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Linc-DYNC2H1-4 Promotes EMT and CSC Phenotypes by Acting As a Sponge of Mir-145 in Pancreatic Cancer Cells

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Linc-DYNC2H1-4 Promotes EMT and CSC Phenotypes by Acting As a Sponge of Mir-145 in Pancreatic Cancer Cells Citation: Cell Death and Disease (2017) 8, e2924; doi:10.1038/cddis.2017.311 OPEN Official journal of the Cell Death Differentiation Association www.nature.com/cddis Linc-DYNC2H1-4 promotes EMT and CSC phenotypes by acting as a sponge of miR-145 in pancreatic cancer cells Yuran Gao1, Zhicheng Zhang2,3, Kai Li1,3, Liying Gong1, Qingzhu Yang1, Xuemei Huang1, Chengcheng Hong1, Mingfeng Ding*,2 and Huanjie Yang*,1 The acquisition of epithelial–mesenchymal transition (EMT) and/or existence of a sub-population of cancer stem-like cells (CSC) are associated with malignant behavior and chemoresistance. To identify which factor could promote EMT and CSC formation and uncover the mechanistic role of such factor is important for novel and targeted therapies. In the present study, we found that the long intergenic non-coding RNA linc-DYNC2H1-4 was upregulated in pancreatic cancer cell line BxPC-3-Gem with acquired gemcitabine resistance. Knockdown of linc-DYNC2H1-4 decreased the invasive behavior of BxPC-3-Gem cells while ectopic expression of linc-DYNC2H1-4 promoted the acquisition of EMT and stemness of the parental sensitive cells. Linc-DYNC2H1-4 upregulated ZEB1, the EMT key player, which led to upregulation and downregulation of its targets vimentin and E-cadherin respectively, as well as enhanced the expressions of CSC makers Lin28, Nanog, Sox2 and Oct4. Linc-DYNC2H1-4 is mainly located in the cytosol. Mechanically, it could sponge miR-145 that targets ZEB1, Lin28, Nanog, Sox2, Oct4 to restore these EMT and CSC-associated genes expressions. We proved that MMP3, the nearby gene of linc-DYNC2H1-4 in the sense strand, was also a target of miR-145. Downregulation of MMP3 by miR-145 was reverted by linc-DYNC2H1-4, indicating that competing with miR-145 is one of the mechanisms for linc-DYNC2H1-4 to regulate MMP3. In summary, our results explore the important role of linc-DYNC2H1- 4 in the acquisition of EMT and CSC, and the impact it has on gemcitabine resistance in pancreatic cancer cells. Cell Death and Disease (2017) 8, e2924; doi:10.1038/cddis.2017.311; published online 13 July 2017 Pancreatic ductal adenocarcinoma (PDAC) is one of the most Reprogramming) was found to upregulate CSC maker Nanog commonly diagnosed cancers and the fourth leading cause of or EMT inducer ZEB1, leading to increased pancreatic cancer cancer-related death.1 Gemcitabine (2′,2′-difluorodeoxy- invasion and tumorigenesis.10,11 LncRNA H19, an imprinted cytidine), a deoxycytidine analog, represents the first line gene was also found to promote PDAC cell invasion and intervention for the treatment of advanced PDAC, and migrationbyincreasingHMGA2-mediatedEMTthroughantag- demonstrates prolonged overall survival time and improved onizing let-7.12 2–4 life quality. However, resistance of cancer cells to gemci- LncRNAs are transcribed from the intergenic regions, tabine, either intrinsic or acquired during treatment, has overlapping (in sense or antisense orientation), or intronic to been frequently observed in patients and is considered as 5 protein-coding genes, among which long intergenic non- the major reason for cancer progression. Emerging evidence coding RNAs (lincRNAs) account for 50% and represent one suggests the association between PDAC chemoresistance of the most mystery groups with functions needed to and the acquisition of epithelial–mesenchymal transition – be defined.13 15 In the present study, we found that the (EMT) phenotype and/or the existence of a sub-population linc-DYNC2H1-4 was upregulated in BxPC-3-Gem cell line of cancer stem-like cells (CSC) within the tumor mass.6,7 These chemoresistant cancer cells are refractory to gemcita- with acquired gemcitabine resistance. Downregulation of bine and highly prone to metastasize.6 linc-DYNC2H1-4 was associated with decreased invasive Recently, long non-coding RNAs (lncRNAs), RNA molecules behavior of BxPC-3-Gem cells while overexpression of linc- with 4200 nt in length, have been reported involved in the DYNC2H1-4 promoted the acquisition of CSC and EMT regulation of CSC and EMT phenotypes in PDAC. Metastasis phenotypes of parental gemcitabine-sensitive BxPC-3 cells. associated lung adenocarcinoma transcript 1 (MALAT1), which Mechanically, linc-DYNC2H1-4 competed with miR-145, was originally discovered in association with metastatic behavior leading to upregulation of its targets Lin28, Nanog, Sox2, of non small cell lung cancer, has been reported to promote Oct4, ZEB1 and MMP3 that are involved in EMT and CSC metastasis in PDAC.8,9 LncRNA ROR (Regulator of regulation. 1School of Life Science and Technology, Harbin Institute of Technology, Harbin, China and 2Department of General Surgery, Fourth Affiliated Hospital of Harbin Medical University, Harbin, China *Corresponding author: H Yang, School of Life Science and Technology, Harbin Institute of Technology, 2 Yikuang Street, Building 2E-303, Harbin, Heilongjiang 150001, China. Tel/Fax: +86-451-86403616; E-mail: [email protected] or M Ding, Department of General Surgery, Fourth Affiliated Hospital of Harbin Medical University, Harbin, China. Tel: +86-451-85939741; E-mail: [email protected] 3These authors contributed equally to this work. Received 17.1.17; revised 13.5.17; accepted 31.5.17; Edited by M Agostini Linc-DYNC2H1-4 promotes pancreatic cancer stemness Y Gao et al 2 Results each selection, we compared the EMT/CSC properties bet- ween BxPC-3-Gem cells and BxPC-3. ZEB1, which initiates Gemcitabine-resistant pancreatic cancer cells exert the EMT program through downregulation of E-cadherin and enhanced EMT and CSC properties. We established a upregulation of vimentin,16 was increased in BxPC-3-Gem gemcitabine-resistant cell line (BxPC-3-Gem) through expos- cells, along with decrease of E-cadherin and increase of ing parental BxPC-3 cells to increased concentrations of vimentin (Figures 1b and c). High expression of ZEB1 and gemcitabine for 16 months. BxPC-3-Gem showed ~ 270-fold vimentin, and low exprssion of E-cadherin, were verified enhanced resistance to gemcitabine compared with BxPC-3 in pancreatic cancer cell lines with intrinsic gemcitabine cells as reflected by IC50 (Figure 1a). As only a small resistance, AsPC-1 and PANC-1 (Figures 1d and e). As EMT population with EMT and/or CSC phenotypes remained after contributes to metastatic behavior of cancer cells,6,7 we 350 300 *** BxPC-3 IC50 (nmol/L) 250 AsPC-1 BxPC-3 13.68 200 PANC-1 120 120 BxPC-3-Gem 3731 *** 100 85 *** 50 80 15 ** 60 1.5 Relative 40 1 20 level expression 0.5 *** Viable cells (%) 0 0 *** 0 ZEB1 E-cadherin Vimentin 25 50 400 100 200 800 12.5 6.25 3200 6400 1600 3.125 Concentration (nmol/L) BxPC-3AsPC-1PANC-1 10 BxPC-3 ** 8 BxPC-3-Gem ZEB1 124kDa 16.323.96 6 *** 4 E-cadherin 97kDa 1.5 10.120.55 Relative 1 Vimentin 54kDa 0.5 *** 124.6213.8 expression levelexpression 0 36kDa ZEB1 E-cadherin Vimentin GAPDH Migration Invasion BxPC-3 BxPC-3 BxPC-3-Gem ZEB1 124kDa 1 1.37 E-cadherin 97kDa 1 0.45 BxPC-3-Gem Vimentin 54kDa 1 2.93 500 300 400 ** 36kDa ** GAPDH 300 200 200 100 100 Cell number 0 Cell number 0 BxPC-3 BxPC-3 BxPC-3-Gem BxPC-3-Gem Figure 1 Gemcitabine-resistant pancreatic cancer cells exhibit enhanced EMT potential. (a) MTTassay after 72 h treatment with gemcitabine in BxPC-3-Gem and parental BxPC-3 cells. (b–e) The expression levels of EMT markers, ZEB1, E-cadherin and vimentin were detected by RT-qPCR (b,d) and western blotting (c and e) in BxPC-3, BxPC-3- Gem, AsPC-1 and PANC-1 cells. GAPDH was used as a loading control (c and e). Bands intensities normalized to GAPDH were shown (f) The migration and invasion abilities of BxPC-3 and BxPC-3-Gem cells were measured by Transwell assay (Scale bar, 200 μm). The data shown were from three independent experiments. **Po0.01; ***Po0.001 versus BxPC-3 Cell Death and Disease Linc-DYNC2H1-4 promotes pancreatic cancer stemness Y Gao et al 3 BxPC-3 BxPC-3-Gem 11 40 *** BxPC-3 *** BxPC-3 AsPC-1 9 BxPC-3-Gem 35 PANC-1 7 30 3 12 *** 10 * Primary 2 *** 8 Relative ** Relative 6 *** 1 4 *** *** expression level expression level 2 * *** *** 0 0 Oct4 Lin28 Nanog Sox2 Oct4 Lin28 Nanog Sox2 Secondary C-3 BxPC-3BxPC-3-Gem BxP AsPC-1 PANC-1 39kDa Oct4 39kDa Oct4 Ternary 1 1.37 11.791.63 Lin28 28kDa 28kDa Lin28 BxPC-3 1 1.49 19.4535.48 200 BxPC-3-Gem Nanog 35kDa Nanog 35kDa *** 150 1 1.3 13.27 13.57 Sox2 34kDa Sox2 34kDa 100 1 1.49 16.13 13.32 50 ** GAPDH 36kDa GAPDH 36kDa per 500 cells * 0 Number ofspheres Primary Secondary Ternary 500 cells 250 cells 125 cells BxPC-3 500 BxPC-3 BxPC-3-Gem 400 *** BxPC-3-Gem BxPC-3 300 200 ** h 40 100 ** 30 * 20 Number ofcolonies 0 500 cells 250 cells 125 cells Tumor 10 BxPC-3-Gem 0 weight (mg) BxPC-3 BxPC-3-Gem Figure 2 Gemcitabine-resistant pancreatic cancer cells exert enhancedcancer stem cell characteristics. (a–d) The expression levels of CSC markers, Oct4, Lin28, Nanog and Sox2 were determined by RT-qPCR (a,c) and western blotting (b,d) in BxPC-3, BxPC-3-Gem, AsPC-1 and PANC-1 cells. The data shown were from three independent experiments. Bands intensities normalized to GAPDH were shown. (e) Pancreatosphere formation. Scale bar, 200 μm. (f) Colony formation. (g) Tumorigenecity in vivo (n = 4). (h) Tumor weights were measured. *Po0.05; **Po0.01; ***Po0.001 versus BxPC-3 determined the cell motility in the two lines through Transwell enhanced in vitro self-renewal capability of BxPC-3-Gem assay.
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