STAT3 Axis

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Accepted Article Preview: Published ahead of advance online publication CRNDE promotes malignant progression of glioma by attenuating miR384/PIWIL4/STAT3 axis

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Jian Zheng, Xiaobai Liu, PingWang, Yixue Xue, Jun Ma, Chengbin Qu, Yunhui Liu

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Cite this article as: Jian Zheng, Xiaobai Liu, PingWang, Yixue Xue, Jun Ma, Chengbin Qu, Yunhui Liu, CRNDE promotes malignant progression of glioma by attenuating miR-384/PIWIL4/STAT3 axis, Molecular Therapy accepted article preview online 08 April 2016; doi:10.1038/mt.2016.71

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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Received 16 November 2015 ; accepted 01 April 2016 ; Accepted article preview online 08 April 2016

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CRNDE promotes malignant progression of glioma by attenuating miR-384/PIWIL4/STAT3 axis

Jian Zheng1,2, Xiaobai Liu1,2, PingWang3,4, Yixue Xue3,4, Jun Ma3,4, Chengbin Qu1,2, Yunhui Liu1,2*

Department of Neurosurgery, Shengjing Hospital of China Medical University,

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Liaoning Research Center for Translational Medicine in Nervous System Disease,

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Shenyang 110004, People’s Republic of China

Department of Neurobiology, College of Basic Medicine, China Medical University,

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Shenyang 110004, People’s Republic of China

Institute of Pathology and Pathophysiology, China Medical University, Shenyang

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Shenyang 110122, People’s Republic of China

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110122, People’s Republic of China

* Corresponding author. Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang 110004, People’s Republic of China. Liaoning Research Center for Translational Medicine in Nervous System Disease, Shenyang 110004, People’s Republic of China Tel.: +86 024 23256666-5506; Fax: +86 024 22958989 Email address: [email protected]

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Abstract Colorectal neoplasia differentially expressed (CRNDE) is the most upregulated long non-coding RNA (lncRNA) in glioma. Herein, the function and potential molecular mechanisms of CRNDE and miR-384 were illustrated in glioma cells. CRNDE overexpression facilitated cell proliferation, migration, and invasion, while inhibited glioma cells apoptosis. Quantitative real-time PCR demonstrated that miR-

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384 was downregulated in human glioma tissues and glioma cell lines. Moreover,

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restoration of miR-384 exerted tumor-suppressive functions. In addition, the

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expression of miR-384 was negatively correlated with CRNDE expression. A binding

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region between CRNDE and miR-384 was confirmed using luciferase assays.

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Moreover, CRNDE promoted cell malignant behavior by decreasing miR-384

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expression. At the molecular level, treatment by CRNDE knockdown or miR-384

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overexpression resulted in a decrease of piwi-like RNA-mediated gene silencing 4

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(PIWIL4) protein. Besides, PIWIL4 was identified as a target of miR-384 and plays

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an oncogenic role in glioma. Similarly, downstream proteins of PIWIL4 such as STAT3, cyclin D1, VEGFA, SLUG, MMP-9, caspase 3, Bcl-2, and bcl-xL were modulated when treated with miR-384 and PIWIL4. Remarkably, CRNDE knockdown combined with miR-384 overexpression led to tumor regression in vivo. Overall, these results depicted a novel pathway mediated by CRNDE in glioma, which may be a potential application for glioma therapy.

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Key words

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Long non-coding RNAs, CRNDE, microRNAs, miR-384, Glioma, PIWIL4

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1. Introduction Gliomas represent the most prevalent and aggressive primary brain tumor in adults, which are characterized by infiltrative growth and early metastasis. Patients suffering from this disease meet a median survival of 15 months despite following advanced clinical therapies[1]. Glioma cells are found to carry heterogeneous genetic molecular aberrations[2]. Hence, it’s urgent to discover efficient dysregulated genes

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as therapeutic targets in glioma treatment.

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Non-coding RNAs (ncRNAs) dysregulation are associated with the progression

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of various tumors and involved in diverse cellular events[3]. Long non-coding RNAs

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(lncRNAs; >200 nt) and microRNAs (miRNAs; ~22 nt) are two members of non-

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coding RNAs[4]. LncRNAs are involved in multiple cellular processes such as

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proliferation, migration, invasion and apoptosis[5]. Abundant evidence has certified

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that lncRNAs play key roles in glioma progression[6]. For exemple, MEG3 attenuated

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proliferation in human glioma cell lines through modulating activation of p53 and

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MDM2 protein[7]. GAS5 inhibited proliferation, migration and invasion, and promoted human glioma cells apoptosis by upregulating Plexin C1[8]. Colorectal neoplasia differentially expressed (CRNDE) is a lncRNA first found in colorectal cancer and located on chromosome 16, exerting oncogenic functions in diverse cancers[9]. Actually, the expression of CRNDE is upregulated in many other tumors including gliomas. Strikingly, CRNDE is the most upregulated lncRNA in glioma,

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and exhibits a 32-fold increase over that in normal brain tissues. However, the indepth mechanism of CRNDE in the regulation of gliomas remains unclear. MiRNAs are characterized by mediating diverse biological processes of cancer cells, by targeting mRNA for deregulation or translational repression[10]. And aberrant expressions of miRNAs are ubiquitous in various tumor cells[11]. Overexpression of miR-27b was clarified to inhibit proliferation, cell adhesion and

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invasion in human colon cancer HCT116 cells by directly targeting ARFGEF1, and

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predicted better prognosis[12]. On the contrary, miR-10b acted as an oncogene in

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medulloblastoma cell lines, promoting cell proliferation and inhibiting apoptosis by

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targeting Bcl-2[13]. Taken together, aberrant expressions of miRNAs are involved in

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various biological responses in cancer cells[14]. It is confirmed that CRNDE harbors

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a miR-384 binding site by Starbase. In addition, miR-384 was found to exhibit a low

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expression in laryngeal carcinoma compared to normal laryngeal tissue[15]. However,

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the expression and function of miR-384 in glioma still remain unclear.

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Argonaute gene family comprises a group of four members: Piwi-like 1 (Hiwi, PIWIL 1), Piwi-like 2 (Hili, PIWIL 2), Piwi-like 3 (PIWIL 3) and Piwi-like 4 (Hiwi 2, PIWIL 4)[16]. Piwi subfamily genes expression are mainly in germ cells, and are involved in various cellular biological processes[17]. Remarkably, PIWIL gene overexpression is frequent in many human tumors[18]. Only PIWIL4 gene is expressed in several human somatic tissues[19]. Importantly, PIWIL4 mRNA was upregulated in several human tumors such as cervical cancer and soft tissue sarcomas

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[20, 21], and the Oncomine database (www.oncomine.org/) contains entries that suggest PIWIL 4 is expressed in brain cancers including glioma[22]. Moreover, using miRNA target prediction software Targetscan and miRanda, PIWIL4 was predicted to be a presumed target of miR-384. However, the expression and function of PIWIL4 in glioma still remain unclear. In this study, we determined the expression of miR-384 and PIWIL4 in human

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glioma tissues and glioma cell lines, and investigated the function of CRNDE,miR-

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384 and PIWIL4 in human glioma cells. Moreover, miR-384 was found to target

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CRNDE in a sequence-specific manner and there is a reciprocal repression between

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miR-384 and CRNDE possibly induced by RNA-induced silencing complex (RISC).

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In addition, the interaction of miR-384 and PIWIL4 were confirmed by luciferase

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assays. These results illustrated a new molecular mechanisms of glioma progression,

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and gave a novel insight into glioma therapy.

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2. Materials and methods

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2.1. Human tissues specimens All human glioma specimens were collected from patients diagnosed with glioma who underwent surgery at the Department of Neurosurgery of Shengjing Hospital, China Medical University, from January 2014 to July 2015. Informed consent was obtained from all patients and the research method was approved by the Ethics Committee of Shengjing Hospital of China Medical University. Parts of specimens were immediately frozen and preserved in liquid nitrogen after surgical

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resection, and the rest were sent for routine neuropathological evaluation. Glioma specimens were divided into two groups: grade I–II glioma group (Low-grade glioma tissues) (n = 15) and grade III–IV glioma group (High-grade glioma tissues) (n = 15) according to the 2007 WHO classification of tumors by neuropathologists. Normal brain tissues (NBTs) were obtained from patients with fresh autopsy material (donation from individuals who died in traffic accident and confirmed to be free of

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any prior pathologically detectable conditions) were used as negative control (n = 15).

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2.2. Cell culture

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Human U87 and U251 glioma cell lines, and human embryonic kidney (HEK)

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293T cells were purchased from Chinese Academy of Medical Sciences (Beijing,

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China). U87 glioma cells and HEK-293T cells were cultured in Dulbecco's modified

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Eagle medium (DMEM)/high glucose supplemented containing 10% FBS, U251 cells

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were cultured in DMEM/F12 medium supplemented containing 10% FBS. All cells

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were maintained in a humidified incubator at 37 °C with 5% CO2. Primary normal

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human astrocytes (NHA) were purchased from the Sciencell Research Laboratories (Carlsbad, CA, USA) and cultured under the instructed condition by the manufacturer. 2.3. Reverse transcription and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from the glioma tissues and NHA, U87, and U251 cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA). The RNA concentration and quality were detected by the 260/280 nm ratio using a Nanodrop Spectrophotometer (ND-100, Thermo, USA). The primers of CRNDE,

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PIWIL4, GAPDH, hsa-miR-384-5p, U6 and 18s rRNA were synthesized from the Applied Biosystems. High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA) were employed to synthesize cDNA from total RNA. TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) was used to generate cDNA from miRNA. qRT-PCR was performed using TaqMan gene expression assays of CRNDE, PIWIL4 and GAPDH or TaqMan

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Universal Master Mix II with TaqMan microRNA assays of miR-384-5p, U6 and 18S

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(Applied Biosystems, Foster City, CA, USA) using the ABI 7500 Fast Real-Time

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PCR System (Applied Biosystems). No significant difference was found in the

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expression of miR-384 normalized to U6 or 18S in glioma tissues and glioma cell

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lines (Figure S1 (a and b)). U6 and GAPDH were selected as endogenous controls for

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miRNA and genes expressions, respectively. Expressions were normalized to

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2.4. Western blot

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endogenous controls and fold change was calculated as 2−∆∆Ct in gene expression.

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Total proteins were extracted form the cells using RIPA buffer with protease inhibitors (Beyotime Institute of Biotechnology) on ice, subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes. Membranes were incubated in Tris-buffered saline (TBS) containing 5% nonfat milk for 2 h at room temperature and then incubated with primary antibodies as follows: PIWIL4 (1:1000, SAB, Chicago, IL), p-STAT3 (1:1000, CST, EUGENE, USA), STAT3 (1:1000, Abcam, UK), cyclin D1 (1:10000, Abcam, UK), VEGFA (1:500, Santa Cruz Biotechnology, USA), SLUG

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(1:1000, CST, EUGENE, USA), MMP-9 (1:200, Santa Cruz Biotechnology, USA), Bcl-2 (1:1000, CST, EUGENE, USA), bcl-xL (1:1000, CST, EUGENE, USA), procaspase3 and cleaved-caspase3 (1:1000, CST, EUGENE, USA), and GAPDH (1:1000, Santa Cruz Biotechnology), followed by incubation with appropriate correlated HRP-conjugated secondary antibody. Then the membranes were incubated with secondary antibodies (Santa Cruz Biotechnology) at room temperature for 2 h.

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Immunoblots were visualized by enhanced chemiluminescence (ECL kit, Santa Cruz

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Biotechnology) and scanned using ChemImager 5500 V2.03 software. The relative

control.

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2.5. Immunohistochemistry Assays

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integrated density values (IDVs) were calculated based on GAPDH as an internal

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The slides of human glioma tissue samples (4 µm thick) were dewaxed,

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rehydrated, and incubated in 0.3% H2O2 for 10 minutes to inhibit endogenous

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peroxidase activity before blocking with 10% normal goat serum (MXB, Fuzhou,

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China) for 30 minutes and incubating overnight at 4 °C with rabbit polyclonal antibody against PIWIL4 (1:200, SAB, Chicago, IL). Slides were washed with PBS three times and then incubated with biotinylated rabbit anti-rabbit IgG for 1 hour at room temperature. After incubation with avidinbiotin-peroxidase complex for 10 minutes, samples were stained with 3,3’-diaminobenzidine. Slides were imaged under a light microscope (Olympus, Japan) at ×100 and ×200 magnification.

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2.6. Cell transfections CRNDE full length (pEX2-CRNDE) plasmid, four short-hairpin CRNDE (shCRNDE) plasmids and their respective non-targeting sequence (negative control, NC); miR-384 agomir (pre-miR-384), miR-384 antagomir (anti-miR-384) and their respective non-targeting sequence (negative control, NC) (pre-NC or anti-NC) were synthesized (GenePharma, Shanghai, China). PIWIL4 full length (with 3′-UTR)

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(PIWIL4 (+) or PIWIL4) plasmid, short-hairpin PIWIL4 (PIWIL4 (-)) plasmid,

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PIWIL4 (without 3′-UTR) (PIWIL4 (non-3′UTR)) plasmid and their respective non-

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targeting sequence (negative control, NC) (PIWIL4 (+)-NC or PIWIL4 (-)-NC) were

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synthesized (Life technology, MA, USA). Cells were seeded into 24-well plates

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(Corning) until they were at 50-70% confluence and then transfected using

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Lipofectamine 3000 reagent (Life Technologies Corporation, Carlsbad, CA, USA)

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according to the manufacturer's instructions. The applicable stably transfected cells

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were selected using G418 screening. The overexpression and the silence efficiency

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were analyzed using qRT-PCR. To determine the effect of CRNDE on glioma, cells were divided into five groups: Control group, pEX2-NC group (tansfected with pEX2-NC plasmid), pEX2-CRNDE group (tansfected with CRNDE full length plasmid), sh-NC group (tansfected with sh-NC plasmid) and sh-CRNDE (transfected with short-hairpin CRNDE plasmid) group. Similarly, to determine the effect of miR384 on glioma, cells were divided into five groups: Control group, pre-NC group (transfected with negative control), pre-miR-384 group (transfected with miR-384

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agomir), anti-NC group (transfected with negative control) and anti-miR-384 (transfected with miR-384 antagomir). To investigate to determine the effect of PIWIL4 on glioma, cells were divide into five groups: Control group, PIWIL4 (+)-NC group (transfected with empty plasmid), PIWIL4 (+) group (transfected with PIWIL4 full length plasmid), PIWIL4 (-)-NC group (transfected with empty plasmid) and PIWIL4 (-) group, (transfected with short-hairpin PIWIL4 plasmid). Further, to

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explore the underlying mechanism of CRNDE regulated the biological behavior of

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glioma cells via decreasing miR-384, cells were divided into five groups: Control

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group, CRNDE (+)+miR-384 (+) group (pEX2-CRNDE stable expressing cells co-

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transfected with pre-miR-384), CRNDE (+)+miR-384 (-) group (pEX2-CRNDE

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stable expressing cells co-transfected with anti-miR-384), CRNDE (-)+miR-384 (-)

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group (sh-CRNDE stable expressing cells co-transfected with pre-miR-384) and

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CRNDE (-)+miR-384 (+) group (sh-CRNDE stable expressing cells co-transfected

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with pre-miR-384). Furthermore, to determine miR-384 hinder the malignant

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progression of glioma cells via targeting to PIWIL4 3′-UTR, cells were dived into four groups: miR-384-NC+PIWIL4-NC group (pre-NC stable expressing cells cotransfected with PIWIL4-NC plasmid), miR-384+PIWIL4-NC group (pre-miR-384 stable expressing co-transfected with PIWIL4-NC), miR-384+PIWIL4 group (premiR-384 stable expressing co-transfected with PIWIL4 (+)) and miR-384+PIWIL4 (non-3′UTR) group (pre-miR-384 stable expression transfected with PIWIL4 (without 3′-UTR) plasmid).

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2.7. Cell proliferation assay Cell Counting Kit-8 assay (CCK-8, Dojin, Japan) was performed to determine U87 and U251 glioma cells proliferation. U87 and U251 cells were seeded in 96-well plates at the density of 2000 cells per well. After cells were transfected 72h, 10 µL of CCK-8 solution was added into each well and cells incubated for 2 h at 37 °C. The absorbance was measured at 450 nm using the SpectraMax M5 microplate reader

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2.8. Cell migration and invasion assay

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(Molecular Devices, USA).

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24-well chambers with 8 µm pore size (Corning, USA) was used for migration

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and invasion of U87 and U251 cells in virto. Cells were resuspended in 100 µL

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serum-free medium at a density of 1×105/mL and seeded in the upper chamber (or

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pre-coated with 500 ng/ml Matrigel solution (BD, Franklin Lakes, NJ, USA) for cell

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invasion assay. After incubation for 48 h, the cells on the upper membrane surface

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were physically removed. Cells that had migrated or invaded to the lower side of the

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membrane were fixed with methanol and stained with 10% Giemsa. Five randomly fields were chosen to count cells for statistics under a microscope and photographs were taken. 2.9. Apoptosis analysis Cell apoptosis was evaluated by Annexin V-PE/7AAD staining (Southern Biotech, Birmingham, AL, USA). After washing with 4 °C PBS twice, cells were collected and stained with Annexin V-PE/7AAD according to the manufacturer’s

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instructions. Then the cells were analyzed by flow cytometry (FACScan, BD Biosciences) and apoptotic fractions were investigated by CELL Quest 3.0 software. 2.10. Reporter vectors construction and luciferase assays CRNDE full length and PIWIL4 3′-UTR sequences were amplified by PCR and cloned into a pmirGlo Dual-luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to construct luciferase reporter vector (CRNDE-Wt and PIWIL4-

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Wt) (GenePharma). The sequence of putative binding site was replaced as indicated

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(CRNDE-Mut and PIWIL4-Mut) to mutate the putative binding site of CRNDE or

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PIWIL4. HEK-293T cells were seeded in 96-well plates and the cells were co-

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transfected with CRNDE-Wt (or CRNDE-Mut) or PIWIL4-Wt (or PIWIL4-Mut) and

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miR-384 or miR-384-NC plasmids when they reached 50-70% confluence. The

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luciferase activities were measured at 48h after transfection by Dual-Luciferase

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reporter assay kit (Promega). The cells were divided in five groups respectively:

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Control group, CRNDE-Wt + miR-384-NC (transfected with CRNDE-Wt and pre-

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miR-384-NC), CRNDE-Wt + miR-384 group (transfected with CRNDE-Wt and premiR-384), CRNDE-Mut + miR-384-NC group (transfected with CRNDE-Mut and pre-miR-384-NC), CRNDE-Mut + miR-384 group (transfected with CRNDE-Mut and pre-miR-384); Control group, PIWIL4-Wt + miR-384-NC (transfected with PIWIL4-Wt and pre-miR-384-NC), PIWIL4-Wt + miR-384 group (transfected with PIWIL4-Wt and pre-miR-384), PIWIL4-Mut + miR-384-NC group (transfected with

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PIWIL4-Mut and pre-miR-384-NC), PIWIL4-Mut + miR-384 group (transfected with PIWIL4-Mut and pre-miR-384). 2.11. RNA immunoprecipitation U87 and U251 cells were lysed by a complete RNA lysis buffer with protease inhibitor and RNase inhibitor from an EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) according to the

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manufacturer's protocol. Whole cell lysate of the control groups and anti-miR-384

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groups were incubated with RIP immunoprecipitation buffer containing magnetic

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beads conjugated with human anti-Argonaute2 (Ago2) antibody (Millipore, Billerica,

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MA, USA), and negative control normal mouse IgG (Millipore, Billerica, MA, USA).

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Samples were incubated with Proteinase K buffer and then immunoprecipitated RNA

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was isolated. The RNA concentration was measured by a NanoDrop (Thermo

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Scientific) and the RNA quality assessed using a bioanalyser (Agilent, Santa Clara,

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CA, USA). Furthermore, purified RNA was obtained and analyzed by qRT-PCR to

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demonstrate the presence of the binding targets using respective primers mentioned above. 2.12. Co-immunoprecipitation Cell lysates were incubated with appropriate amounts of antibodies for at least 1 h at 4°C. Antibody-protein complexes were precipitated with protein A-Agarose immunoprecipitation reagent (Santa Cruz Biotechnology, USA) according to the

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manufacturer's instructions. Anti-PIWIL4 (Santa Cruz Biotechnology, USA) and antiSTAT3 (Abcam, UK) were used to perform the immunoprecipitation experiments. 2.13. Tumor xenografts in nude mice The stable expression U87 and U251 cells were used for in vivo study. Lentivirus encoding miR-384-5p was generated using pLenti6.3/V5eDEST Gateway Vector Kit (Life Technologies Corporation, Carlsbad, CA, USA). The miR-384-5p

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and short-hairpin RNA targeting human CRNDE were ligated into the

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pLenti6.3/V5eDEST vector and LV3-CMV-GFP-Puro vector (GenePharma,

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Shanghai, China), respectively. And then pLenti6.3/V5eDEST-miR-384 and LV3-

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CMV-GFPPuro-sh-CRNDE vectors were generated. The ViraPower Packaging Mix

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was used to generate Lentivirus in 293FT cells. After infection, the stable expressing

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cells of miR-384 and sh-CRNDE were picked. The lentiviruses of miR-384 were

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transduced in sh-CRNDE stably transfected cells to generate miR-384+sh-CRNDE

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cells. All experiments with nude mice were performed strictly in accordance with a

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protocol approved by the Administrative Panel on Laboratory Animal Care of the Shengjing Hospital. Four-week-old BALB/C athymic nude mice were obtained from the National Laboratory Animal Center (Beijing, China). The animals were free to autoclaved food and water during the study. The nude mice were divided into four groups: Control group (only U87 or U251), sh-CRNDE group (sh-CRNDE stable expression U87 or U251 cells), miR-384 group (miR-384 stable overexpression U87 or U251 cells) and sh-CRNDE + miR-384 group (CRNDE inhibition and miR-384

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ACCEPTED ARTICLE PREVIEW overexpression stable U87 and U251 cells). 3 × 105 cells were subcutaneously injected in the right flanks of the mice. Tumor volume was measured every four days when the tumors were obviously identified and the volume was calculated by the formula: volume (mm3) = length × width2/2. 40 days after injection, mice were sacrificed and tumors were isolated. For survival analysis in orthotopic inoculations, 3 × 105 cells were stereotactically implanted into the right striatum of the mice. The

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number of survived nude mice was recorded and survival analysis was determined

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using Kaplane-Meier survival curve.

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2.14. Statistical analysis

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Data are presented as mean + standard deviation (SD). All statistical analyses

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were evaluated by SPSS 18.0 statistical software with the Student’s t-test or one-way

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3. Results

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ANOVA. Differences were considered to be significant when P < 0.05.

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3.1 CRNDE exerted oncogenic function in glioma cells

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CRNDE was known as the most upregulated lncRNA in glioma[9]. To determine the effects of CRNDE on glioma cells, the stable overexpression of CRNDE and knockdown of CRNDE U87 and U251 cell lines were established. As shown in Figure 1(a), overexpression of CRNDE resulted in a significant increased proliferation in U87 and U251 cells compared to pEX2-NC group. Transwell assays were used to investigate the effect of CRNDE on glioma cells. Figure 1(b) showed that migrating and invading U87 and U251 cell numbers were obviously decreased in sh-CRNDE

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group than in respective sh-NC group. To clarify whether knockdown of CRNDE caused apoptosis in glioma cells, flow cytometry analysis was conducted. As shown in Figure 1(c), knockdown of CRNDE increased the apoptosis ratio of glioma cells compared to sh-NC group. These results inferred that CRNDE functioned as an oncogene in glioma cells. 3.2 miR-384 was downregulated in glioma tissues and glioma cell lines, and

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functioned as tumor supperessor

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The expressions of miR-384 in glioma tissues and glioma cell lines were

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measured by quantative Real-time PCR (qRT-PCR). MiR-384 was significantly

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decreased in glioma tissues and glioma cell lines than in normal brain tissues and

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normal human astrocytes, and the expression of miR-384 was negative correlated with

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the progression of glioma pathological grade (Figure 2(a and b)). This implied miR-

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384 play a tumor suppressor role in glioma cells. CCK-8 assay indicated that

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overexpression of miR-384 inhibited the proliferation of U87 and U251 cells than in

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pre-NC group (Figure 2(c)). The migration and invasion U87 and U251 numbers were apparently decreased in anti-miR-384 groups than in respective anti-NC group (Figure 2(d)). Flow cytometry analysis was conducted to determine the effect of overexpression of miR-384 in glioma cells. Restoration of miR-384 increased the glioma cells apoptosis, while inhibition of miR-384 hindered the glioma cells apoptosis (Figure 2(e)). We proposed miR-384, in contrast to CRNDE, exerted tumorsuppressive function in glioma cells.

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3.3 CRNDE is a target of miR-384 Accumulate evidence showed that lncRNA might be a competing endogenous RNA (ceRNA) or a molecular sponge in inflecting the expression and biological functions of miRNA[23]. Using bioinformatics databases (Starbase, RNAhybrid), CRNDE is a putative target of several miRNAs (Table 1), and we proposed that CRNDE might harbor one miR-384 binding site. To quantify our prediction that miR-

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384 could target to CRNDE, we first detect the expression of miR-384 in pEX2-

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CRNDE and sh-CRNDE U87 and U251 cells respectively by qRT-PCR. The

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expression of miR-384 was decreased in pEX2-CRNDE group compared to pEX2-NC

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group (Figure 3(a)), while the expression of miR-384 was increased in sh-CRNDE

CRNDE-Mut groups.

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group than in sh-NC group (Figure 3(a)), while found no significant difference in

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Dual-luciferase gene reporter assays were conducted to determine the binding

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site of CRNDE and miR-384. The luciferase activity in the CRNDE-Wt+pre-miR-384

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group was significantly attenuated than that in the Control group (Figure 3(d)), while the luciferase activity in the CRNDE-Mut group was not affected. To confirm whether CRNDE and miR-384 are in the expected RNA-induced silencing complex (RISC) complex, RNA-binding protein immunoprecipitation (RIP) assay was carried out. qRT-PCR was performed to measure RNA levels in immunoprecipitates. The expression of CRNDE and miR-384 were both increased in anti-Ago2 group compared with anti-normal group, in the anti-miR-384 group, the

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expressions of CRNDE and miR-384 immunoprecipitated with Ago2 were lower than those in the control group respectively (Figure 3 (e-h)). Taken together, these results inferred that CRNDE could impaired miR-384 expression in RISC manner, and there might be a reciprocal repression feedback loop between CRNDE and miR-384. 3.4 CRNDE increased while miR-384 decreased the expression of PIWIL4 The above results illustrated CRNDE and miR-384 could modulate the biological

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behaviors of U87 and U251 cells, but the underlying molecular mechanisms remain

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unclear. Bioinformatics database (Targetscan, miRanda) predicted that several genes

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are downstream genes of miR-384 including PIWIL4 (Tables 2 and 3). We first

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measured the effect of CRNDE or miR-384 on mRNA and protein levels of PIWIL4

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by qRT-PCR and western blot, and found PIWIL4 expression was significantly

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affected among servral downstream genes of miR-384. The mRNA expression was

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higher in pEX2-CRNDE group than that in pEX2-NC group, while the mRNA

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expression was impaired in pre-miR-384 group than that in pre-NC group (Figure 3(i

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and j)). Similarly, the expression of PIWIL4 protein was raised in pEX2-CRNDE group than that in pEX2-NC group, while the expression of PIWIL4 protein in premiR-384 showed the contrary result (Figure 3(k and l)). The location and expression of PIWIL4 protein in gliomas were investigated using immunohistochemistry and western blot. Immunohistochemistry analysis showed that PIWIL4 located in cytoplasm, and was upregulated in human glioma tissues compared with human nontumorous tissues (Figure 3(m)). Similarly, the

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expression of PIWIL4 was significantly increased in low- or high-grade glioma tissues than that in human normal tissues (Figure 3(n)). We also detected the expression of PIWIL4 in glioma cell lines and normal human astrocytes cells. The expression PIWIL4 protein was higher in U87 and U251 cells versus that in normal human astrocytes cells (Figure 3(o)). 3.5 Overexpression of miR-384 largely reversed CRNDE-induced oncogenetic

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effects on glioma cells

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To investigate whether overexpression of miR-384 could rescue CRNDE-

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induced oncogenetic influences on U87 and U251 cells, miR-384 upregulation by

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CRNDE inhibition was rescued using anti-miR-384 prior to the determination of cell

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biological behaviors. The proliferation of glioma cells in CRNDE (+)+miR-384 (-)

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group was robustly increased versus with that in CRNDE (+)+miR-384 (+) group,

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whereas the proliferation of glioma cells in CRNDE (-)+miR-384 (+) group was

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vigorously decreased than that in CRNDE (-)+miR-384 (-) group (Figure 4(a)).

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Further, the apoptosis of glioma cells in CRNDE (+)+miR-384 (-) group was reduced than that in CRNDE (+)+miR-384 (+) group, whereas knockdown of CRNDE and overexpression of miR-384 largely elevated the apoptosis ratio of glioma cells (Figure 4(c)). Transwell assays revealed that overexpression of CRNDE combined with knockdown of miR-384 in glioma cells exhibit significant increased migrating and invading cells, compared with overexpression of CRNDE combined with overexpression of miR-384 (Figure 4(b)). Indicating that CRNDE could promote

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glioma cells malignant biological behaviors by decreasing miR-384 expression. To explore the molecular mechanism, the expression of PIWIL4 protein in glioma cells was determined by western blot. Inhibition of miR-384 in glioma cells, which stably knockdown of CRNDE, remarkably rescued the expression of PIWIL4 (Figure 4(d)). These results implied that miR-384 played a crucial role in CRNDE-induced promotion effects on glioma cells.

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3.6 PIWIL4 promoted progression of glioma cells by inducing phosphorylation

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of STAT3

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Having confirmed PIWIL4 was a target of miR-384, and the expression of

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PIWIL4 was increased in glioma tissues and glioma cell lines, we predicted PIWIL4

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play an oncogenetic role on glioma cells. CCK-8 assay revealed that glioma cells in

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PIWIL4 (+) group exhibit a higher proliferation than that in PIWIL4 (+)-NC group

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(Figure 5(a)). Migration and invasion glioma cells in PIWIL4 (+) group were

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increased compared with that in PIWIL4 (+)-NC group (Figure 5(b)). Further, the cell

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apoptosis in PIWIL4 (+) group was hindered versus with that in PIWIL4 (+)-NC group (Figure 5(c)). These results indicated that PIWIL4 exerted oncogenic role by promoting malignant biological behaviors of glioma cells. However, the underlying molecular mechanism remains unclear. Both PIWIL2 and PIWIL4 belong to PIWI subfamily, and as previous reported, signal transducer and activator of transcription 3 (STAT3) could be phosphorylated by PIWIL2 [24]. Hence, we hypothesized PIWIL4 could

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induce STAT3 phosphorylation. As previous reported, STAT3 was confirmed to be upregulated in glioma tissues and promoted glioma progression[25]. To ensure whether STAT3 was phosphorylated by PIWIL4, western blot analysis was performed. Phosphorylation of STAT3 (p-STAT3) was enhanced in PIWIL4 (+) group than that in PIWIL4 (+)-NC group (Figure 5(d)). Furthermore, coimmunoprecipitation was performed to examine whether PIWIL4 interacts with the

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STAT3 in the glioma U87 and U251 cell lines. When PIWIL4 was

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immunoprecipitated from U87 and U251 homogenates, the precipitate also contained

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STAT3 (Figure 5(e)). Similarly, the immunocomplex precipitated by the antibody

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specific to the STAT3 contained PIWIL4. These results indicated that PIWIL4

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promoted glioma cells progression by inducing phosphorylation of STAT3.

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3.7 Overexpression of miR-384 hindered PIWIL4-induced promotion of

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proliferation, migration and invasion proteins expression, and upregulated the

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apoptosis proteins expression by targeting PIWIL4 3′-UTR

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The above results showed that CRNDE and miR-384 could modulate the expression of PIWIL4, and we predicted PIWIL4 was a potential target gene of miR384 according to bioinformatics database (Targetscan, miRanda). To further confirm our prediction, dual-luciferase reporter assay was performed. The luciferase activity was obviously decreased in PIWIL4 (+)-Wt+ miR-384 group compared with PIWIL4 (+)-Wt+ miR-384-NC group; whereas the luciferase activity between PIWIL4 (+)-

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Mut+ miR-384 and PIWIL4 (+)-Mut+ miR-384-NC groups showed no significant difference (Figure 6(b)). To uncover whether PIWIL4 could reverse miR-384-meidiated inhibition malignant progression of glioma cells, we assessed cells proliferation, migration invasion and apoptosis which stably expressed miR-384+PIWIL4 (non-3′UTR). The proliferation of glioma cells in miR-384+PIWIL4 (non-3′UTR) group was

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significantly increased than that in miR-384+PIWIL4 group (Figure 6(c)). Moreover,

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migration and invasion cells were rescued in miR-384+PIWIL4 (non-3′UTR) group

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compared with that in miR-384+PIWIL4 group (Figure 6(d)). In addition, flow

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cytometry analysis results showed that PIWIL4 (non-3′UTR) apparently reversed

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promotion effect of apoptosis on glioma cells mediated by miR-384 (Figure 6(e)).

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Having confirmed that PIWIL4 was a functional target gene of miR-384, we

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detected downstream protein of STAT3 by western blot as well. As previously

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reported, cyclin D1, VEGFA, SLUG, MMP-9, Bcl-2, bcl-xL and caspase 3 are

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involved in regulating glioma cells proliferation, migration, invasion and apoptosis[26-30]. Meanwhile, STAT3 could promote tumor cells progression via modulating these genes[31-37]. The expressions of p-STAT3, cyclin D1, VEGFA, SLUG, MMP-9, Bcl-2 and bcl-xL were robustly rescued in miR-384+PIWIL4 (non3′UTR) group than that in miR-384+PIWIL4 group; the expression of cleaved caspase 3 in miR-384+PIWIL4 (non-3′UTR) group was blocked versus with that in miR384+PIWIL4 group (Figure 6(f)).

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3.8 Knockdown of CRNDE combined with overexpression of miR-384 restrained tumor growth and exhibit high survival in nude mice The in vivo research showed that CRNDE inhibition, miR-384 overexpression, or CRNDE inhibition combined with miR-384 overexpression produced lower tumors than control (Figure 7(b)). In addition, CRNDE inhibition combined with miR-384 overexpression led to the smallest tumor volume. The survival analysis showed that

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CRNDE inhibition, miR-384 overexpression or CRNDE inhibition combined with

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miR-384 overexpression produced longer survival than control. CRNDE inhibition

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combined with miR-384 overexpression produced the longest survival (Figure 7(b)).

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4. Discussion

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compared with Control group.

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And the tumor volume in CRNDE (-)+miR-384 (+) group was much reduced

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The present study demonstrated that overexpression of CRNDE promoted cell

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proliferation, migration and invasion, while inhibited apoptosis of glioma cells.

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Further, miR-384 had a low expression in human glioma tissues and glioma cell lines. Restoration of miR-384 inhibited cell proliferation, migration and invasion, while facilitated apoptosis of glioma cells. Moreover, overexpression of CRNDE increased the expression of PIWIL4 via down-regulating miR-384 which could negatively modulated PIWIL4 through targeting to its 3′-UTR. PIWIL4 had a high expression level in human glioma tissues and glioma cell lines. Furthermore, PIWIL4 facilitated malignant progression of glioma via inducing phosphorylation of STAT3. This

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

process was consistent with the upregulated proteins of tumorgenesis and downregulated proteins of tumor suppressor, such as cyclin D1, VEGFA, SLUG, MMP-9, Bcl-2, bcl-xL and caspase 3, which are major molecular members of proliferation, migration, invasion and apoptosis. Remarkably, the in vivo study showed that nude mice carrying tumors with knockdown of CRNDE and overexpression of miR-384 exhibited the lowest volume, as well as the longest

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survival.

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Recently, emerging evidence on lncRNA has indicated that lncRNA

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dysregulation is ubiquitous in heterogeneous tumors, and affects the malignant cells

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by promoting growth robustly, leading to continuous and unrestrained tumor

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growth[38]. Notwithstanding, the mechanisms of lncRNA affecting tumor cells are

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anfractuous. Long non-coding RNA Hox transcript antisense intergenic RNA

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(HOTAIR) is upregulated in glioma, and is found to stimulate glioblastoma cell cycle

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progression under an E2H2 dependent manner[39]. Meanwhile, HOTAIR exerted an

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oncogenetic role in glioma by decreasing miR-326[40]. CRNDE was first found in colorectal neoplasia, in which its expression was extremely upregulated. Notably, CRNDE-h, a transcript isoform, could be detected in patient plasma with encouraging application as a biomarker. Moreover, overexpression of CRNDE contributes to the progression of colorectal carcinoma by promoting proliferation, migration and invasion[41]. Further, CRNDE is the most upregulated lncRNA in glioma, and overexpression of CRNDE resulted in facilitating glioma proliferation and invasion

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through mTOR signaling[42]. Consistent with this, we showed CRNDE could promote glioma cell progression in a novel and detailed manner. Meanwhile, we previously reported CRNDE could promote the malignant biological behaviors of glioma stem cells by negatively regulating miR-186[43]. However, the comprehensive mechanisms in which CRNDE regulates glioma remain largely unclear. The present study showed that CRNDE promoted cell proliferation,

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migration and invasion, while hindered apoptosis of U87 and U251 glioma cells. In

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and exerted a pivotal role in glioma progression.

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this regard, CRNDE might be involved in the regulation of the function glioma cells

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Emerging evidences have confirmed lncRNAs may act as endogenous miRNAs

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sponges to bind to miRNAs and supervise their function[44]. To ascertain whether

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CRNDE harbors a miR-384 binding site, bioinformatics analysis was employed to

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explore the potential correlations between them and found out that the expression of

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miR-384 was obviously decreased in U87 and U251 glioma cell lines which stably

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overexpressed CRNDE, while miR-384 expression was not affected in the cells with CRNDE-Mut. Contrarily, overexpression of miR-384 reduced the expression of CRNDE, suggesting CRNDE and miR-384 may form a reciprocal repression feedback loop. Luciferase reporter assays confirmed our hypothesis that miR-384 binds to CRNDE in a sequence-specific manner. Moreover, the results of RIP assays supported the involvement of RISC complex in the reciprocal repression process. Consistent with the present findings, in breast cancer, GAS5 acted as a tumor

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suppressor and acted as an endogenous sponge of miR-21, which downregulated the expression of miR-21, while overexpression of miR-21 reduced the expression of GAS5[45]. HOTAIR was illustrated to promote malignancy of renal carcinoma cells, and executed the oncogenic function in part through the repression of miR-141 in RISC complex[46]. MiR-384, a novel conserved miRNA, was downregulated by 5-fold in laryngeal

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carcinoma by microarray[15]. Our present study demonstrated that miR-384 had a

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low expression level and negatively correlated with the histopathological grade in

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human glioma tissues as well as in glioma U87 and U251 cell lines, suggesting miR-

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384 might play a tumor suppressor role in glioma. To further explore the potential

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function of reduced miR-384 in glioma, we determined overexpression and inhibition

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of miR-384 on cell proliferation, migration, invasion and apoptosis in U87 and U251

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cells. Our results showed overexpression of miR-384 inhibited cell proliferation,

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migration and invasion, while inducing apoptosis in U87 and U251 cell lines. These

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results indicated miR-384 exerted tumor suppressor role in glioma by inhibiting proliferation and metastasis of U87 and U251, which may be a potential therapeutic target in glioma treatment. However, the underlying mechanisms still need to be explored. To quantify the hypothesis that CRNDE exerted oncogenetic functions through down-regulating the expression of miR-384, stably overexpressed miR-384 and silenced CRNDE of U87 and U251 glioma cells were established. The results

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

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indicated that overexpression of miR-384 in glioma cells, which stably inhibited CRNDE, largely rescued the promotion effect of inhibition of CRNDE exerted. Moreover, overexpression of miR-384 largely reversed the expression of PIWIL4 in glioma cells upregulated by CRNDE. Further more, the in vivo study showed that deletion of CRNDE combined with overexpression of miR-384 diminished the tumor volume, and produced the longest survival. Collectively, CRNDE exerted oncogenic

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role via down-regulating miR-384 in glioma cells, but the underlying mechanisms

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under PIWIL4 remained unknown.

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MiRNAs are involved in cellular events by targeting downstream messenger

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RNA 3′-UTR[47]. As bioinformatics analysis and luciferase assay indicated, PIWIL4

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was confirmed as one of the direct targets of miR-384 in affecting the malignant

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biological characteristics of glioma. Argonaute protein family was first discovered in

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plants, the members of which contained the PAZ (Piwi-Argonaute-Zwille) and PIWI

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domains[16]. In humans, four members formed the PIWI-like family, PIWIL1,

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PIWIL2, PIWIL3 and PIWIL4. Previous reports showed PIWIL 1-4 are highly expressed and exerted oncogene function in various tumors such as tumorous colon tissues [48]. Remarkably, among PIWI-like family members, PIWIL4 plays oncogenetic roles in tumorigenesis and exhibits a ubiquitous expression pattern in various types of cancers, such as colon, cervical, ovarian cancers and brain cancers [17, 21, 49]. Moreover, PIWIL4 expression was the most significantly changed among the genes that targeted by miR-384. However, the expression and function of

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

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PIWIL4 in glioma remain largely unclear. Our results showed that PIWIL4 located in cytoplasm and the expression was higher in human glioma tissues than that in normal brain tissues, and was higher in U87 and U251 glioma cell lines than in astrocytes. Hence, we hypothesized that PIWIL4 played an oncogenic role in glioma. To further explore the effect of overexpression of PIWIL4 on glioma cells, we examined cell proliferation, migration, invasion and apoptosis. Our results showed that

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overexpression of PIWIL4 promoted cell proliferation, migration and invasion, while

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inhibited apoptosis of glioma cells. However, the molecular mechanism in PIWIL4

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promotion in glioma tumorgenesis remained unclear. There are several reasons why

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STAT3 is classified as an oncogene. Activation of STAT3 enhanced growth while

m

inhibited apoptosis of an arm of tumor cell such as human prostate cancer cells[50,

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51]. Silencing of STAT3 attenuates proliferation while induces apoptosis in glioma

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cells[52]. In addition, STAT3 is involved in multiple vital pathways related to tumor

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progression. PI3K/Akt1/IL-6/STAT3 pathway stimulates growth and regulates stem

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cell-like properties in lung tumor initiating cells[53]. LncRNA UCA1 facilitated malignant progression of bladder cancer cells through mTOR-STAT3/microRNA-143 pathway[54]. Further, PIWIL2 reduced p53 by inducing phosphorylation of STAT3 in hepatocellular carcinoma cell[24]. Consistent with the previous reports, our results showed that overexpression of PIWIL4 largely induced phosphorylation of STAT3 in glioma cells. Moreover, the result of co-immunoprecipitation indicated that there was a physical interaction between PIWIL4 and STAT3. In addition, the detection of

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

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phosphorylation of STAT3 was performed so as to determine whether overexpression of miR-384 hindered phosphorylation of STAT3 by downregulating PIWIL4. Our results showed that overexpression of miR-384 reduced the phosphorylation of STAT3 by down-regulating of PIWIL4. Notoriously, phosphorylation of STAT3 facilitated cell proliferation, migration and invasion, while inhibited apoptosis in various tumor cells. An arm of previous

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reports have explored the in-depth oncogenic function of STAT3 in tumor cells on

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molecular lever. Previous studies demonstrated that cycnlin D1 was highly expressed

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in human glioma, and played key roles in glioma cells proliferation[55, 56].

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Moreover, phosphorylation of STAT3 was found to have an obvious effect on tumor

m

cell proliferation via the interaction with cyclin D1[31, 57]. In order to ascertain

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whether cyclin D1 was involved in the inhibited cell proliferation induced by

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overexpression of miR-384 via down-regulating PIWIL4, the expression of cyclin D1

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was measured. The results indicated overexpression of miR-384 decreased the

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expression of cyclin D1 via downregulating PIWIL4, SLUG, MMP-9 and VEGFA were defined as oncogenes and involved in epithelial-mesenchymal transition, migration and invasion in human glioma[58-60]. Intriguingly, the activation of STAT3 was found to have a marked influence on cell migration and invasion through interplay with SLUG, MMP-9 and VEGFA, respectively[32-34]. Likewise, the expressions of SLUG, MMP-9 and VEGFA were detected. Consistent with the previous results, overexpression of miR-384 largely reduced the overexpression of

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

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SLUG, MMP-9 and VEGFA induced by PIWIL4. In addition, overexpression of miR384 led to an increase in the expression of apoptotic protein cleaved caspase 3 induced by the knockdown of PIWIL4. Contrarily, overexpression of miR-384 resulted in a reduction of the expression of anti-apoptopic proteins Bcl-2 and bcl-xL through downregulating PIWIL4. Notoriously, cleaved caspase 3 is the strongest proapoptotic enzyme in the cysteine protease family and directly led to apoptosis in

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cells[61]. Bcl-2 and bcl-xL played key roles in regulating tumor cells apoptosis by

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controlling mitochondrial permeability[62]. Remarkably, phosphorylation of STAT3

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blocked cell apoptosis by regulating the expressions of caspase 3, Bcl-2 and bcl-xL in

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tumor cells[35-37]. The above results gave a novel insight into the molecular

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mechanism which CRNDE and miR-384 might involved in. The mechanism

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presented in Figure 8.

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underlying tumorgenesis of human glioma cell lines by CRNDE is schematically

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In conclusion, our study revealed CRNDE promoted cell proliferation, migration

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and invasion , while inhibited apoptosis in glioma cell lines. MiR-384 functioned as tumor suppressor by decreasing PIWIL4 in glioma cell lines. The significance of interaction among CRNDE, miR-384, PIWIL4 and STAT3 was highlighted for the first time. More importantly, therapeutic target to CRNDE/miR-384/PIWIL4/STAT3 may be promising for the treatment of human glioma.

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Acknowledgements This work is supported by grants from the Natural Science Foundation of China (81172197, 81272564, 81372484 and 81573010), Liaoning Science and Technology Plan Project (No. 2015225007), Shenyang Science and Technology Plan Projects (Nos. F15-199-1-30 and F15-199-1-57) and outstanding scientific fund of Shengjing hospital (No. 201304).

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Conflict of Interest

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The authors declare no conflict of interest.

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References 1.

Thorne, AH, Meisen, WH, Russell, L, Yoo, JY, Bolyard, CM, Lathia, JD, et al. (2014). Role of cysteine-rich 61 protein (CCN1) in macrophage-mediated oncolytic herpes simplex virus clearance. Mol Ther 22: 1678-1687.

2.

Berges, R, Balzeau, J, Peterson, AC, and Eyer, J (2012). A Tubulin Binding Peptide Targets Glioma Cells Disrupting Their Microtubules, Blocking

Liz, J, and Esteller, M (2016). lncRNAs and microRNAs with a role in cancer

cr

3.

ip

t

Migration, and Inducing Apoptosis. Mol Ther 20: 1367-1377.

Wang, P, Liu, YH, Yao, YL, Li, Z, Li, ZQ, Ma, J, et al. (2015). Long non-

an

4.

us

development. Biochimica et biophysica acta 1859: 169-176.

m

coding RNA CASC2 suppresses malignancy in human gliomas by miR-21.

Gupta, RA, Shah, N, Wang, KC, Kim, J, Horlings, HM, Wong, DJ, et al.

ep

5.

te d

Cellular signalling 27: 275-282.

cc

(2010). Long non-coding RNA HOTAIR reprograms chromatin state to

A

promote cancer metastasis. Nature 464: 1071-U1148. 6.

Ramos, AD, Attenello, FJ, and Lim, DA (2015). Uncovering the roles of long noncoding

RNAs

in

neural

development

and

glioma

progression.

Neuroscience letters. 7.

Wang, P, Ren, Z, and Sun, P (2012). Overexpression of the long non-coding RNA MEG3 impairs in vitro glioma cell proliferation. Journal of cellular biochemistry 113: 1868-1874.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

8.

Zhao, X, Wang, P, Liu, J, Zheng, J, Liu, Y, Chen, J, et al. (2015). Gas5 Exerts Tumor-suppressive Functions in Human Glioma Cells by Targeting miR-222. Mol Ther.

9.

Kiang, KM, Zhang, XQ, and Leung, GK (2015). Long Non-Coding RNAs: The Key Players in Glioma Pathogenesis. Cancers 7: 1406-1424.

10.

Frampton, AE, Castellano, L, Colombo, T, Giovannetti, E, Krell, J, Jacob, J, et

ip

t

al. (2015). Integrated molecular analysis to investigate the role of microRNAs

Rupaimoole, R, Calin, GA, Lopez-Berestein, G, and Sood, AK (2016).

us

11.

cr

in pancreatic tumour growth and progression. Lancet 385 Suppl 1: S37.

Matsuyama, R, Okuzaki, D, Okada, M, and Oneyama, C (2015). miR-27b

te d

12.

m

Cancer discovery.

an

miRNA Deregulation in Cancer Cells and the Tumor Microenvironment.

ep

suppresses tumor progression by regulating ARFGEF1 and the focal adhesion

Pal, R, and Greene, S (2015). microRNA-10b Is Overexpressed and Critical

A

13.

cc

signaling. Cancer science.

for Cell Survival and Proliferation in Medulloblastoma. PloS one 10. 14.

Ding, J, Huang, S, Wu, S, Zhao, Y, Liang, L, Yan, M, et al. (2010). Gain of miR-151 on chromosome 8q24.3 facilitates tumour cell migration and spreading through downregulating RhoGDIA. Nature cell biology 12: 390399.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

15.

Wang, P, Fu, T, Wang, X, and Zhu, W (2010). Primary study of miRNA expression pattens in laryngeal carcinoma by microarray. Journal of Clinical Otorhinolaryngology Head and Neck Surgery 24: 535-538.

16.

Sasaki, T, Shiohama, A, Minoshima, S, and Shimizu, N (2003). Identification of eight members of the Argonaute family in the human genome. Genomics 82: 323-330.

t

Li, L, Yu, C, Gao, H, and Li, Y (2010). Argonaute proteins: potential

ip

17.

Suzuki, R, Honda, S, and Kirino, Y (2012). PIWI Expression and Function in

Sugimoto, K, Kage, H, Aki, N, Sano, A, Kitagawa, H, Nagase, T, et al.

m

19.

an

Cancer. Frontiers in genetics 3: 204.

us

18.

cr

biomarkers for human colon cancer. BMC cancer 10: 38.

te d

(2007). The induction of H3K9 methylation by PIWIL4 at the p16Ink4a locus.

Greither, T, Koser, F, Kappler, M, Bache, M, Lautenschlager, C, Gobel, S, et

cc

20.

ep

Biochem Biophys Res Commun 359: 497-502.

A

al. (2012). Expression of human Piwi-like genes is associated with prognosis for soft tissue sarcoma patients. BMC cancer 12. 21.

Su, C, Ren, ZJ, Wang, F, Liu, M, Li, X, and Tang, H (2012). PIWIL4 regulates cervical cancer cell line growth and is involved in down-regulating the expression of p14ARF and p53. FEBS Lett 586: 1356-1362.

22.

Al-Janabi, O, Wach, S, Nolte, E, Weigelt, K, Rau, TT, Stohr, C, et al. (2014). Piwi-like 1 and 4 gene transcript levels are associated with clinicopathological

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

parameters in renal cell carcinomas. Biochimica et biophysica acta 1842: 686690. 23.

Poliseno, L, Salmena, L, Zhang, J, Carver, B, Haveman, WJ, and Pandolfi, PP (2010). A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465: 1033-1038.

24.

Lu, Y, Zhang, K, Li, C, Yao, Y, Tao, D, Liu, Y, et al. (2012). Piwil2

ip

t

suppresses p53 by inducing phosphorylation of signal transducer and activator

Abou-Ghazal, M, Yang, DS, Qiao, W, Reina-Ortiz, C, Wei, J, Kong, LY, et al.

us

25.

cr

of transcription 3 in tumor cells. PLoS One 7: e30999.

an

(2008). The incidence, correlation with tumor-infiltrating inflammation, and

m

prognosis of phosphorylated STAT3 expression in human gliomas. Clinical

te d

cancer research : an official journal of the American Association for Cancer

Paternot, S, and Roger, PP (2009). Combined inhibition of MEK and

cc

26.

ep

Research 14: 8228-8235.

A

mammalian target of rapamycin abolishes phosphorylation of cyclindependent kinase 4 in glioblastoma cell lines and prevents their proliferation. Cancer research 69: 4577-4581. 27.

Seystahl, K, Tritschler, I, Szabo, E, Tabatabai, G, and Weller, M (2015). Differential regulation of TGF-beta-induced, ALK-5-mediated VEGF release by SMAD2/3 versus SMAD1/5/8 signaling in glioblastoma. Neuro-oncology 17: 254-265.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

28.

Lee, KH, Ahn, EJ, Oh, SJ, Kim, O, Joo, YE, Bae, JA, et al. (2015). KITENIN promotes glioma invasiveness and progression, associated with the induction of EMT and stemness markers. Oncotarget 6: 3240-3253.

29.

Asuthkar, S, Velpula, KK, Chetty, C, Gorantla, B, and Rao, JS (2012). Epigenetic regulation of miRNA-211 by MMP-9 governs glioma cell apoptosis, chemosensitivity and radiosensitivity. Oncotarget 3: 1439-1454.

t

Pont, LM, Naipal, K, Kloezeman, JJ, Venkatesan, S, van den Bent, M, van

ip

30.

cr

Gent, DC, et al. (2015). DNA damage response and anti-apoptotic proteins

us

predict radiosensitization efficacy of HDAC inhibitors SAHA and LBH589 in

Kesanakurti, D, Chetty, C, Dinh, DH, Gujrati, M, and Rao, JS (2013). Role of

m

31.

an

patient-derived glioblastoma cells. Cancer Lett 356: 525-535.

te d

MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with

Steder, M, Alla, V, Meier, C, Spitschak, A, Pahnke, J, Furst, K, et al. (2013).

cc

32.

ep

alpha5beta1 integrin in glioma. Oncogene 32: 327-340.

A

DNp73 exerts function in metastasis initiation by disconnecting the inhibitory role of EPLIN on IGF1R-AKT/STAT3 signaling. Cancer cell 24: 512-527. 33.

Natesh, K, Bhosale, D, Desai, A, Chandrika, G, Pujari, R, Jagtap, J, et al. (2015). Oncostatin-M differentially regulates mesenchymal and proneural signature genes in gliomas via STAT3 signaling. Neoplasia 17: 225-237.

34.

Siveen, KS, Nguyen, AH, Lee, JH, Li, F, Singh, SS, Kumar, AP, et al. (2014). Negative regulation of signal transducer and activator of transcription-3

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

signalling cascade by lupeol inhibits growth and induces apoptosis in hepatocellular carcinoma cells. Br J Cancer 111: 1327-1337. 35.

Mohan, CD, Bharathkumar, H, Bulusu, KC, Pandey, V, Rangappa, S, Fuchs, JE, et al. (2014). Development of a novel azaspirane that targets the Janus kinase-signal transducer and activator of transcription (STAT) pathway in hepatocellular carcinoma in vitro and in vivo. J Biol Chem 289: 34296-34307.

t

Kim, SM, Lee, JH, Sethi, G, Kim, C, Baek, SH, Nam, D, et al. (2014).

ip

36.

cr

Bergamottin, a natural furanocoumarin obtained from grapefruit juice induces

us

chemosensitization and apoptosis through the inhibition of STAT3 signaling

Kim, D, Lee, IH, Kim, S, Choi, M, Kim, H, Ahn, S, et al. (2014). A specific

m

37.

an

pathway in tumor cells. Cancer Lett 354: 153-163.

te d

STAT3-binding peptide exerts antiproliferative effects and antitumor activity

Hu, XW, Feng, Y, Zhang, DM, Zhao, SHD, Hu, ZY, Greshock, J, et al.

A

38.

cc

2151.

ep

by inhibiting STAT3 phosphorylation and signaling. Cancer Res 74: 2144-

(2014). A Functional Genomic Approach Identifies FAL1 as an Oncogenic Long Noncoding RNA that Associates with BMI1 and Represses p21 Expression in Cancer. Cancer Cell 26: 344-357. 39.

Zhang, K, Sun, X, Zhou, X, Han, L, Chen, L, Shi, Z, et al. (2015). Long noncoding RNA HOTAIR promotes glioblastoma cell cycle progression in an EZH2 dependent manner. Oncotarget 6: 537-546.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

40.

Ke, J, Yao, YL, Zheng, J, Wang, P, Liu, YH, Ma, J, et al. (2015). Knockdown of long non-coding RNA HOTAIR inhibits malignant biological behaviors of human glioma cells via modulation of miR-326. Oncotarget 6: 21934-21949.

41.

Ellis, BC, Molloy, PL, and Graham, LD (2012). CRNDE: A Long NonCoding RNA Involved in CanceR, Neurobiology, and DEvelopment. Frontiers in genetics 3: 270.

t

Wang, YL, Wang, YT, Li, JF, Zhang, YZ, Yin, HL, and Han, B (2015).

ip

42.

cr

CRNDE, a long-noncoding RNA, promotes glioma cell growth and invasion

Zheng, J, Li, XD, Wang, P, Liu, XB, Xue, YX, Hu, Y, et al. (2015). CRNDE

an

43.

us

through mTOR signaling. Cancer Lett 367: 122-128.

m

affects the malignant biological characteristics of human glioma stem cells by

Liz, J, and Esteller, M (2015). lncRNAs and microRNAs with a role in cancer

ep

44.

te d

negatively regulating miR-186. Oncotarget 6: 25339-25355.

Zhang, Z, Zhu, Z, Watabe, K, Zhang, X, Bai, C, Xu, M, et al. (2013).

A

45.

cc

development. Biochimica et biophysica acta.

Negative regulation of lncRNA GAS5 by miR-21. Cell death and differentiation 20: 1558-1568. 46.

Chiyomaru, T, Fukuhara, S, Saini, S, Majid, S, Deng, GR, Shahryari, V, et al. (2014). Long Non-coding RNA HOTAIR Is Targeted and Regulated by miR141 in Human Cancer Cells. J Biol Chem 289: 12550-12565.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

47.

Min, AJ, Zhu, C, Peng, SP, Rajthala, S, Costea, DE, and Sapkota, D (2015). MicroRNAs as Important Players and Biomarkers in Oral Carcinogenesis. Biomed Res Int.

48.

Navarro, A, Tejero, R, Vinolas, N, Cordeiro, A, Marrades, RM, Fuster, D, et al. (2015). The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer. Oncotarget 6: 31544-31556.

t

Chen, C, Liu, J, and Xu, G (2013). Overexpression of PIWI proteins in human

ip

49.

cr

stage III epithelial ovarian cancer with lymph node metastasis. Cancer

Lin, J, Zhou, J, Zhong, X, Hong, Z, and Peng, J (2015). Inhibition of the signal

an

50.

us

biomarkers : section A of Disease markers 13: 315-321.

m

transducer and activator of transcription 3 signaling pathway by Qianliening

te d

capsules suppresses the growth and induces the apoptosis of human prostate

Resemann, HK, Watson, CJ, and Lloyd-Lewis, B (2014). The Stat3 paradox: a

cc

51.

ep

cells. Molecular medicine reports 11: 2207-2214.

A

killer and an oncogene. Molecular and cellular endocrinology 382: 603-611. 52.

Rahaman, SO, Harbor, PC, Chernova, O, Barnett, GH, Vogelbaum, MA, and Haque, SJ (2002). Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells. Oncogene 21: 8404-8413.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

53.

Malanga, D, De Marco, C, Guerriero, I, Colelli, F, Rinaldo, N, Scrima, M, et al. (2015). The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells. Oncotarget.

54.

Li, Z, Li, X, Wu, S, Xue, M, and Chen, W (2014). Long non-coding RNA UCA1 promotes glycolysis by upregulating hexokinase 2 through the mTORSTAT3/microRNA143 pathway. Cancer science 105: 951-955.

t

Cuevas, P, Diaz-Gonzalez, D, and Dujovny, M (2003). Antiproliferative

ip

55.

cr

action of neomycin is associated with inhibition of cyclin D1 activation in

Zhang, X, Zhao, M, Huang, AY, Fei, Z, Zhang, W, and Wang, XL (2005). The

an

56.

us

glioma cells. Neurological research 25: 691-693.

m

effect of cyclin D expression on cell proliferation in human gliomas. Journal

te d

of clinical neuroscience : official journal of the Neurosurgical Society of

Darvin, P, Baeg, SJ, Joung, YH, Sp, N, Kang, DY, Byun, HJ, et al. (2015).

cc

57.

ep

Australasia 12: 166-168.

A

Tannic acid inhibits the Jak2/STAT3 pathway and induces G1/S arrest and mitochondrial apoptosis in YD-38 gingival cancer cells. International journal of oncology 47: 1111-1120. 58.

Yang, HW, Menon, LG, Black, PM, Carroll, RS, and Johnson, MD (2010). SNAI2/Slug promotes growth and invasion in human gliomas. BMC cancer 10: 301.

© 2016 The American Society of Gene & Cell Therapy. All rights reserved

ACCEPTED ARTICLE PREVIEW

59.

Pan, HC, Jiang, Q, Yu, Y, Mei, JP, Cui, YK, and Zhao, WJ (2015). Quercetin promotes cell apoptosis and inhibits the expression of MMP-9 and fibronectin via the AKT and ERK signalling pathways in human glioma cells. Neurochemistry international 80: 60-71.

60.

McIntyre, A, Patiar, S, Wigfield, S, Li, JL, Ledaki, I, Turley, H, et al. (2012). Carbonic anhydrase IX promotes tumor growth and necrosis in vivo and

ip

t

inhibition enhances anti-VEGF therapy. Clinical cancer research : an official

Galluzzi, L, Kepp, O, and Kroemer, G (2012). Caspase-3 and prostaglandins

us

61.

cr

journal of the American Association for Cancer Research 18: 3100-3111.

Volkmann, N, Marassi, FM, Newmeyer, DD, and Hanein, D (2014). The

m

62.

an

signal for tumor regrowth in cancer therapy. Oncogene 31: 2805-2808.

te d

rheostat in the membrane: BCL-2 family proteins and apoptosis. Cell death

A

cc

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and differentiation 21: 206-215.

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Table 1 Nam

StarBase (v2.0) predicted the miRNAs that target CRNDE mirAccession

e hsa-

MIMAT000044

miR-

8

GeneNam

TargetSite

BioComple

ClipReadNu

e

s

x

m

CRNDE

1

1

8

CRNDE

1

1

136-

MIMAT000107

miR-

5

8

us

cr

hsa-

ip

t

5p

A

cc

ep

te d

m

an

384

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Table 2 TargetScan (Release 7.0) predicted some of the RNAs target by miR-384 Target gene

Representative

Gene name

transcript

Site

Representative Cumulative Total

counts

miRNA

weighted

context+

context++

+ score

score DIRC1

ENST00000308100.4 disrupted in renal

1

hsa-miR-384

-0.64

-0.64

1

hsa-miR-384

-0.56

-0.56

hsa-miR-384

-0.55

-0.73

1

hsa-miR-384

-0.40

-0.40

1

hsa-miR-384

-0.32

-0.32

1

hsa-miR-384

-0.31

-0.31

1

hsa-miR-384

-0.30

-0.30

2

hsa-miR-384

-0.29

-0.29

ip

ENST00000357325.5 hepatoma-derived growth factor ENST00000370500.5 general

2

an

transcription factor

m

IIB

ENST00000299001.6 piwi-like RNA-

te d

PIWIL4

us

GTF2B

cr

HDGF

t

carcinoma 1

mediated gene

ENST00000303958.2 proteolipid protein

cc

PLP1

ep

silencing 4

ELL3

A

1

ENST00000319359.3 elongation factor RNA polymerase II-like 3

TXNIP

ENST00000369317.4 thioredoxin interacting protein

ZNF624

ENST00000311331.7 zinc finger protein 624

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ACCEPTED ARTICLE PREVIEW ANKRD20A2 ENST00000377601.2 ankyrin repeat

1

hsa-miR-384

-0.29

-0.31

1

hsa-miR-384

-0.29

-0.29

domain 20 family, member A2 ENST00000344843.7 mitochondrial ribosomal protein

cc

ep

te d

m

an

us

cr

ip

t

L20

A

MRPL20

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Table 3 miRanda (August 2010 Release) predicted some of the RNAs target by miR-384 Target gene

NM Number

mirSVR score

SOX30

NM_178424

-1.21

MCTP1

NM_024717,

-1.20

NM_001002796 NM_006467

-1.13

PIWIL4

NM_152431

CAMK2N1

NM_018584

PTHLH

NM_198965, NM_198966

USO1

NM_003715

-0.96

CDK14

NM_012395

-0.92

RHPN2

NM_033103

-0.83

NM_002053

-0.78

ip

t

POLR3G

cr

-1.07

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m

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us

-1.06

A

cc

ep

GBP1

-1.05

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Figure legends Fig. 1. Effect of CRNDE on proliferation, apoptosis, migration and invasion of U87 and U251 glioma cells. (a) CCK-8 assay was used to determine the proliferation effect of CRNDE on U87 and U251 cells. (b) Quantification number of migration and invasion cells with overexpression or knockdown of CRNDE. Representative images and accompanying statistical plots were presented. (c) Flow cytometry analysis of U87 and U251 cells with the expression of CRNDE changed. (Data are presented as the mean + SD

ip

t

(n=5, each group). *P<0.05 vs. pEX2-NC group; #P<0.05 vs. sh-NC group. Scale bars represent 40 µm).

cr

Fig. 2. miR-384 expression in glioma tissues and glioma cell lines, overexpression of miR-384 inhibited

us

the malignant progression of glioma cells.

an

(a) Expression levels of miR-384 in glioma tissues of different grades and normal brain tissues (NBTs).

m

(Data are presented as the mean + SD (n=15, each group). **P<0.01 vs. NBTs group; ##P<0.01 vs. Grade

te d

I group; ∆∆P<0.01 vs. Grade II group; ΨΨP<0.01 vs. Grade III group). (b) Expression levels of miR-384 in human normal astrocytes and glioma cell lines. (Data are presented as the mean + SD (n=5, each

ep

group). **P<0.01 vs. Normal human astrocytes group). (c) CCK-8 assay was applied to evaluate the

cc

proliferation effect of miR-384 on U87 and U251 cells. (d) Quantification number of migration and

A

invasion cells with different expression levels of miR-384. Representative images and accompanying statistical plots were presented. Scale bars represent 40 µm. (e) Flow cytometry analysis of U87 and U251 cells with the expression of miR-384 changed. (Data are presented as the mean + SD (n=5, each group). *P<0.05 vs. pre-NC group; #P<0.05 vs. anti-NC group). Fig. 3. Overexpression of CRNDE inhibited miR-384 expression, downregulation of CRNDE or overexpression of miR-384 inhibited the expression of PIWIL4. (a) qRT-PCR analysis for CRNDE (Wt and Mut) regulated miR-384 expression in U87 and U251 cells. (Data are presented as the mean + SD (n=5, each group). **P<0.01 vs. pEX-NC group; ##P<0.01 vs. shNC group). (b) qRT-PCR analysis for miR-384 impaired CRNDE expression in U87 and U251 cells.

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(Data are presented as the mean + SD (n=5, each group). **P<0.01 vs. pre-NC group; ##P<0.01 vs. antiNC group). (c) The predicted miR-384 binding sites in the 3′-UTR of CRNDE (CRNDE-Wt) or and the designed mutant sequence (CRNDE-Mut) were indicated. (d) Luciferase reporter assay of HEK 293T cells co-transfected with pEX2-CRNDE or pEX2-CRNDE-NC and miR-384-3′UTR-Wt (miR-384 WT) or the miR-384-3′UTR-Mut (miR-384 Mut). (Data are presented as the mean + SD (n=5, each group)). (e-h) miR-384 was identified in CRNDE-RISC complex. CRNDE and miR-384 expression levels were detected using qRT-PCR. (Data represent mean + SD (n = 5, each group). *P < 0.05, **P < 0.01 vs. anti-

t

normal IgG group of respective group, #P<0.05, ##P<0.01 vs. anti-Ago2 in control group). (i and j) qRT-

cr

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PCR and western blot analysis for CRNDE regulating PIWIL4 expression in U87 and U251 cells. The

us

relative expression of PIWIL4 was shown using GAPDH as an endogenous control. The IDVs of PIWIL4 was shown using GAPDH as an endogenous control. (Data are presented as the mean + SD

an

(n=5, each group). *P<0.05 vs. pEX2-NC group; #P<0.05 vs. sh-NC group). (k and l) qRT-PCR and

m

western blot analysis for miR-384 regulating PIWIL4 expression in U87 and U251. The relative

te d

expression of PIWIL4 was shown using GAPDH as an endogenous control. The IDVs of PIWIL4 was

ep

shown using GAPDH as an endogenous control. (Data are presented as the mean + SD (n=5, each

cc

group). *P<0.05 vs. pre-NC group; #P<0.05 vs. anti-NC group). (m) Immunohistochemistry of PIWIL4

A

protein in nontumorous brain, low-grade glioma, and high-grade glioma tissues. Original magnification: 100×, 200×. Scale bar = 50 µm. (n) PIWIL4 protein expression levels in nontumorous brain tissues and glioma tissues using GAPDH as an endogenous control. Representative protein expression and their integrated light density values (IDVs) of PIWIL4 in nontumorous brain tissues, low-grade glioma tissues (World Health Organization [WHO] I-II), and high-grade glioma tissues (WHO III-IV) are shown. (Data are presented as the mean + SD (n=15, each group). **P<0.01 vs. NBTs group; ##P<0.01 vs. Low-grade glioma tissues group). (o) PIWIL4 protein expression levels in Astrocytes, U87 and U251 cells and using GAPDH as an endogenous control. Representative protein expression and their IDVs in human

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normal astrocytes, U87 and U251 are shown. (Data are presented as the mean + SD (n=15, each group). **

P<0.01 vs. human normal astrocytes group).

Fig. 4. Effect of CRNDE and miR-384 on proliferation, migration, invasion and apoptosis on U87 and U251 cells and overexpression CRNDE elevated levels of the expression of PIWIL4 by downregulating miR-384. (a) CCK-8 assay was applied to evaluate the proliferation effect of CRNDE and miR-384 on U87 and U251 cells. (Data are presented as the mean + SD (n=5, each group). *P<0.05 vs. CRNDE(+)+miR-

t

384(+) group; #P<0.05 vs. CRNDE(-)+miR-384(-) group; ▲P<0.05 vs. Control group). (b)

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Quantification of migration and invasion cells with the expression of CRNDE and miR-384 changed.

us

Representative images and accompanying statistical plots were presented. (Data are presented as the mean + SD (n=5, each group). *P<0.05 vs. CRNDE(+)+miR-384(+) group; #P<0.05 vs. CRNDE(-

an

)+miR-384(-) group; ▲P<0.05 vs. Control group. Scale bars represent 40 µm). (c) Flow cytometry

m

analysis of U87 and U251 with the expression of CRNDE and miR-384 changed. (d) Western blot

te d

analysis for CRNDE and miR-384 regulating IDVs of PIWIL4, they are shown using GAPDH as

ep

endogenous control. (Data are presented as the mean + SD (n=5, each group). *P<0.05 vs.

A

group).

cc

CRNDE(+)+miR-384(+) group; #P<0.05 vs. CRNDE(-)+miR-384(-) group; ▲P<0.05 vs. Control

Fig. 5. PIWIL4 played an oncogenic role in glioma cells. (a) CCK-8 assay was used to determine the proliferation effect of PIWIL4 on U87 and U251 cells. (b) Quantification number of migration and invasion cells with overexpression or knockdown of PIWIL4. Representative images and accompanying statistical plots were presented. Scale bars represent 40 µm. (c) Flow cytometry analysis of U87 and U251 cells with the expression of PIWIL4 changed. (Data are presented as the mean + SD (n=5, each group). *P<0.05 vs. PIWIL4(+)-NC group; #P<0.05 vs. PIWIL4(-)-NC group). (d) Western blot analysis for PIWIL4 regulating IDVs of p-STAT3 and STAT3,

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they are shown using GAPDH as endogenous control. Data are presented as the mean + SD (n=5, each group). *P<0.05 vs. PIWIL4(+)-NC group; #P<0.05 vs. PIWIL4(-)-NC group. Fig. 6. miR-384 inhibited glioma cell malignant behaviors by regulating cyclin D1, VEGFA, SLUG, MMP-9, Bcl-2, bcl-xL and caspase 3 through targeting PIWIL4 3′-UTR. (a) The predicted miR-384 binding sites in the 3′-UTR of PIWIL4 (PIWIL4-Wt) or and the designed mutant sequence (PIWIL4-Mut) were indicated. (b) Luciferase reporter assay of HEK 293T cells transfected with PIWIL4-3’UTR-Wt (PIWIL4-Wt) (or the PIWIL4-3′UTR-Mut (PIWIL4-Mut)) and the

t

indicated miRNAs. (Data are presented as the mean + SD (n=5, each group). *P<0.05 vs. PIWIL4-Wt +

cr

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miR-384-NC group). (c) CCK-8 assay was applied to evaluate the proliferation effect of miR-384 and

us

miR-PIWIL4 on U87 and U251 cells. (Data are presented as the mean + SD (n=5, each group). Data are

an

presented as the mean + SD (n=5, each group). *P＜0.05 vs. miR-384+PIWIL4 group; #P＜0.05 vs.

m

miR-384+PIWIL4-NC group. (d) Quantification of migration and invasion cells with the expression of

te d

miR-384 and PIWIL4 changed. Representative images and accompanying statistical plots were

ep

presented. (Data are presented as the mean + SD (n=5, each group). *P＜0.05 vs. miR-384+PIWIL4

cc

group; #P＜0.05 vs. miR-384+PIWIL4-NC group. Scale bars represent 40 µm). (e) Flow cytometry

A

analysis of U87 and U251 with the expression of miR-384 and PIWIL4 changed. (f) Western blot analysis of p-STAT3, STAT3, cyclin D1, VEGFA, SLUG, MMP-9, Bcl-2, bcl-xL and caspase 3 regulated by miR-384 and PIWIL4 in U87 and U251 cells, they are shown using GAPDH as endogenous control. (Data are presented as the mean + SD (n=5, each group). *P<0.05 vs. PIWIL4(+)-NC group; #

P<0.05 vs. PIWIL4(-)-NC group).

Fig. 7. In vivo study. (a) The stable expressing cells were used for the in vivo study. The nude mice carrying tumors from respective groups were shown. The sample tumor from respective group was shown. (b) Tumor volume

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was calculated every four days after injection, and the tumor was excised after 40 days. *P < 0.05 vs. Control group; #P < 0.05 vs. miR-384 group; ▲P < 0.05 vs. sh-CRNDE group. The survival curves of nude mice injected into the right striatum (n=15). P < 0.05 (miR-384 or sh-CRNDE group vs. Control group); P < 0.01 (miR-384+ sh-CRNDE group vs. Control group). Fig. 8. The schematic cartoon of the mechanism of CRNDE as an oncogene negative regulation of miR384 in glioma cells. Fig. S1. miR-384 expression normalized to 18S in glioma tissues and glioma cell lines, overexpression

t

of miR-384 inhibited the malignant progression of glioma cells.

ip

(a) Expression levels of miR-384 normalized to 18S in glioma tissues of different grades and normal

us

cr

brain tissues (NBTs). (Data are presented as the mean + SD (n=15, each group). **P<0.01 vs. NBTs

an

group; ##P<0.01 vs. Grade I group; ∆∆P<0.01 vs. Grade II group; ΨΨP<0.01 vs. Grade III group). (b) Expression levels of miR-384 normalized to 18S in human normal astrocytes and glioma cell lines.

m

(Data are presented as the mean + SD (n=5, each group). **P<0.01 vs. Normal human astrocytes group).

te d

Fig. S2. Oncogenic effects being directly mediated by CRNDE. Western blot analysis of p-STAT3,

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STAT3, cyclin D1, VEGFA, SLUG, MMP-9, Bcl-2, bcl-xL and caspase 3 in CRNDE overexpression

cc

U87 and U251 cells, they are shown using GAPDH as endogenous control. (Data are presented as the

A

mean + SD (n=5, each group). *P<0.05 vs. pEX2-NC group). Fig. S3. miR-384 expression in PIWIL4 overexpression cell lines.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

Figure 8

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