Linc00152 Promotes Malignant Progression of Glioma Stem Cells By

Yu et al. Molecular Cancer (2017) 16:110 DOI 10.1186/s12943-017-0677-9

RESEARCH Open Access Linc00152 promotes malignant progression of glioma stem cells by regulating miR-103a-3p/FEZF1/CDC25A pathway Mingjun Yu1,2,3, Yixue Xue4,5,6, Jian Zheng1,2,3, Xiaobai Liu1,2,3, Hai Yu1,2,3, Libo Liu4,5,6, Zhen Li1,2,3 and Yunhui Liu1,2,3*

Abstract Background: Glioma is one of the most frequent intracranial malignant tumors. LncRNAs have been identified as new modulators in the origination and progression of glioma. Methods: Quantitative real-time PCR were conducted to evaluate the expression of linc00152 and miRNA-103a-3p in glioma tissues and cells. Western blot were used to determine the expression of FEZF1 and CDC25A in glioma tissues and cells. Stable knockdown of linc00152 or over-expression of miR-103a-3p in glioma stem cells (GSCs) were established to explore the function of linc00152 and miR-103a-3p in GSCs. Further, luciferase reports were used to investigate the correlation between linc00152 and miR-103a-3p. Cell Counting Kit-8, transwell assays, and flow cytometry were used to investigate the function of linc00152 and miR-103a-3p in GSC malignant biological behaviors. ChIP assays were employed to ascertain the correlations between FEZF1 and CDC25A. Results: Linc00152 was up-regulated in glioma tissues as well as in GSCs. Knockdown of linc00152 inhibited cell proliferation, migration and invasion, while promoted GSC apoptosis. Linc00152 regulated the malignant behavior of GSCs by binding to miR-103a-3p, which functions as a tumor suppressor. In addition, knockdown of linc00152 down-regulated forebrain embryonic zinc finger protein 1 (FEZF1), a direct target of miR-103a-3p which played an oncogenic role in GSCs. FEZF1 elevated promoter activities and up-regulated expression of the oncogenic gene cell division cycle 25A (CDC25A). CDC25A over-expression activated the PI3K/AKT pathways, which regulated the malignant behavior of GSCs. Conclusions: Linc00152/miR-103a-3p/FEZF1/CDC25A axis plays a novel role in regulating the malignant behavior of GSCs, which may be a new potential therapeutic strategy for glioma therapy. Keywords: Long non-coding RNA, linc00152, miR-103a-3p, Glioma stem cells, FEZF1, CDC25A

Background formation, self-renewal and multi-directional differentiation Glioma is one of the most frequent intracranial malig- [4]. Therefore, illustration of the molecular mechanisms nant tumors. Due to the highly invasive growth pattern underlying GSCs offers new orientation toward the gloma and frequent resistance to therapies, the prognosis for treatments. patients with glioma is dismal [1]. Glioma stem cells Lately, long non-coding RNAs (lncRNAs), which are (GSCs), a neoplastic subpopulation within glioma, are defined as non-coding RNAs more than 200 nucleotides responsible for therapy resistance [2] and poor survival in length, are involved in various genetic phenomena, rate of glioma [3]. GSCs have characteristics of neurosphere including transcriptional, post-transcriptional and epi- genetic regulations [5]. Moreover, lncRNAs have been * Correspondence: [email protected] identified as new modulators in the origination and 1 Department of Neurosurgery, Shengjing Hospital of China Medical progression of glioma [6]. Mounting evidence has also University, Shenyang 110004, People’s Republic of China 2Liaoning Clinical Medical Research Center in Nervous System Disease, demonstrated that lncRNAs involved in the regulation Shenyang 110004, People’s Republic of China Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yu et al. Molecular Cancer (2017) 16:110 Page 2 of 18

of gene expression in stem cell biology and in tumori- them. Our findings will give a new direction for the genesis [7]. treatment of glioma. Long intergenic non-coding RNA 152 (Linc00152) was found aberrant expression in numerous cancers. Linc00152 Results was distinctly up-regulated in gastric cancer and its expres- Isolation and identification of GSCs sion was positively correlated with tumor invasion depth, AsshowninAdditionalfile1:FigureS1A,GSCs-87 lymph node metastasis and poor survival [8]. Moreover, (or GSCs-251) isolated from glioma cells were cul- linc00152 could modulate the expression of miR-193a-3p tured in the serum-free medium and formed cell to increase disease recurrence through acting as a compet- spheres. Immunofluorescence analysis indicated that ing endogenous RNA in colon cancer [9]. However, the most cells within spheres were positive to neural stem expression and possible functional role of linc00152 in cell lineage markers Nestin and CD133 (Additional GSCs remains uncharted. file 1: Figure S1B). During the differentiation assay, MicroRNAs (miRNAs) are small non-coding RNAs single-cell suspensions of spheres were differentiated ′ that regulate gene expression by binding to the 3 -UTRs and stained with GFAP and β-tubulin III, implying of mRNAs [10]. MiRNAs are reported to be involved that isolated GSCs-87 and GSCs-251 possessed the in a variety of human carcinogenesis, including glioma potential for differentiation toward astrocytic and [11]. MiR-103a-3p was illuminated to participate in neuronal lineages (Additional file 1: Figure S1C). the proliferation, migration and invasion process in ovarian carcinoma [12]. It is predicted that miR-103a- 3p has putative binding sites with linc00152 by Star- Linc00152 expression was up-regulated in glioma tissues base (http://starbase.sysu.edu.cn/). Moreover, miR- and GSCs while miR-103a-3p was down- regulated in glioma 103a-3p was identified as prognostic biomarker of tissue and GSCs colon cancer, breast cancer and pleural mesothelioma The linc00152 and miR-103a-3p expression levels in [13–15], etc. However, limited knowledge is available normal brain tissues(NBTs), grade I-II (low grade) and concerning whether miR-103a-3p affected the bio- grade III-IV (high grade) glioma tissues, as well as in logical processes of GSCs. GSCs and non stem cells (non-GSCs) were evaluated by Forebrain embryonic zinc finger protein 1(FEZF1) is qRT-PCR. As shown in Fig. 1a-b, linc00152 expression highly conserved transcription factor that encoded a levels elevated with the pathological grades of glioma tis- transcriptional repressor which contributed to the em- sues and were apparently up-regulated in GSCs compared bryonic migration of gonadotropin-releasing hormone with non-GSCs. However, expression of miR-103a-3p was neurons into the brain [16]. In addition, FEZF1 is re- negatively correlated with the progression of glioma quired for olfactory development and knock down pathological grade and was distinctly down-regulated in FEZF1 in mice, olfactory neurons fail to mature [17]. GSCs compared with non-GSCs (Fig. 2a-b). We predicted FEZF1 as a presumed target of miR-103a- 3p by miRNA target prediction software Target Scan Knockdown of linc00152 repressed malignant biological (http://www.targetscan.org/). Studies have shown that properties of GSCs FEZF1 played a oncogenic role through DNA demethyl- Following the evaluation of linc00152 expression in GSCs, ation [18] in gastric cancer. Nevertheless, the role of we further explored the possible effect of linc00152 on the FEZF1 in GSCs remains ambiguous. proliferation, migration, invasion and apoptosis of GSCs. Cell division cycle 25A (CDC25A), a member of the Stable linc00152 silenced constructs were used to appraise CDC25 family of phosphatases, removes the inhibitory the function of linc00152 on GSCs. The RT-PCR assay phosphorylation in cyclin-dependent kinases (CDKs) was applied to demonstrate the linc00152 knockdown [19], and regulates the progression from G1 to the S (Fig. 1c). The Cell Counting Kit-8 (CCK-8) assay suggested phase of the cell cycle [20]. It is discovered that the that GSCs proliferation was declined in the sh-linc00152 promoter of CDC25A contains sequence of CnnCAnCn group than that in the sh-NC group (Fig. 1d). The limiting core which is a putative consensus FEZF1 binding site dilution assay implied that knockdown of linc00152 [21, 22]. Furthermore, CDC25A was identified as an hindered the sphere-formation ability of GSCs (Fig. 1e). oncogene, and over-expression of CDC25A promoted The migration and invasion of GSCs assay showed that tumorigenesis [23]. the numbers of migrated and invaded cells in the sh- The major purpose of this study was to explore the linc00152 group were significantly decreased compared expression of linc00152, miR-103a-3p, FEZF1 and with the sh-NC group (Fig. 1f). Flow cytometry analysis CDC25A in glioma tissues and GSCs. We also investi- implied that silence of linc00152 markedly increased gated the roles in regulating the malignant behavior of apoptosis of GSCs (Fig. 1g). Taken together, linc00152 GSCs and the potential molecular pathways among functioned as an oncogene in GSCs. Yu et al. Molecular Cancer (2017) 16:110 Page 3 of 18

Fig. 1 linc00152 functioned as an oncogene in GSCs. a Expression of linc00152 in glioma tissues of different grades and normal brain tissues (NBTs). (Data are presented as the mean ± SD (n = 10, each group). **P < 0.01 vs. NBTs group; *P < 0.05 vs. low grade group; #P < 0.05 vs. low grade group. b Expression of linc00152 in non-GSCs and GSCs (Data are presented as the mean ± SD (n =5,eachgroup).**P <0.01vs. non-GSCs-U87 group; ##P < 0.01 vs. non-GSCs-U251 group. c Relative expression of linc00152 after GSCs transfected with sh-linc00152 plasmids and scrambled vectors (NC). d CCK-8 assay was conducted to explore the proliferation effect of linc00152 on GSCs. e Limiting dilution assay showing the impact of linc00152 on the sphere-formation ability of GSCs. f Transwell assays for assessing GSC migration and invasion. Typical images and accompanying statistical plots were presented. g Flow cytometry detected the apoptosis of GSCs with the knockdown of linc00152. Data are presented as the mean ± SD (n =5,eachgroup).*P < 0.05 vs. sh-NC group. Scale bars represent 20 μm. The photographs were taken at 200 × magnification Yu et al. Molecular Cancer (2017) 16:110 Page 4 of 18

Fig. 2 miR-103a-3p manifested an anti-oncogene in GSCs. a Expression of miR-103a-3p in glioma tissues of different grades and NBTs (Data are presented as the mean ± SD (n = 10, each group). **P < 0.01 vs. NBTs group; *P < 0.05 vs. NBTs group; #P < 0.05 vs. low grade group. b Expression of miR-103a-3p in non-GSCs and GSCs (Data are presented as the mean ± SD (n =5,eachgroup).**P < 0.01 vs. non-GSCs-U87 group; ##P <0.01vs. non-GSCs-U251 group. c Relative expression of miR-103a-3p after GSCs transfected with pre-miR-103a-3p, anti-miR-103a-3p as well as their scrambled vectors (NC). d Effect of miR-103a-3p on the proliferation of GSCs. e Effect of miR-103a-3p on the sphere-formation ability of GSCs. f Effect of miR-103a-3p on the migration and invasion of GSCs. g The apoptotic rates after GSCs transfected with pre-miR-103a-3p and anti-miR-103a-3p. *P < 0.05 vs. pre-NC group; #P < 0.05 vs. anti-NC group. Scale bars, 20 μm. The photographs were taken at 200 × magnification miR-103a-3p manifested a tumor suppressor Flow cytometry analysis suggested that over-expression Had appraised the miR-103a-3p expression in GSCs, we of miR-103a-3p markedly promoted apoptosis of GSCs assessed the effect of miR-103a-3p on GSCs and transfected (Fig. 2g). The results above inferred that miR-103a-3p, miR-103a-3p-agomir (pre-miR-103a-3p) and miR-103a-3p- in contrast to linc00152, appeared as tumor-suppressive antagomir (anti-miR-103a-3p) into GSCs, respectively. The function in GSCs. RT-PCR assay was applied to detect the transfection ef- ficiency of miR-103a-3p (Fig. 2c). As shown in Fig. 2d, Linc00152 is a direct target of miR-103a-3p pre-miR-103a-3p group emerged a significantly declined Increasing evidence implied that lncRNAs could be proliferation in GSCs compared to the pre-NC group. The competing endogenous RNAs (ceRNAs) or molecular limiting dilution assay implied that over-expression of sponges in affecting the expression and biological func- miR-103a-3p hindered the sphere-formation ability of tions of miRNAs [24]. Using a bioinformatics database GSCs (Fig. 2e). Transwell assays implied that migrated (Starbase), linc00152 harbor one conjectural binding site and invaded cell numbers were remarkably decreased in of miR-103a-3p. To certify the prediction that miR- pre-miR-103a-3p group than in pre-NC group (Fig. 2f). 103a-3p could directly bind to linc00152, we detected Yu et al. Molecular Cancer (2017) 16:110 Page 5 of 18

the expression of miR-103a-3p in sh-linc00152 GSCs expression was negatively correlated with linc00152 by qRT-PCR. MiR-103a-3p expression was raised in the expression. The best fit slope was −4.243 (P <0.01).Dual- sh-linc00152 group whereas not in the sh-NC group. In luciferase gene reporter assays were employed to assess comparison, linc00152 expression declined in the pre- the binding sites of linc00152 and miR-103a-3p. The lucif- miR-103a-3p group comparing with the pre-NC group erase activity was dramatic decline in the linc00152- (Fig. 3a). In addition, the correlation between expression wt + miR-103a-3p group than in the Control group of linc00152 and miR-103a-3p in glioma tissues was (Fig. 3d), while there was no distinction between the detected by qRT-PCR. As shown in Fig. 3b, miR-103a-3p linc00152-mut + miR-103a-3p group and the Control

Fig. 3 Reciprocal restrain between miR-103a-3p and linc00152, down-regulated of linc00152 hindered the malignant behavior of GSCs by target miR-103a-3p. a qRT-PCR analysis revealed the negative correlation between miR-103a-3p and linc00152 expression in glioma cells. **P < 0.01 vs. pre-NC group; ##P < 0.01 vs. anti-NC group; **P < 0.01 vs. sh-NC group. b Linear regression analysis was done to each individual linc00152 and miR- 103a-3p expression, the slope was −4.243, **P < 0.01. c Schematic representation of the putative binding site for linc00152 and miR-103a-3p, and the contrivable mutant sequence indicated for the reporter assay. d The relative luciferase activities were restrained in the HEK-293 T cells co-transfected with vector linc00152-wt and miR-103a-3p. *P < 0.05. vs. linc00152-wt + miR-103a-3p-NC group; #P < 0.05. vs. linc00152-mut + miR-103a-3p-NC group. e The CCK-8 assay was conducted to evaluate the effects of linc00152 and miR-103a-3p on the proliferation of GSCs. f-g The capacities of migration and invasion were detected through transwell assays on GSCs. h Flow cytometry analysis of GSCs in groups according to linc00152 and miR-103a-3p expression. *P < 0.05 vs. sh-NC + pre-NC group; #P < 0.05 vs. sh-linc00152 + pre-NC groups;&P < 0.05 vs. sh-NC + pre-miR-103a-3p group; ▲P < 0.05 vs. sh-NC + anti-NC group; ★P < 0.05 vs. sh-linc00152 + anti-NC group;▼P < 0.05 vs. sh-NC + anti-miR-103a -3p group. Scale bars, 20 μm. For a, c, d, e and g, data are presented as the mean ± SD (n = 5, each group) Yu et al. Molecular Cancer (2017) 16:110 Page 6 of 18

group in luciferase activity, indicating the presence of a downstream genes of miR-103a-3p. Then, the expression binding site between linc00152 and miR-103a-3p. In level of FEZF1 in GSCs was explored, which was trans- summary, linc00152 is a direct target of miR-103a-3p fected with sh-linc00152, pre-miR-103a-3p or anti-miR- and there might be a reciprocal repression feedback 103a-3p. As shown in Fig. 4a and c, expression of FEZF1 loop between linc00152 and miR-103a-3p. was declined in the sh-linc00152 group comparing with the sh-NC group. On the contrary, cells in anti-miR-103a- MiR-103a-3p involved in the tumor-suppressive effects of 3p group manifested higher levels of FEZF1 than those in linc00152 knockdown in GSCs anti-NC group (Fig. 4b and d). Ulteriorly, to explore the tumor-suppressive effects of In addition, FEZF1 expression was lower in the sh- linc00152 knockdown were mediated by miR-103a-3p, linc00152 + pre-NC group, sh-NC + pre-miR-103a-3p GSCs were divided into nine groups: the Control group, group and sh-linc00152 + pre-miR-103a-3p group com- the sh-NC + pre-NC group (cells stably expressing sh-NC, paring with the NC group, repectively (Fig. 4e). The co-transfected with pre-NC), sh-linc00152 + pre-NC group data above indicated that FEZF1 was involved in the (cells stably expressing sh-linc00152, co-transfected with linc00152/miR-103a-3p dependent malignant progres- pre-NC), sh-NC + pre-miR-103a-3p group (cells stably ex- sion of GSCs. pressing sh-NC, co-transfected with pre-miR-103a-3p), sh- linc00152 + pre-miR-103a-3p group (cells stably expressing FEZF1 was up-regulated in glioma tissues and GSCs, and sh-linc00152, co-transfected with pre-miR-103a-3p), facilitated GSC proliferation, migration and invasion and sh-NC + anti-NC group (cells stably expressing sh-NC, receded GSC apoptosis co-transfected with anti-NC), sh-linc00152 + anti-NC After verified FEZF1 was a target of miR-103a-3p, we in- group (cells stably expressing sh-linc00152, co-transfected vestigated the protein levels of FEZF1 in NBTs, glioma with anti-NC), sh-NC + anti-miR-103a-3p group (cells tissues and GSCs by Western blot. As shown in Fig. 5a-b, stably expressing sh-NC, co-transfected with anti-miR- FEZF1 protein levels elevated with the pathological grades 103a-3p) and sh-linc00152 + anti-miR-103a-3p group of glioma tissues and were apparently up-regulated in (cells stably expressing sh-linc00152, co-transfected with GSCs compared with non-GSCs. In consequence, we con- anti-miR-103a-3p). CCK8 assay indicated that GSCs jectured that FEZF1 facilitated the malignant progression proliferation was significantly decreased in the sh- of GSCs and detected the effect of FEZF1 on the prolifera- linc00152 + pre-NC group, sh-NC + pre-miR-103a-3p tion, migration, invasion and apoptosis of GSCs. As shown group and sh-linc00152 + pre-miR-103a-3p group than in Fig. 5c, the CCK8 assay implied that proliferation of the sh-NC + pre-NC group, respectively (Fig. 3e). More- GSCs was raised in the FEZF1(+) groups compared with over, the numbers of GSC migration and invasion were the FEZF1(+)-NC group. Transwell assays indicated that dramatically declined in the sh-linc00152 + pre-NC group, migration and invasion capacity of GSCs in the FEZF1(+) sh-NC + pre-miR-103a-3p group and sh-linc00152 + pre- group were stronger than that in the FEZF1(+)-NC group miR-103a-3p group than in the sh-NC + pre-NC group, (Fig. 5d). Further, up-regulated FEZF1 dramatically sup- respectively (Fig. 3f-g). In addition, GSCs of sh- pressed apoptosis in GSCs compared with the NC group linc00152 + pre-NC group, sh-NC + pre-miR-103a-3p (Fig. 5e). group and sh-linc00152 + pre-miR-103a-3p group manifested higher apoptosis rates compared with the miR-103a-3p hindered FEZF1-induced malignant behavior sh-NC + pre-NC group, respectively (Fig. 3h). The data on GSCs by targeting its 3′-UTR above implied that miR-103a-3p mediated the tumor To further affirm whether FEZF1 is a direct target of suppressive effects of linc00152 knockdown in GSCs. miR-103a-3p, luciferase assay was carried out. Luciferase activity was dramatically declined in cells co-transfected FEZF1 was involved in the linc00152/miR-103a-3p with pre-miR-103a-3p and FEZF1-wt (Fig. 4g), illustrated dependent malignant progression of GSCs that FEZF1 was a direct target of miR-103a-3p. Never- The above results uncovered that linc00152 and miR- theless, there was no significant difference between 103a-3p have remarkable impacts on the biological be- FEZF1-mut + pre-miR-103a-3p group and FEZF1- haviors of GSCs, but the latent molecular mechanisms mut + miR-103a-3p-NC group, suggesting the specific remain blurry. Bioinformatics database (Targetscan) binding site of miR-103a-3p in the FEZF1- 3′-UTR. speculated that several genes would be downstream Furthermore, to explore whether miR-103a-3p sup- targets of miR-103a-3p, including FEZF1. Next, we ap- pressed GSC malignant evolution were mediated by praised the impact of linc00152 or miR-103a-3p on FEZF1, down-regulated FEZF1 by pre-miR-103a-3p was mRNA and protein levels of FEZF1 by qRT-PCR and rescued using FEZF1 prior to the assessment of the cell western blot, and noticed expression level of FEZF1 proliferation, migration, invasion and apoptosis. CCK8 was prominently affected among the predicted assay indicated that miR-103a-3p over-expression Yu et al. Molecular Cancer (2017) 16:110 Page 7 of 18

Fig. 4 FEZF1 was a target gene of miR-103a-3p, the expression of FEZF1 was regulated by both linc00152 and miR-103a-3p. a qRT-PCR analysis of the effect of linc00152 on FEZF1 mRNA expression in glioma cells. **P < 0.01 vs. sh-NC group. b qRT-PCR analysis of the effect of miR-103a-3p on FEZF1 mRNA expression in glioma cells. **P < 0.01 vs. pre-NC group; ##P < 0.01 vs. anti-NC group. c Western blot analysis on FEZF1 in linc00152-knockdown GSCs, with GAPDH as an endogenous control. *P < 0.05 vs. sh-NC group. d Western blot analysis for expression between miR-103a-3p and FEZF1 in GSCs, with GAPDH as an endogenous control. *P < 0.05 vs. pre-NC group; #P < 0.05 vs. anti-NC group. e Western blot analysis for FEZF1 in GSCs co-transfected with sh-linc00152 and pre-miR-103a-3p or anti-miR-103a-3p, with GAPDH as an endogenous control. *P < 0.05 vs. sh-NC+ pre-NC group. #P < 0.05 vs. sh-linc00152 + pre-NC group; &P < 0.05 vs. sh-NC + pre-miR-103a-3p group; ▲P <0.05vs. sh-NC + anti-NC group; ★P < 0.05 vs. sh-linc00152 + anti-NC group; ▼P < 0.05 vs. sh-NC + anti-miR-103a -3p group. For a-e, data are presented as the mean ± SD (n = 5, each group). f Schematic representation of the putative binding site for miR-103a-3p and FEZF1, and the contrivable mutant sequence for the reporter assay. g The relative luciferase activities were hindered in the HEK-293 T cells co-transfected with vector pre-miR-103a-3p and FEZF1-wt. *P < 0.05. vs. FEZF1-wt + pre-NC group restrained the proliferation of GSCs, whereas FEZF1 miR-103a-3p over-expression impaired the migrating and over-expression accelerated the proliferation of GSCs. invading capacity of GSCs, while FEZF1 over-expression FEZF1 over-expression rescued the inhibitory effect of showed the contrary result. FEZF1 over-expression rescued miR-103a-3p over-expression on the proliferation of the impaired migration and invasion of GSCs induced by GSCs (Fig. 6b). Besides, transwell assay revealed that miR-103a-3p over-expression (Fig. 6c). Further, flow Yu et al. Molecular Cancer (2017) 16:110 Page 8 of 18

Fig. 5 (See legend on next page.) Yu et al. Molecular Cancer (2017) 16:110 Page 9 of 18

(See figure on previous page.) Fig. 5 FEZF1 was up-regulated in glioma tissues, and played an oncogenic role in GSCs. a FEZF1 protein expression in non-tumorous brain tissues, low-grade glioma tissues (WHO I-II), and high-grade glioma tissues (WHO III-IV), with GAPDH as an endogenous control. Data are presented as the mean ± SD (n = 10, each group). **P or ##P<0.01 vs. non-tumorous brain tissue group. b Western blot analysis of FEZF1 expression in non-GSCs and GSCs, with GAPDH as an endogenous control. **P < 0.01 vs. non-GSC group. c CCK8 assay was performed to evaluate the effect of FEZF1 on the proliferation of GSCs. d Quantification of GSC migration and invasion upon FEZF1 over-expression or down-regulation. Representative images and accompanying statistical plots are presented. e Flow cytometry analysis of the effects of FEZF1 on GSCs. Data are presented as the mean ± SD (n =5,eachgroup).*P < 0.05 vs. FEZF1 (+)-NC group; #P < 0.05 vs. FEZF1(−)-NC group. Scale bars, 20 μm. The photographs were taken at 200 × magnification. f Effect of FEZF1 on the CDC25A protein expression, with GAPDH as an endogenous control. Data are presented as the mean ± SD (n =5,eachgroup).*P < 0.05 vs. FEZF1(+)-NC group, #P < 0.05 vs. FEZF1(+)-NC group. g Effect of FEZF1 on the CDC25A mRNA expression. Data are presented as the mean ± SD (n =5,eachgroup).*P < 0.05 vs. FEZF1(+)-NC group, #P < 0.05 vs. FEZF1(+)-NC group. h Schematic depiction of the CDC25A reporter constructs used and the luciferase activity. The Y-bar shows the position of the deletions on the DNA fragments. X-bar shows the constructed plasmid activity after normalization with the co-transfected reference vector (pRL-TK), and relative to the activity of pEX3 empty vector,which the activity was set to 1. Data representmeans ± SD (n =5,each).i Schematic representation of the CDC25A promoter region 3000 bp upstream of the transcription start site (TSS) which designated as +1. ChIP PCR products for putative binding sites and an upstream region not expected to associate with FEZF1 are depicted with bold lines. Immunoprecipitated DNA was amplified by PCR. Normal rabbit IgG was used as a negative control cytometry analysis implied that over-expression of miR- whereas FEZF1 over-expression increased the expression 103a-3p promoted apoptosis of GSCs, whereas over- (Fig. 6a and e). expression of FEZF1 suggested the opposite result. To verify whether FEZF1 is necessary for the promoter FEZF1 over-expression rescued the enhancement of activity of CDC25A in GSCs, a suite of constructs were miR-103a-3p over-expression on the apoptisis of GSCs generated and luciferase assays were applied. The pro- (Fig. 6d). These results revealed that FEZF1 mediated moter sequences of CDC25A were set according to the the tumor-suppressive effects of miR-103a-3p over- database of DBTSS HOME (http://dbtss.hgc.jp/). By expression on GSCs. analyzing these DNA sequences in the 3000 bp region upstream of the transcription start site (TSS) and its 100 bp downstream sequence, we confirmed two puta- FEZF1 up-regulated the promoter activities and bound to tive FEZF1 binding sites in CDC25A (Fig. 5i). So a suite the promoters of CDC25A in GSCs of DNA fragments were constructed and ligated into The results above revealed that FEZF1 exerted onco- the pGL3 basic vector. Wild-type, deletion construct genic role by facilitating malignant biological behaviors and putative FEZF1 binding sites were indicated. After of GSCs. However, the underlying molecular mechanism co-transfection with pEX3-FEZF1, deletion of the −707 remains indistinct. Both FEZF1 and FEZF2 belong to the site region significantly decreased the promoter activities forebrain embryonic zinc finger (Fez) family [25], and as of CDC25A. The results indicated that the responsive previous report, CnnCAnCn core, which was found in FEZF1-binding sites resided within −707siteregionof promoter region of CDC25A by DBTSS database (http:// CDC25A (Fig. 5h). dbtss.hgc.jp/), was identified as putative consensus FEZF2 To explore whether FEZF1 directly associated with the binding site [21]. In addition, FEZF1 and FEZF2 were re- promoters of CDC25A in GSCs, ChIP assay was applied. ported to have the same target gene [22]. Therefore, we As a negative control, PCR was performed to amplify hypothesized FEZF1 could bind with CDC25A. the 2000 bp upstream region of the putative FEZF1 Recent research identified CDC25A as an oncogene binding site which was not supposed to associate with and over-expression of CDC25A promoted a variety of FEZF1. Our results showed that there was direct associ- tumors. CDC25A was observed high expression in lung ation of FEZF1 with putative binding site 2 of CDC25A. cancer [26, 27] and high expression of CDC25A pre- There was no association of FEZF1 with putative binding dicted poor prognosis in human lung adenocarcinoma site 1 of CDC25A and the control regions (Fig. 5i). These [28]. Moreover, up-regulation of CDC25A was reported results revealed that FEZF1 could up-regulate the pro- to enhance cell proliferation, which possibly mediated moter activities and bind to the promoter of CDC25A. chemoresistance in human AML [29]. To confirm whether CDC25A was regulated by FEZF1 in GSCs, qRT-PCR and CDC25A accelerated malignant biological behaviors of western blot analysis was carried out. GSCs by promoting the PI3K/AKT pathway As shown in Fig. 5f-g, expression of CDC25A protein Up-regulated of CDC25A was discovered in human gli- and mRNA were enhanced in FEZF1(+) group comparing oma specimens and depletion of CDC25A suppressed with FEZF1 (+)-NC group. In GSCs co-transfected with cell proliferation and induced apoptosis in glioma cell miR-103a-3p and FEZF1, miR-103a-3p over-expression lines [30]. The results above implied a tumor promotion declined mRNA and protein expression of CDC25A, of CDC25A in glioma. However, the effect of CDC25A Yu et al. Molecular Cancer (2017) 16:110 Page 10 of 18

Fig. 6 miR-103a-3p inhibited malignant progression of GSCs by binding to the FEZF1 3′-UTR. a Effect of miR-103a-3p and FEZF1 co-transfection on the CDC25A mRNA expression. b The CCK-8 assay was used to assess the effects of miR-103a-3p and FEZF1 on GSC proliferation. c Transwell assay was employed to detect the effects of miR-103a-3p and FEZF1 on GSC migration and invasion. Representative images and accompanying statistical plots are presented. d Flow cytometry analysis of GSCs in groups according to miR-103a-3p and FEZF1(+) expression. e Effect of miR-103a-3p and FEZF1 co-transfection on the CDC25A, p-AKT and p-PI3K protein expression, with GAPDH as an endogenous control. *P < 0.05 vs. pre-NC + FEZF1 -NC group, #P < 0.05 vs. pre- miR-103a-3p + FEZF1-NC group. Scale bars, 20 μm. The photographs were taken at 200 × magnification. Data are presented as the mean ± SD (n =5,eachgroup)

in GSCs was fuzzy. Hence, we detected the protein level The results above illustrated CDC25A promoted the of CDC25A in GSCs. As shown in Fig. 7a, CDC25A pro- proliferation, migration and invasion in GSCs, and tein levels elevated dramatically in GSCs compared with refrained the apoptosis of GSCs. To investigate the the non-GSCs. Furthermore, we evaluated the effect of underlying molecular mechanism, we assessed the ac- CDC25A on the proliferation, migration, invasion and tivity of PI3K/AKT pathway by Western blot assays. As apoptosis of GSCs. As shown in Fig. 7b-c, over-expression shown in Fig. 7e, over-expression of CDC25A prompted of CDC25A promoted the proliferation, migration and in- thep-PI3Kandp-AKTexpressioninGSCs.Besides,in vasion capacity of GSCs. Further, up-regulated CDC25A GSCs co-transfected with miR-103a-3p and FEZF1, distinctly diminished apoptosis in GSCs compared with miR-103a-3p over-expression declined expression of the NC group (Fig. 7d). CDC25A, P-AKT and P-PI3K, whereas FEZF1 over- Yu et al. Molecular Cancer (2017) 16:110 Page 11 of 18

Fig. 7 CDC25A was up-regulated in GSCs, and played an oncogenic role in GSCs. a Western blot analysis of CDC25A expression in non-GSCs and GSCs, with GAPDH as an endogenous control. *P < 0.05 vs. non-GSCs-U87 group, #P < 0.05 vs. non-GSCs-U251 group. b CCK8 assay was performed to evaluate the effect of CDC25A on the proliferation of GSCs. c Quantification of GSC migration and invasion upon CDC25A overexpression or downregulation. Representative images and accompanying statistical plots are presented. d Flow cytometry analysis of the effects of CDC25A on GSCs. e Western blot analysis of the PI3K/AKT pathway regulated by CDC25A in GSCs. Data are presented as the mean ± SD (n =5,eachgroup).*P <0.05vs. CDC25A (+)-NC group; #P < 0.05 vs. CDC25A (−)-NC group. Scale bars, 20 μm. The photographs were taken at 200 × magnification expression increased the expression (Fig. 6e). These re- (Fig. 8a-c). Moreover, mice in the linc00152(−) + miR-103a- sults demonstrated that CDC25A accelerated malignant 3p(+) group had the longest survival time (Fig. 8d). biological behaviors of GSCs by promoting the PI3K/ AKT pathway. Dicussion In this study, we elaborated that expression of the lncRNA linc00152 was elevated in glioma tissues and GSCs. Linc00152 knockdown combined with over-expressed Linc00152 could directly bind to miR-103a-3p and nega- miR-103a-3p suppressed tumor growth and manifested tively regulated the expression of miR-103a-3p. This im- high survival in nude mice plied a significant mechanism of linc00152 pathway in the To further certify the above findings, an in vivo tumor model glioma intervention. Knockdown of linc00152 restrained was employed. The results indicated that the linc00152 proliferation, migration and invasion, and facilitated apop- knockdown, miR-103a-3p over-expression, and linc00152 tosis in GSCs. On the contrary, miR-103a-3p manifested knockdown combined with over-expressed miR-103a-3p carcinostatic role in GSCs and suppressed the prolifera- groups had smaller tumors compared with the linc00152(−)- tion, migration and invasion, prompted apoptosis of GSCs. NC + miR-103a-3p(+)-NC group (Fig. 8a-c). In addition, In addition, FEZF1 was identified as a direct target of tumors in the linc00152(−) + miR-103a-3p(+) group mani- miR-103a-3p and mediated the tumor-suppressive effects fested the smallest volume compared to the other groups of miR-103a-3p. Moreover, inhibition of miR-103a-3p Yu et al. Molecular Cancer (2017) 16:110 Page 12 of 18

Fig. 8 Tumor xenografts study of tumor growth and survival rates in nude mice. a Thenudemicecarryingtumorsofrespectivegroupswereshown.b Sample tumors from respective group were shown. c Tumor growth curves of five groups in nude mice (n = 8). Tumor growth was monitored for up to 40 days. *P < 0.05 vs. linc00152(−)-NC + miR-103a-3p(+)-NC group, #P < 0.05 vs. linc00152(−)group,▲P < 0.05 vs. miR-103a-3p(+) group. d Survival curves of nude mice injected into the right striatum. Mice were monitored for up to 50 days (n =8).e The schematic cartoon of the mechanism of linc00152 as an oncogene by regulating miR-103a-3p/FEZF1/CDC25A pathway in glioma stem cells Yu et al. Molecular Cancer (2017) 16:110 Page 13 of 18

promoted the expression of CDC25A by up-regulating Mechanistically, miR-103a-3p was identified as a direct FEZF1, which positively control the promoter activities target of linc00152 by dual-luciferase assay, suggesting via binding to the promoter of CDC25A. Further, PI3K/ the potential reciprocal repression feedback loop AKT pathway was blocked when CDC25A was sup- between linc00152 and miR-103a-3p. In addition, our pressed. Experiment in vivo showed that knockdown of results showed that ablation of linc00152 inhibited cell linc00152 combined with over-expression of miR-103a-3p proliferation, migration and invasion as well as inducing generated the smallest tumor and led to the longest apoptosis in GSCs by elevating miR-103a-3p, implying survival time in nude mice. that linc00152 knockdown impaired the malignant Glioblastoma (GBM) is one of the most common and behavior of GSCs by up-regulating miR-103a-3p. More- lethal primary malignant brain tumor in adults [31, 32]. over, the in vivo studies showed that nude mice in the GBM harbor a subpopulation of self-renewing, therapy- sh-linc00152 + pre-miR-103a-3p group manifested the resistant cells, GSCs, which can flourish in the stresses smallest tumors and highest survival compared to the present in an unfavorable tumor microenvironment [33]. other groups, suggesting that these were likely to achieve In consequence, therapies targeting GSCs have become synergistic anti-tumor effects. Collectively, linc00152 a hotspot in treatment of glioma [34]. A growing num- acted as an oncogenic gene by restraining miR-103a-3p ber of evidence has implied that lncRNAs play an im- in GSCs, but the further mechanisms under miR-103a- portant role in the occurrence and progression of 3p were blurred. tumors recently. Exploring the potential molecular We discovered that the protein levels of FEZF1 had mechanisms of lncRNAs in GSCs may provide promis- adverse changes to the alteration of miR-103a-3p. ing therapeutic strategies in glioma. Luciferase reporter assays certified that miR-103a-3p Our present data revealed that linc00152 was posi- regulated gene expression post-transcriptionally by bind- tively correlated with the histopathological grade in ing to the 3’UTR of FEZF1. In addition, our research human glioma tissues and elevated in GSCs. In addition, showed that FEZF1 expression was raised in glioma knockdown of linc00152 inhibited cell proliferation, tissues as well as in GSCs. Over-expression of FEZF1 migration and invasion, as well as promoted apoptosis in facilitated cell proliferation, migration and invasion, and GSCs, which indicated that linc00152 may serve as an inhibited cell apoptosis, implying that FEZF1 functions oncogene in GSCs. Previous researches also showed the as an oncogene in GSCs. Consistent with our results, same oncogenic function of linc00152. Cai Q reported FEZF1 was reported over-expressed in gastric cancer tis- linc00152 was up-regulated in gallbladder cancer and sues and played a significant role in the progression and correlated negatively with the overall survival time in metastasis of gastric cancer by activation of the K-ras gallbladder cancer patients [35]. In addition, linc00152 oncogene [44]. What’s more, miR-103a-3p and FEZF1 was identified as increased expression in gastric cancer co-overexpression significantly rescued the inhibition [36] and regarded as a novel biomarker for predicting effect induced by up regulating miR-103a-3p alone, re- gastric cancer [37]. Our results illustrated that linc00152 vealing that miR-103a-3p inhibits the malignant behavior acted as a oncogene in GSCs for the first time, but the of GSCs by declined FEZF1 expression. underlying mechanisms need to be investigated. CDC25A is an identified cell division cycle protein, LncRNAs finely regulate gene expression through a which is required to enter S time [45], and the over- variety of ways, including transcriptional regulation, spli- expression of this phosphatase accelerates the entrance cing and translation, post-transcriptional regulation and to S time [46]. Our research showed that expression of epitogenetic modulation, etc. [38]. To detect the poten- CDC25A was remarkably increased in GSCs. Moreover, tial oncogenic mechanism of linc00152 in glioma, over-expression of CDC25A facilitated cell proliferation, bioinformatics analysis software was applied to identify migration and invasion, while inhibited apoptosis in miR-103a-3p as a novel target of linc00152. MiR-103a- GSCs. Consistent with our study, Wang XQ [47] re- 3p situated in 5q34, and was associate with tumor, Par- ported expression of CDC25A elevated in hepatocellular kinson disease and pregnancy-related complications carcinoma, which was significantly correlated with HCC [39–41]. Our study suggested that miR-103a-3p was tumor-node-metastasis staging and venous invasion. In declined in glioma tissues and GSCs compared with addition, CDC25A was reported over-expressed and NBTs and glioma cells, respectively. Furthermore, over- suppressed by miR-21 through a defined sequence in its expression of miR-103a-3p inhibited proliferation, mi- 3′-UTR in colon cancer [48]. gration and invasion, as well as promoted apoptosis in Moreover, we discovered that CDC25A was involved GSCs. This was consistent with the previous studies in a FEZF1-induced oncogenic effect in GSCs. Over- showing that miR-103a-3p was low expressed in gastric expression of FEZF1 could elevate CDC25A expression, cancer [42] as well as bladder carcinoma [43], and while FEZF1 knockdown showed contrary effects. played a crucial role in cancer suppression. Luciferase assays and ChIP assays showed that FEZ1 Yu et al. Molecular Cancer (2017) 16:110 Page 14 of 18

increased promoter activity and bound to the promoter brain tissues(NBTs) were used as the negative control region of CDC25A, indicated that CDC25A was involved group (n = 30). They were acquired from patients’ fresh in FEZF1 regulation in GSCs. autopsy material (donation from individuals who died in Taken together, we revealed that the linc00152-miR- traffic accident and confirmed to be free of any prior 103a-3p-FEZF1-CDC25A axis manifested an important pathologically detectable conditions). role in human glioma. Briefly, the miR-103a-3p over- expression induced by the knockdown of linc00152 Cell culture could down-regulate the expression FEZF1, decreased Human glioma cell lines (U87, U251) and human em- FEZF1 hindered the promoter activities of CDC25A and bryonic kidney (HEK) 293 T cells were purchased from inhibited malignant biological behavior in GSCs. the Chinese Academy of Medical Sciences (Beijing, We further explored the molecular mechanisms of China). U87 glioma cells and HEK-293 cells were cul- CDC25A oncogenic functions. Recent studies showed tured in Dulbecco’s modified Eagle medium (DMEM)/ that CDC25A promoted PKM2-dependent β-catenin high glucose with 10% fetal bovine serum (FBS, Gibco, transactivation, which promotes the Warburg effect and Carlsbad, CA, USA), U251 cells were cultured in tumorigenesis [49]. In addition, CDC25A was reported DMEM/F12 medium with 10% FBS. All cells were main- up-regulated in chondrosarcoma cells and played an im- tained in a humidified incubator at 37 °C with 5% CO2. portant role in cell cycle progression and tumorigenesis of chondrosarcoma as an activator of CDK complexes Isolation and identification of GSCs [50]. Moreover, the activation of PI3K/AKT pathway GSCs were seperated as described previously [54, 55]. In participated in the glioma progression and various a nutshell, GSCs were cultured in DMEM/F-12 medium biological effects including proliferation, migration, inva- (Life Technologies Corporation, Grand Island, NY, USA) sion and apoptosis [51, 52]. The PI3K/AKT pathway in- appended to basic fibroblast growth factor (bFGF, 20 ng/ hibitors were discovered to exhibit efficacy against mL, Life Technologies Corporation, Carlsbad, CA, USA), glioblastoma in clinically relevant mouse models [53]. In epidermal growth factor (EGF, 20 ng/mL, Life Technolo- this work, we revealed that CDC25A over-expression gies Corporation, Gaithersburg, MD, USA) and 2% B27 activated the PI3K/AKT pathways in GSCs and knock- (Life Technologies Corporation, Grand Island, NY, down of CDC25A manifested the opposite effects. USA). The serum-free medium was replaced every 2 days Therefore, the reduced CDC25A induced by FEZF1 until the spheres were visible under microscopy. The knocked down could attenuate the activity of PI3K/AKT spheres were centrifuged for 3 min at 1000 r.p.m. and pathways to inhibit the malignant behaviors of GCSs. harvested, then trypsinized into single-cell suspensions The mechanism underlying tumor suppressive function and plated into a 96-well plate for the limiting dilution of human glioma cells by linc00152 knockdown is assay and colon sphere formation by limiting dilution as schematically presented in Fig. 8e. described previously [56, 57]. For differentiation assay, sphere cells were plated onto glass coverslips coated with poly-L-ornithine (BD Biosciences, Franklin Lakes, Conclusions NJ, USA) in medium containing 10% FBS for the differ- In summary, our research verified linc00152 facilitated entiation assay. For immunostaining of undifferentiated cell proliferation, migration and invasion, while sup- spheres, cells were incubated with antibodies against pressed apoptosis in GSCs. MiR-103a-3p functioned as Nestin and CD133 (1:100, Santa Cruz Biotechnology, tumor suppressor by decreasing FEZF1 in GSCs. FEZF1 Santa Cruz, CA, USA). For immunostaining of differen- was downstream targets of miR-103a-3p, and CDC25A tiated spheres, cells were stained with antibodies against was a target of FEZF1. The significance of interaction GFAP (1:100, Abcam, Cambridge, MA, USA) and beta- among linc00152, miR-103a-3p, FEZF1 and CDC25A tubulin III (1:100, Santa Cruz Biotechnology). The was highlighted for the first time. In addition, linc00152- primary antibody complexes were visualized with anti- miR-103a-3p-FEZF1-CDC25A axis might represent a rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 555 promising therapeutic strategy for the treatment of secondary antibodies (Beyotime Institute of Biotechnol- human glioma. ogy, Jiangsu, China). Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). Methods Patients and glioma specimens Cell transfection and generation of stable transfected Grades of glioma were identified by neuropathologists cells according to WHO classification. Glioma specimens Short-hairpin RNA directed against human linc00152 plas- were divided into two groups: grade I–II glioma group mid (sh-linc00152), short-hairpin RNA directed against (n = 10) and grade III–IV glioma group (n = 10). Normal human FEZF1 plasmid (FEZF1(−)), short-hairpin RNA Yu et al. Molecular Cancer (2017) 16:110 Page 15 of 18

directed against human CDC25A plasmid (CDC25A(−)) group (FEZF1 stable transfected cells co-transfected with and their respective non-targeting sequence (negative con- miR-103a-3p). trol, NC) were synthesized (GenePharma, Shanghai, China). FEZF1 full length (FEZF1(+)) plasmid, CDC25A RNA and miRNA isolation and quantitative RT-PCR full length (CDC25A(+)) plasmid, and their respective Total RNA was isolated from the glioma tissues, NBTs non-targeting sequence (negative control, NC) were syn- and cells using Trizol reagent (Life Technologies Cor- thesized (GenScript, Piscataway, NJ, USA). Glioma cells poration, Carlsbad, CA, USA). The RNA concentration were transfected in 24-well plates using Opti-MEM and was determined by 260/280 nm absorbance using a Lipofectamine 3000 reagents (Invitrogen, CA, USA) ac- Nanodrop Spectrophotometer (ND-100, Thermo, USA). cording to the manufacturer’s instructions. Stable cell lines One-Step SYBR PrimeScript RT-PCR Kit (Perfect Real were selected applying Geneticin (G418; Invitrogen, CA, Time) (Takara Bio, Inc., Japan) was used for qRT-PCR USA). Four weeks later, G418-resistant cell clones were detection of linc00152 and mRNA of FEZF1 and established. The over-expression and the silence efficien- CDC25A. cDNA from miRNAs was generated by using cies were evaluated by quantitative real-time PCR (qRT- a TaqMan miRNA Reverse Transcription kit (Applied PCR). And then GSCs were isolated as previously Biosystems, Foster City, CA, USA). qRT-PCR was car- described. To investigate the effect of linc00152 on GSCs, ried out by using TaqMan Universal Master Mix II with cells were divided into three groups: control group, sh-NC Taq-Man microRNA assays of miR-103a-3p and U6 group (tansfected with empty plasmid) and sh-linc00152 (Applied Biosystems, Foster City, CA, USA). U6 and group. To investigate the effect of FEZF1 on GSCs, cells GAPDH were employed as endogenous controls for were divided into five groups: control group, FEZF1(+)-NC miRNA and gene expression detection. Expressions were group, FEZF1(+) group, FEZF1(−)-NC group and normalized to endogenous controls and relative quantifi- −ΔΔ FEZF1(−) group. To investigate the effect of CDC25A on cation (2 Ct) method was used for fold changes’ GSCs, cells were divided into five groups: control group, calculating. CDC25A(+)-NC group, CDC25A(+) group, CDC25A(−)- NC group and CDC25A(−) group. Cell proliferation assay Cell proliferation was measured by conducting Cell Cell transfection of miRNAs Counting Kit-8 (CCK-8, Beyotime Institute of Biotech- MiR-103a-3p agomir (pre-miR-103a-3p), miR-103a-3p nology, Jiangsu, China) assay. After transfection efficacy antagomir (anti-miR-103a-3p) and their respective was measured, GSCs were dissociated with Accutase negative control molecules (pre-NC and anti-NC) were (Life Technologies Corporation, Carlsbad, CA, USA), synthesized (GenePharma, Shanghai, China). To investi- resuspended, and seeded in 96-well plates at 2000 cells gate the effect of miR-103a-3p on GSCs, cells were per well. Cells per well were added 10 μl CCK-8 solution divided into five groups: control group, pre-NC group, and cultured for 2 h at 37 °C. The absorbance was pre-miR-103a-3p group, anti-NC group and anti-miR- recorded at 450 nm on a SpectraMax M5 microplate 103a-3p group. To investigate whether linc00152 reader (Molecular Devices, USA). mediated regulation of miR-103a-3p expression could regulate the behavior of GSCs, cells were divided into Limiting dilution assay five groups: control group, sh-NC + pre-NC group (sh- Different amounts (2, 5, 10, 20, 50, 100, 200, 500) of NC stable transfected cells co-transfected with pre-NC), GSCs were seeded into a 96-well plate. Serum-free con- sh-linc00152 + pre-miR-103a-3p group (sh-linc00152 ditional culture medium (25 μl) was added to each well stable transfected cells co-transfected with pre-miR- every 2 days. The percentage of wells without neuro- 103a-3p), sh-NC + anti-NC group (sh-NC stable spheres was enumerated. Then, a linear regression transfected cells co-transfected with anti-NC) and sh- model was used to compare the slope of each group. linc00152 + anti-miR-103a-3p group (sh-linc00152 stable transfected cells co-transfected with anti-miR- Cell migration and invasion assay 103a-3p). To investigate the mechanism of FEZF1 Cells were resuspended in 200 μL serum-free medium at regulated by miR-103a-3p to effect the behavior of a density of 2 × 105 cells/ml and placed in the upper GSCs, cells were divided into five groups: control group, chamber (or precoated with 80 μL of Matrigel solution pre-NC + FEZF1-NC group (FEZF1-NC stable trans- (BD, Franklin Lakes, NJ, USA) for cell invasion assay) of fected cells co-transfected with pre-NC), pre-miR-103a- the 24-well insert with 8 mm pore size. 600 μL of 10% 3p + FEZF1-NC group (FEZF1-NC stable transfected FBS medium was added to the lower chamber. After in- cells co-transfected with pre-miR-103a-3p), pre- cubation for 24 h, Cells had migrated or invaded to the NC + FEZF1 group (FEZF1 stable transfected cells co- lower side of the membrane were fixed and stained with transfected with pre-NC) and pre-miR-103a-3p + FEZF1 20% Giemsa. Stained cells were counted under a Yu et al. Molecular Cancer (2017) 16:110 Page 16 of 18

microscope in five randomly chosen fields and the aver- Chromatin immunoprecipitation (Chip) assay age number was calculated. The Chip assay was carried out using the Simple Chip Enzymatic Chromatin IP kit (Cell Signaling Technology, ’ Apoptosis detection Danvers, MA) following the manufacturer s instructions. Apoptosis was detected by Annexin V-APC/7-AAD (BD Cells were cross-linked with 1% formaldehyde for Biosciences). Cells were harvested added allophycocyanin 10 min and then dealed with glycine for 5 min at room (APC) and 7-aminoactinomycin D (AAD) following the temperature. Cells were lysed with cold buffer contain- instructions of the manufacturer. Cell samples were ing PMSF and resuspended with cold PBS. Chromatin analyzed by flow cytometry (FACScan, BD Biosciences), was digested by micrococcal nuclease and incubated for and apoptotic fractions were recorded. 20 min at 37 °C with frequent mixture. Lysates (2%) were employed as an input reference. Other immunoprecipitation samples were incubated overnight with Western blot analysis normal rabbit IgG or anti-FEZF1 antibodies (Santa Cruz Total proteins were isolated from cells with RIPA buffer Biotechnology, CA, USA) at 4 °C with vibration. Protein on ice and were further analysed by SDS-PAGE and G agarose beads were used to collect the chromatinim- electrophoretically transferred to polyvinylidene difluor- mune complex, and beads were scoured with low salt ide membranes. After non-specific binding was pre- chip buffer and high salt chip buffer. DNA crosslinks were vented by 5% nonfat milk at room temperature for 2 h, reversed by 5 mol/l NaCl and proteinase K at 65 °C for membranes were embraced overnight at 4 °C by primary 2 h to purify. DNA was amplified by PCR applying the fol- antibodies as below: FEZF1 (1:200, Santa Cruz Biotech- lowing DNA fragments: putative binding site 1 using the nology),CDC25A(1:1000, Abcam, EUGENE, USA), PI3K, primers 5′-GGCTAAGAAGTGGTGGGAAAG-3′ and p-PI3K, AKT, p-AKT (1:1000, Cell Signaling Technol- 5′-AGGGAGGGAATGCAATGAC-3′,generatinga ogy, Beverly, MA, USA) and GAPDH (1:1000, Santa 211 bp product; putative binding site 2 using the primers Cruz Biotechnology). Then, incubated 2 h at room 5′-GCTTTCTTCTTCCCCTCTCA-3′ and 5′-CCGACC- temperature with HRP-conjugated secondary antibodies. TACACCTCTTACCC-3′, generating a 195 bp product; Immunoblots were visualized by ECL chemilumines- control using the primers 5′-TCTACCTCCTTCAGGGC cence detection system and the relative integrated dens- TCA-3′ and 5′-TTTGGCCTTATCCTGTGGAC-3′,gen- ity values were calculated by FLuor Chem2.0 software. erating a 171 bp product.

Dual-luciferase reporter assays Tumor xenografts in nude mice Potential miR-103a-3p binding sites of linc00152 and In vivo study, the stable expressing cells were applied. FEZF1 3′-UTR were predicted by bioinformatics tool Lentivirus encoding miR-103a-3p and its non-targeting se- Starbase (http://starbase.sysu.edu.cn/) and Target Scan quence (negative control, NC) were generated using (http://www.targetscan.org/). In brief, the fragment of pLenti6.3/V5eDEST Gateway Vector Kit (Life Technologies linc00152 possessing the assumptive miR-103a-3p bind- Corporation, Carlsbad, CA, USA). The miR-103a-3p and ing sites was cloned into a pmirGLO Dual-Luciferase short-hairpin RNA targeting human linc00152 were ligated Vector (Promega, Madison, WI, USA) to construct the into the pLenti6.3/V5eDEST vector and LV3-CMV-GFP- reporter vector linc00152-wild-type (linc00152-Wt) Puro vector (GenePharma, Shanghai, China), respectively. (GenePharma, Shanghai, China). Analogously, the corre- And then pLenti6.3/V5eDEST-miR-103a-3p and LV3- sponding mutant of hypothetic miR-103a-3p binding CMV-GFPPuro-sh-linc00152 vectors were generated. The sites was manufactured to form the reporter vector ViraPower Packaging Mix was used to generate Lentivirus linc00152-mutated-types (linc00152-Mut) (GenePharma, in 293FT cells. After infection, the stable expressing cells of Shanghai, China). The 3′-UTR fragment of FEZF1 gene miR-103a-3p (miR-103a-3p(+)), sh-linc00152 (linc00152(−)) and its mutant of the theoretical miR-103a-3p binding were picked. The lentiviruses of miR-103a-3p(+) were site were cloned into a pmirGLO Dual-Luciferase Vector transduced in linc00152(−) stably transfected cells to gener- to form the reporter vector FEZF1-3′-UTR-wild-type ate linc00152(−) + miR-103a-3p(+) cells. (FEZF1-3′-UTR-Wt) and FEZF1-3′-UTR-mutated-type The nude mice were divided into five groups: control (FEZF1-3′-UTR-Mut) (GenePharma, Shanghai,China), group, linc00152(−)-NC + miR-103a-3p(+)-NC group, respectively. The pmirGLO vector(wild type fragments linc00152(−) group, miR-103a-3p(+) group and or mutated type fragments) and indicated miRNAs were linc00152(−) + miR-103a-3p(+) group. transfected into HEK 293 T cells using Lipofectamine For subcutaneous implantation, every mouse was sub- 3000. Relative luciferase activities were measured 48 h cutaneously injected with 3 × 105 cells in the right flank. after transfection and firefly luciferase activity was Tumor volume was measured every 4 days using the normalized by renilla luciferase activity. formula: volume (mm3) = length × width2/2. The Yu et al. Molecular Cancer (2017) 16:110 Page 17 of 18

subcutaneous tumor-bearing mice were executed 40 days Neurosurgery, Shengjing Hospital of China Medical University. Informed after injection. For survival analysis in orthotopic trans- consent was obtained from all patients and the research approaches were 5 approved by the Ethics Committee of Shengjing Hospital of China Medical plantation, 3 × 10 cells were stereotactically transplanted University (NO.2016PS366K). into the right striatum of the mice. The number of survived Four-week-old athymic nude mice were purchased from the Cancer Institute nude mice was recorded every day and survival analysis of the Chinese Academy of Medical Science (Beijing, China). All animal handling procedures were strictly abided by the Guide for the Care and Use was conducted applying KaplaneMeier survival curve. of Laboratory Animals, and approved by the Animal Care Committee of Shengjing Hospital. Statistical analysis Author details SPSS 18.0 statistical software was used for statistical 1Department of Neurosurgery, Shengjing Hospital of China Medical analysis. All data are presented as the mean ± standard University, Shenyang 110004, People’s Republic of China. 2Liaoning Clinical deviation (SD) from at least three independent replicates. Medical Research Center in Nervous System Disease, Shenyang 110004, People’s Republic of China. 3Key Laboratory of Neuro-oncology in Liaoning Statistical analysis of data was performed using the Stu- Province, Shenyang 110004, People’s Republic of China. 4Department of dent’s t-test. Differences were considered to be statistically Neurobiology, College of Basic Medicine, China Medical University, Shenyang 5 significant when P <0.05. 110122, People’s Republic of China. Key Laboratory of Cell Biology, Ministry of Public Health of China, China Medical University, Shenyang 110122, People’s Republic of China. 6Key Laboratory of Medical Cell Biology, Ministry Additional file of Education of China, China Medical University, Shenyang 110122, People’s Republic of China.

Additional file 1: Figure S1. Isolation and identification of GSCs. A.a,c: Received: 4 March 2017 Accepted: 7 June 2017 Cells grew into spheres in serum-free medium. b, d: Spheres generated again from a single cell. B. Individual undifferentiated GSCs-87 and GSCs- 251 stained for Nestin (green) and CD133 (red) by immunofluorescence analysis. C. Cell spheres were differentiated and then stained for GFAP References (green) and β-tubulin III (red) by immunofluorescence analysis. Nuclei 1. Zheng J, Liu X, Xue Y, Gong W, Ma J, Xi Z, et al. TTBK2 circular RNA (blue) were labeled with DAPI. Images are representative of independent promotes glioma malignancy by regulating miR-217/HNF1beta/Derlin-1 experiments (n = 5). Scale bars represent 20 μm. (TIFF 35029 kb) pathway. J Hematol Oncol. 2017;10:52. 2. Tso JL, Yang S, Menjivar JC, Yamada K, Zhang Y, Hong I, et al. Bone morphogenetic protein 7 sensitizes O6-methylguanine methyltransferase Abbreviations expressing-glioblastoma stem cells to clinically relevant dose of 3’UTR: 3’untranslated region; CCK-8: Cell counting kit-8; CDC25A: Cell division temozolomide. Mol Cancer. 2015;14:189. cycle 25A; ceRNA: Competing endogenous RNA; DMEM: Dulbecco’s modified 3. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem Eagle Medium; FBS: Fetal bovin serum; FEZF1: Forebrain embryonic zinc cells promote radioresistance by preferential activation of the DNA damage finger protein 1; GSCs: Glioma stem cells; linc00152: Long intergenic response. Nature. 2006;444:756–60. non-coding RNA 152; mut: Mutation type; NBT: Normal brain tissue; 4. Cheng P, Wang J, Waghmare I, Sartini S, Coviello V, Zhang Z, et al. FOXD1- qRT-PCR: Real-time quantitative polymerase chain reaction; wt: Wild type ALDH1A3 signaling is a determinant for the self-renewal and Tumorigenicity of Mesenchymal Glioma stem cells. 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Long intergenic 201304). non-coding RNA 00152 promotes tumor cell cycle progression by binding to EZH2 and repressing p15 and p21 in gastric cancer. Oncotarget. 2016;7: Availability of data and materials 9773–87. The datasets during and/or analyzed during the current study are available 9. Yue B, Cai D, Liu C, Fang C, Yan D. Linc00152 functions as a competing from the corresponding author on reasonable request. endogenous RNA to confer Oxaliplatin resistance and holds prognostic values in Colon cancer. Mol Ther. 2016;24:2064–77. Authors’ contributions 10. Liu L, Bi N, Wu L, Ding X, Men Y, Zhou W, et al. MicroRNA-29c functions as a YL contributed to the experiment design, manuscript draft, and data analysis. tumor suppressor by targeting VEGFA in lung adenocarcinoma. Mol Cancer. MY contributed to the experiment implementation, manuscript draft and 2017;16:50. data analysis. YX designed the experiments. JZ, XL, and HY performed the 11. 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