Directed Mutagenesis kit (Agilent, Santa Clara, CA, USA) to generate the mutant DANCR reporter plasmid pmirGLO-DANCR- mut. The pmirGLO-HSP27-wt and pmirGLO-HSP27- Table 1 Correlation of DANCR expression and mut reporter plasmids were constructed using the same clinicopathological features in CRC method. The above plasmids were used for subsequent Features No. of cases DANCR p value luciferase reporter assays. Similarly, DANCR fragments containing miR-577 binding sites were amplified and High Low cloned into the KpnI and XhoI restriction sites (Promega, Age at diagnosis 0.571* USA) of the pcDNA3.1 vector to synthesize pcDNA3.1- DANCR-wt, and pcDNA3.1-DANCR-mut was also con- ≤ 50 22 11 10 structed using the QuikChange Site-Directed Mutagen- >50 25 11 14 esis kit (Agilent, USA). Four shRNAs against the human Gender 0.664* DANCR gene (NR_145129.1) (DANCR shRNA) and a Female 24 13 11 non-targeting control sequence (NC shRNA) were ligated into the pLKO.1 plasmid vector (Sigma, USA). These Male 23 11 12 plasmids were used to construct DANCR up-regulation Location 0.671& and down-regulation cell models. Meanwhile, the same Colon 26 12 14 method was applied to construct the wild-type and Rectal 21 11 10 mutant HSP27 overexpression plasmids pcDNA3.1- & HSP27-wt and pcDNA3.1-HSP27-mut, respectively. Oli- Clinical stage 0.043 gonucleotides encoding the miR-577 precursor and anti- I936miR-577 precursor were ligated into the pGPH1/GFP/ II 10 3 7 Neo (pre-miR-577) and pGPU6/GFP/Neo plasmid (anti- III 17 12 5 miR-577) vectors (GenePharma, Shanghai, China), respectively. Plasmids carrying a non-targeting control IV 12 9 3 sequence were used as a negative control (pre-miR-577- T classification 0.078* NC and anti-miR-577-NC). These plasmids were used to T1+T2 16 6 10 construct the miR-577 up-regulation and down- T3+T4 31 20 11 regulation cell models. N classification 0.016& Cell transfection No 14 4 10 All of the plasmids used for the construction of the Yes 33 22 11 DANCR or miR-577 up-regulation and down-regulation M classification 0.003& cell models were studied in an up-regulation setting. For transient cell transfection, the CRC cell lines HT29 and M0 15 4 11 LOVO at 60–80% confluence were prepared for trans- M1 32 23 9 fection. The plasmids were transfected into HT29 and Liver metastasis LOVO cells using Lipofectamine 2000 (Invitrogen, ’ No 19 6 13 0.007* Carlsbad, CA, USA) according to the manufacturer s protocol. For stable cell transfection, cells were selected in Yes 28 20 8 culture medium containing 0.4 mg/ml geneticin (G418; Pathologic differentiation 0.990* Invitrogen).