Mir-506 Acts As a Tumor Suppressor by Directly Targeting the Hedgehog Pathway Transcription Factor Gli3 in Human Cervical Cancer

ORIGINAL ARTICLE miR-506 acts as a tumor suppressor by directly targeting the hedgehog pathway transcription factor Gli3 in human cervical cancer

S-Y Wen1,2,3,8, Y Lin2,8, Y-Q Yu4, S-J Cao5, R Zhang6, X-M Yang3,JLi3, Y-L Zhang3, Y-H Wang3, M-Z Ma3, W-W Sun5, X-L Lou5, J-H Wang7, Y-C Teng1 and Z-G Zhang3

Although significant advances have recently been made in the diagnosis and treatment of cervical carcinoma, the long-term survival rate for advanced cervical cancer remains low. Therefore, an urgent need exists to both uncover the molecular mechanisms and identify potential therapeutic targets for the treatment of cervical cancer. MicroRNAs (miRNAs) have important roles in cancer progression and could be used as either potential therapeutic agents or targets. miR-506 is a component of an X chromosome- linked miRNA cluster. The biological functions of miR-506 have not been well established. In this study, we found that miR-506 expression was downregulated in approximately 80% of the cervical cancer samples examined and inversely correlated with the expression of Ki-67, a marker of cell proliferation. Gain-of-function and loss-of-function studies in human cervical cancer, Caski and SiHa cells, demonstrated that miR-506 acts as a tumor suppressor by inhibiting cervical cancer growth in vitro and in vivo. Further studies showed that miR-506 induced cell cycle arrest at the G1/S transition, and enhanced apoptosis and chemosensitivity of cervical cancer cell. We subsequently identified Gli3, a hedgehog pathway transcription factor, as a direct target of miR-506 in cervical cancer. Furthermore, Gli3 silencing recapitulated the effects of miR-506, and reintroduction of Gli3 abrogated miR-506- induced cell growth arrest and apoptosis. Taken together, we conclude that miR-506 exerts its anti-proliferative function by directly targeting Gli3. This newly identified miR-506/Gli3 axis provides further insight into the pathogenesis of cervical cancer and indicates a potential novel therapeutic agent for the treatment of cervical cancer.

Oncogene (2015) 34, 717–725; doi:10.1038/onc.2014.9; published online 10 March 2014

INTRODUCTION diseases and malignant carcinomas. miR-506 expression has been Cervical carcinoma represents the third most common female shown to be upregulated in kidney allografts bearing interstitial cancer, with recently increasing incidence.1 The long-term (5-year) fibrosis and tubular atrophy and in primary biliary cirrhosis.10,11 survival of advanced cervical cancer patients only improved However, the expression pattern of miR-506 is complicated, even 3% by combined chemotherapy and radiotherapy, as compared contradictory, in different types of malignant carcinoma, indicat- with radiotherapy along.2 Therefore, it is urgent to uncover ing the complex role of miR-506 in cancer development. miR-506 the molecular mechanisms of cervical carcinoma and identify has been demonstrated to act as an oncogene in melanomas12 potential therapeutic targets to improve treatment. and can confer chemoresistance in colon cancer.13 In contrast, MicroRNAs (miRNAs) are non-coding RNA molecules of 20–24 miR-506 functions as a tumor suppressor in ovarian cancer14 and nucleotides that modulate the translational efficiency or stability suppresses malignant transformation in lung cancer.15 Using three of target messenger RNAs.3 Accumulating evidence has indicated different miRNA target prediction algorithms (PicTar, TargetScan that miRNAs are critical regulators of cancer progression that are and miRanda), we identified thousands of cancer-related genes involved in cancer cell proliferation, differentiation, apoptosis and that may be potential targets of miR-506. However, only two of – metastasis.4 8 miR-506 was identified by Bentwich et al.9 as a these genes, PPARα13 and SNAI2,14 have been shown to be direct component of an X chromosome-linked miRNA cluster in the targets of miR-506 by experimental analysis. primate testis. The genomic location of miR-506 is conserved in The zinc-finger protein Gli3, a mediator of Sonic hedge- mouse, dog, primate and ground squirrel. The biological function hog signaling, is also predicted to be a potential target of miR-506 has not been well studied. Currently, only a limited of miR-506. Several studies have shown that Gli3 has an number of reports exist regarding the roles of miR-506 in benign important role in cancer progression by stimulating cancer cell

1Department of Obstetrics & Gynecology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, PR China; 2Department of Obstetrics & Gynecology, International Peace Maternity & Child Health Hospital of the China Welfare Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China; 3State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China; 4Department of Pathophysiology, College of Basic Medical Science, China Medical University, Shenyang, PR China; 5Department of Obstetrics and Gynecology, Shanghai Songjiang District Central Hospital, Shanghai, PR China; 6Department of Obstetrics and Gynecology, Shanghai Fengxian District Central Hospital, Shanghai, PR China and 7Department of Obstetrics and Gynecology, Southern Medical University, Guangzhou, PR China. Correspondence: Dr Y-C Teng, Department of Obstetrics & Gynecology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600 Yisan Road, Shanghai 200233, PR China or Dr Z-G Zhang, State Key Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Room 428, Wenxuan Building of Medicine, 800 Dongchuan Road, Shanghai 200240, PR China. E-mail: [email protected] or [email protected] 8These authors contributed equally to this work. Received 27 June 2013; revised 2 February 2014; accepted 3 February 2014; published online 10 March 2014 miR-506 suppresses cervical cancer growth S-Y Wen et al 718 proliferation.16,17 In gynecological cancers, only one study proliferation. We selected two cervical cancer cell lines, Caski and has reported high expression of Gli3 in endometrial SiHa, to perform overexpression and/or knockdown experiments. adenocarcinoma.18 Although both miR-506 and Gli3 have been The proliferation rate of Caski cells stably transfected with implicated in cancer progression, the molecular mechanisms by sh-miR-506 was clearly increased compared with negative control which they affect cancer development, particularly cervical cells (Figure 2a). In contrast, the proliferation rate of SiHa cells carcinoma, remain unclear. transiently transfected with miR-506 mimics was significantly In this study, we found that miR-506 was downregulated in decreased compared with vector control cells (Figure 2b). To approximately 80% of our cervical cancer samples and was further evaluate the effect of miR-506 on cervical cancer cell inversely correlated with Ki-67, indicating that miR-506 expression proliferation in vivo, Caski cells stably expressing sh-miR-506 or may be related with cervical cell proliferation. Gain-of-function negative control cells were subcutaneously injected into nude and loss-of-function studies demonstrated that miR-506 acts as a mice. The mice were killed 4 weeks later, and the weights of the tumor suppressor by affecting cervical cancer growth, apoptosis resulting tumors were measured. The tumor weights in the and chemosensitivity. Furthermore, the hedgehog pathway miR-506-silenced group were significantly higher than those in the transcription factor Gli3 was identified as a direct functional negative control group (Figures 2c and d). These results indicate target of miR-506 in cervical cancer. that miR-506 suppresses cervical cancer cell proliferation both in vitro and in vivo. RESULTS miR-506 expression is downregulated and inversely correlates miR-506 induces cell cycle arrest of cervical cancer cells at with Ki-67 expression in human cervical cancer the G1/S transition fi To evaluate the expression and significance of miR-506 in cervical As miR-506 signi cantly suppressed cervical cancer cell prolifera- cancer, we first evaluated miR-506 expression in 50 matched pairs tion, we next aimed to determine whether miR-506 affects cell of cervical cancer and corresponding non-tumorous cervical cycle progression of cervical cancer cells. Cell cycle distribution tissues using real-time quantitative reverse transcriptase–PCR with analysis of Caski cells treated with nocodazole showed that fi normalization against an endogenous control (U6 RNA). miR-506 silencing of miR-506 resulted in a signi cant decrease in the expression was clearly downregulated in cervical cancer tissues cellular population in G0/G1 phase but a sharp increase in S phase compared with non-tumorous cervical tissues (Figures 1a and b). (Figure 3a). Consistent with this result, miR-506 mimics noticably miR-506 expression was inversely correlated with Ki-67 in these induced G1 phase arrest of both Caski and SiHa cells treated with clinical samples (Figure 1c), suggesting that miR-506 may have a nocodazole (Figures 3b and c). critical role in regulating cell proliferation. However, the specific function of miR-506 in human cervical cancer remained elusive. miR-506 induced apoptosis and enhanced chemosensitivity of cervical cancer cells miR-506 inhibits cervical cancer cell proliferation and xenograft We further investigated whether miR-506 has any effects on tumor growth cervical cancer cell apoptosis and chemosensitivity. For cell As miR-506 expression inversely correlated with Ki-67 in cervical apoptosis assay, the cervical cancer cells were stained cancer, we speculated that miR-506 may affect cervical cancer cell with propidium iodide and annexin V, and analyzed by flow

Figure 1. miR-506 expression was downregulated and inversely correlated with Ki-67 in human cervical cancer. (a, b) Expression of mature miR-506 in 50 matched pairs of primary cervical cancer samples and their corresponding non-tumorous cervix uteri samples. The expression level of miR-506 was detected using quantitative real-time–PCR and normalized against an endogenous control (U6 RNA). The data were ÀΔCt(miR-506-U6) analyzed using the ΔΔCt approach and expressed as either the miR-506/U6 ratio [2 ](a) or log2(fold change) (ΔΔCt [cervical cancer/non-tumorous]) (b). (c) Spearman correlation analysis of miR-506 expression and Ki-67 in 50 cervical cancer tissues. An inverse correlation between miR-506 and Ki-67 was observed (Po0.001, R2 = 0.3155).

Figure 2. miR-506 inhibited cervical cancer cell proliferation in vitro and in vivo.(a) By Cell Counting Kit-8 assay, the proliferation of Caski cells stably transfected with sh-miR-506 was accelerated compared with vector control. (b) The proliferation of SiHa cells transiently transfected with miR-506-mimics was inhibited compared with negative control (NC). (c) The morphologic characteristics of tumors in mice inoculated with miR-506 knockdown Caski cells or with vector control cells are shown. (d) The xenograft tumor weights of miR-506-silenced group were signiﬁcantly higher than those of negative control group. (*Po0.05; **Po0.01; ***Po0.001. Scale bar = 1 cm.)

Figure 3. miR-506 induced cell cycle arrest of cervical cancer cells at the G1/S transition. (a)Byﬂow cytometric analysis, miR-506 silencing by stable transfection with sh-miR-506 resulted in a signiﬁcant decrease in the cellular population in G0/G1 phase but a sharp increase in S phase in Caski cells treated with nocodazole. (b, c) Transient transfection of miR-506 mimics induced G1 arrest of Caski and SiHa cells treated with nocodazole. **Po0.01, ***Po0.001.

© 2015 Macmillan Publishers Limited Oncogene (2015) 717 – 725 miR-506 suppresses cervical cancer growth S-Y Wen et al 720 cytometry. The results showed that silencing of miR-506 Gli3 is a downstream target of miR-506 in cervical cancer cells significantly inhibited apoptosis of Caski cell (Figure 4a), miRNAs usually exert their functions by suppressing the whereas miR-506 mimics noticeably promoted apoptosis of expression of target mRNAs; therefore, we next aimed to identify Caski and SiHa cells (Figures 4b and c). To confirm the effects the miR-506 target genes in cervical cancer. By searching in the of miR-506 on cell apoptosis, we further measured the PicTar (http://pictar.mdcberlin.de/), TargetScan (http://www. Tar- caspase activity of cervical cancer cells. The results indicated getscan.org/) and miRanda (microrna.org and miRbase) that silencing of miR-506 significantly decreased caspase-3/7 databases, we found that Gli3 was a potential target gene of activities of Caski cell (Figure 4d), whereas miR-506 miR-506. Despite possessing a very long 3′ untranslated region mimics increased caspase-3/7 activities of Caski and SiHa cells (UTR), Gli3 could bind only seven miRNA families: miR-143/1721/ (Figures 4e and f). 4770, miR-203, miR-200bc/429/-548a, miR-7/7ab, miR-140, To investigate the effects of miR-506 on chemosensitivity of miR-194 and miR-124/506. Among these, we chose four cervical cancer cells, two chemotherapy drugs, cisplatin and miRNAs with highly conserved binding sites to test their ability paclitaxel, were used in the study. The results showed to bind to the 3′ UTR of Gli3 using a luciferase reporter system. that miR-506 mimics significantly enhanced chemosensitivity The results indicated that miR-506 could bind more efficiently to of Caski and SiHa cells to these two chemotherapy drugs the 3′ UTR of the Gli3 mRNA than other miRNAs (Supplementary (Figures 4g–j). Figure 1). In addition, overexpression of miR-506 clearly inhibited

Figure 4. miR-506 enhanced apoptosis and chemosensitivity in cervical cancer cells. (a) Caski cells by stable transfection with sh-miR-506 were stained with 50 μg/ml propidium iodide and Annexin V-fluorescein isothiocyanate (FITC), and analyzed by flow cytometry. miR-506 silencing resulted in a significant decrease of apoptotic cells. (b, c) Using the same method as above, transient transfection of miR-506 mimics resulted in a significant increase of apoptosis in Caski and SiHa cells. (d–f) Caspase-3/7 activities of Caski cells stably transfected with sh-miR-506 or transiently transfected with miR-506 mimics, and Caspase-3/7 activities of SiHa cells transiently transfected with miR-506 mimics. The data were normalized to controls. (g–j) The chemosensitivity of Caski and SiHa cells to cisplatin and paclitaxel was increased by transient transfection with miR-506 mimics.

Oncogene (2015) 717 – 725 © 2015 Macmillan Publishers Limited miR-506 suppresses cervical cancer growth S-Y Wen et al 721 expression of the Gli3 protein as measured by western Gli3 silencing recapitulates the effects of miR-506 in cervical blotting (Figure 5a), and TargetScan analysis indicated that the cancer cells miR-506 binding site in the Gli3 3′ UTR is highly conserved Gli3 regulates a variety of biological behaviors by targeting different among different species (Supplementary Figure 5A). We fused genes in diverse tumors, but the effects of Gli3 on cervical cancer either the wild-type 3′ UTR of Gli3 or a mutant version to the cells has not previously been described. To explore the function of firefly luciferase gene to create a real-time reporter for Gli3 gene Gli3, three specific small interfering RNAs (siRNAs) against Gli3 expression (Supplementary Figure 5B). The plasmids containing mRNA were synthesized. si-Gli3-1 and si-Gli3-3 siRNAs significantly mutant or wild-type Gli3 3′ UTR was co-transfected with a reduced the expression of Gli3 protein (Supplementary Figure 3). Renilla reporter vector into 293T cells transiently transfected Cell proliferation analysis indicated that both si-Gli3-1 and si-Gli3-3 with miR-506 mimics or negative control cells. As shown in clearly suppressed the proliferation of both Caski and SiHa cells Figure 5b, miR-506 clearly reduced the luciferase activity (Figures 6a and b). Both si-Gli3-1 and si-Gli3-3 blocked the G1/S of the wild-type Gli3 reporter (57% reduction), but did not affect phase transition of the cell cycle in Caski and SiHa cells treated with the mutant Gli3 reporter, suggesting that miR-506 can bind nocodazole (Figures 6c and d). In addition, silencing of Gli3 also directly to the Gli3 3′ UTR. Furthermore, we examined the significantly promoted apoptosis of Caski and SiHa cells (Figures 6e relationship between miR-506 and Gli3 in 50 cervical cancer and f). Furthermore, the chemosensitivity of Caski and SiHa cells to tissues and found that miR-506 expression was inversely cisplatin and paclitaxel was increased by silencing of Gli3 (Figures correlated with Gli3 expression in these clinical samples 6g–j). The suppression of proliferation, blockade of cell cycle (Figure 5c). progression, enhancement of apoptosis and chemosensitivity As the miRNAs belonging to the same family share the similar observed by silencing of Gli3 were similar to the phenotype seed sequence, for example, miR-506 and miR-124, they may also induced by forced expression of miR-506 in cervical cancer cells. share the common targets. To exclude the possibility that the These results suggest that miR-506 down-regulates Gli3 to suppress super-physiologic levels of miR-506 by transfection may mimic the cervical cancer cell proliferation. actions of miR-124, which is also downregulated in cervical cancer (Supplementary Figure 2), we tested whether miR-124 could target Gli3. As shown in Figure 5b, miR-124 only reduced 22% of Reintroduction of Gli3 rescues miR-506-induced effects on cervical the luciferase activity of the wild-type Gli3 reporter, which is much cancer cell proliferation and apoptosis less than that of miR-506. Furthermore, the functional assay If Gli3 indeed serves as a functional target of miR-506, showed that miR-124 had no significant effects on Caski cells reintroduction of Gli3 into miR-506-expressing cells should proliferation (Supplementary Figure 4). Together, these data antagonize the effects of miR-506. To address this hypo- indicate that the inhibitory effects of miR-506 on cervical cancer thesis, we constructed two lentiviral expression vectors of Gli3 growth through directly targeting Gli3 are not due to mimicking containing wild-type 3′ UTR (Gli3 WT) or mutant 3′ UTR (Gli3 MT), the actions of other miRNAs, for example, miR-124 sharing similar which were transfected into miR-506-expressing cells. As shown seed sequence with miR-506. in Figures 7a and b, reintroduction of Gli3 MT, but not Gli3 WT,

Figure 5. Gli3 is a downstream target gene of miR-506 in cervical cancer cells. (a) The protein levels of Gli3 in Caski and SiHa cells were signiﬁcantly dwonregulated by transient transfection of miR-506 mimics, as detected by western blotting (upper). The diagrams below the blotting represent densitometry analysis of the protein levels. (b) The pGL3 -Gli3 3′ UTR-wild or pGL3 -Gli3 3′ UTR-mutant vector was transiently transfected into 293T cells with negative control (NC) or hsa-miR-506-3p mimics or hsa-miR-124-3p mimics. The Renilla luciferase expression construct was co-transfected and used as an internal control. The relative luciferase activity was analyzed. (c) Spearman correlation analysis of the expression of miR-506 and Gli3 in 50 cervical cancer tissues. An inverse correlation between miR-506 and Gli3 was observed (**Po0.01, ***Po0.001, R2 = 0.26).

© 2015 Macmillan Publishers Limited Oncogene (2015) 717 – 725 miR-506 suppresses cervical cancer growth S-Y Wen et al 722 significantly enhanced the proliferation of miR-506-expressing by miR-506 (Figures 7c and d). Furthermore, reintroduction of Gli3 cervical cancer cells. In addition, enforced expression of Gli3 MT, MT, but not Gli3 WT, significantly decreased apoptosis of miR-506- but not Gli3 WT, clearly counteracted the G1 phase arrest induced expressing cervical cancer cells (Figures 7e and f). These findings

Figure 6. Gli3 silencing recapitulated the effects of miR-506 in cervical cancer cells. (a, b) By Cell Counting Kit-8 assay, the proliferation of Caski and SiHa cells was significantly inhibited by transient transfection with Gli3 siRNAs. (c, d) Silencing of Gli3 induced G1 arrest of Caski (c) or SiHa (d) cells treated with nocodazole. (e, f) Caski and SiHa cells by transient transfection with Gli3 siRNAs were stained with 50 μg/ml propidium iodide and Annexin V-fluorescein isothiocyanate (FITC), and analyzed by flow cytometry. The apoptosis of Caski and SiHa cells was significantly accelerated by transient transfection with Gli3 siRNAs. (g–j) Cells were treated for 48 h with cisplatin (0–40 μM) and paclitaxel (0–160 nM) and the viability of cells were measured by Cell Counting Kit-8 for chemosensitivity. The chemosensitivity of Caski and SiHa cells to cisplatin and paclitaxel was significantly decreased by transient transfection with si-Gli3-1 or si-Gli3-3 compared with negative control (NC). *Po0.05; **Po0.01; ***Po0.001.

Figure 7. Reintroduction of Gli3 abrogated miR-506-induced growth arrest and apoptosis of cervical cancer cells. (a, b) Proliferation of Caski and SiHa cells transiently transfected with miR-506 mimics, Gli3 WT, Gli3 MT, negative control or vector was measured by Cell Counting Kit-8 assay. (c, d) Flow cytometric analysis of cell cycle distribution of Caski cells and SiHa cells, which both were treated with nocodazole. (e, f) Caski and SiHa cells transiently transfected with miR-506 mimics, Gli3 WT, Gli3 MT, negative control or vector were stained with 50 μg/ml propidium iodide and Annexin V-fluorescein isothiocyanate (FITC), and analyzed by flow cytometry for cell apoptosis. *Po0.05; **Po0.01; ***Po0.001, NS, not significant. demonstrated that Gli3 reintroduction abrogates the miR-506- cases and inversely correlated with Ki-67 expression (an index of induced cellular behaviors, suggesting that Gli3 is a functional cell proliferation), suggesting that miR-506 may negatively regulate mediator of miR-506 in cervical cancer cells. cervical cancer cell growth. Through in vitro and in vivo miR-506 gain-of-function and loss-of-function studies, we further confirmed that miR-506 acts as a tumor suppressor in cervical cancer. DISCUSSION miRNAs usually exert their functions by suppressing the Sustained proliferative signaling is one of the hallmarks of cancer. expression of target mRNAs;3 therefore, we aimed to identify Discovering the molecules involved in cancer cell proliferation and miR-506 target genes in cervical cancer using the TargetScan, understanding the related mechanisms are critical for developing PicTar and miRanda databases. All of three databases suggested effective therapeutic strategies to improve the survival and that Gli3 could be a miR-506 target gene. Using a luciferase prognosis of patients suffering from cancer. Non-coding RNAs, reporter system, we found that miR-506 caused a decrease in the especially miRNAs, have been reported to be key factors at all luciferase activity of a wild-type Gli3 3′ UTR reporter but did not stages of cancer development (including initiation, progression and affect the luciferase activity of a ‘seed region’ mutant Gli3 3′ UTR metastasis) by regulating processes such as cancer cell prolifera- reporter. Transfection of miR-506 mimics inhibited Gli3 expression tion, differentiation, apoptosis, adhesion and migration.4 miR-124- in cervical cancer cells. Furthermore, miR-506 expression was /124ab, which shares a common conserved seed region with inversely correlated with Gli3 expression in human cervical cancer miR-506, often acts as a tumor suppressor by inhibiting cell tissues. These data indicate that miR-506 directly regulates Gli3 proliferation through the regulation of different genes in gene expression via binding to the 3′ UTR of Gli3 mRNA. The specific malignant tumor types, including colorectal cancer,19 miR-506 binding site on the 3′ UTR of Gli3 is conserved in many pancreatic cancer,20 hepatocellular carcinoma,21 glioblastoma,22 species, including Homo sapiens, Pan troglodytes and Macaca medulloblastoma,23,24 prostate cancer25 and cervical cancer.26 mulatta, suggesting that the interaction between miR-506 and However, contradictory roles for miR-506 have been observed in Gli3 may have a fundamental biological role. different types of cancer. For example, miR-506 has been reported Gli transcription factors, the central effectors of hedgehog to act as an oncogene in melanomas12 but functions as a tumor signaling, have important roles in organ development and cancer suppressor in ovarian cancer.14 In this study, we found that miR-506 progression.27 Recent studies have shown that Gli3 regulates expression was downregulated in the majority of cervical cancer diverse cell behaviors not only through hedgehog pathway

© 2015 Macmillan Publishers Limited Oncogene (2015) 717 – 725 miR-506 suppresses cervical cancer growth S-Y Wen et al 724 signaling but also through cross-talk with other signaling were performed using an All-in-One miRNA quantitative real-time–- pathways.28 The hedgehog activator Gli3 has been shown to PCR detection kit (GeneCopoeia, Guangzhou, China). promote anchorage-independent growth and tumorigenecity of colorectal cancer through upregulation of adhesion-related genes, 29 Plasmid constructs irrespective of p53. However, another study has shown that For construction of the lentivirus vector expressing antagomir of miR-506- abrogation of Gli3 expression suppresses colon cancer growth ′ 16 3p, two oligonucleotides (sense: 5 -GATCCTCTACTCAGAAGGGTGCCTTATT through activation of p53. Gli3 has also been reported to regulate TTTG-3′ and antisense: 5′-AATTCAAAAATAAGGCACCCTTCTGAGTAGAG-3′) angiogenesis and endothelial cell migration in ischemic tissue were synthesized and annealed before cloned into the pGreenPuro vector repair in a manner dependent on Akt and ERK1/2 activation.30 (System Biosciences, Mountain View, CA, USA)31 and the empty vector was Recent studies have shown that Gli3 regulates diverse cell used as the control. We produced the lentiviruses, determined their behaviors not only through hedgehog pathway signaling but also titers and transduced cultured cells according to previously described 32 through cross-talk with other signaling pathways, such as the Wnt/ methods. To construct the Gli3 expression plasmids, the full-length ′ ′ β-catenin pathway.28 These data suggest complex roles for Gli3 in construct with wild-type 3 UTR or mutant 3 UTR of Gli3 gene was cloned into the PEZ-Lv105 letivirus vector (GeneCopoeia). For luciferase assays, various physiological and pathological conditions. the full-length wild-type UTR of Gli3 was amplified by PCR (Gli3-3′UTR-F: In this study, our data clearly demonstrated that miR-506 5′-GCCGGCATAGACGCGTGCTTTAGGAAAAAAAGACTGC-3′; Gli3-3′UTR-R: suppresses human cervical cancer growth in vitro and in vivo by 5′-ATCCTTTATTAAGCTTTTCCATACAAAGAATTTTTTATTG-3′) and cloned into directly targeting Gli3. miR-506 has been reported to be involved the pMIR-REPORT luciferase reporter vector (Invitrogen, Carlsbad, CA, USA) in diverse biological behaviors via the regulation of different at the 3′ end of the Firefly luciferase coding sequence. The mutant of the target genes. By directly targeting the transcription factor Gli3-3′UTR was constructed by replacing miR-506 binding site with their SNAI2, miR-506 prevents transforming growth factor-β-induced complimentary sequence. All constructs were verified by sequencing. epithelial–mesenchymal transition and suppresses cell migration 14 and invasion. miR-506 also confers resistance to hydroxycamp- Transfection tothecin by suppressing PPARα expression in an hydroxy- 13 Stable transfections were performed using Lipofectamine 2000 (Invitrogen) camptothecin-resistant colon cancer cell line. Furthermore, according to the manufacturer’s instructions. All of the siRNAs and miR-506 has also been demonstrated to be involved in the miRNA mimics were synthesized by GenePharma (Shanghai, China). They pathogenesis of primary biliary cirrhosis by targeting the were transiently transfected with siRNA mate (GenePharma).The siRNAs À À 11 μ Cl /HCO3 Anion exchanger 2 gene. were used at a concentration of 20 M, miRNA mimics were used at a In this study, the only miR-506 target we identified as involved concertration of 20 nM and the sequences were as follows: in regulating cervical cancer cell proliferation, cell cycle progres- siGli3:(sense: 5′-GCCCAGCAGAAUACUAUCATT-3′; ′ ′ sion, apoptosis and chemosensitivity was hedgehog pathway antisense: 5 -UGAUAGUAUUCUGCUGGGCTT-3 ); siGli3 (sense: 5′-GUCUCGUGCUUCAGAAUUATT-3′; transcription factor Gli3. However, our analysis of three miRNA antisense: 5′-UAAUUCUGAAGCACGAGACTT-3′). target databases identified many cancer-related genes that could be potential miR-506 targets. We speculate that, in addition to Gli3, other potential targets of miR-506 may also be involved in Luciferase reporter assay these processes. Further investigation should determine whether Luciferase reporter assays were performed in the 293 T cell. Cells at 70% other targets and their related signaling pathways are involved in confluence in a 96-well plate were transfected with 0.1 μg firefly and 0.01 miR-506-mediated cervical cancer progression. μg Renilla using 0.25 μl of transfection reagent. Cell extracts were prepared In conclusion, we have demonstrated that miR-506 acts as a 48 h after co-transfection, and the luciferase activity was measured using a Luciferase Reporter Assay System kit (Promega, Madison, WI, USA). The tumor suppressor in cervical cancer by suppressing cancer growth, mean values were measured, and the firefly luciferase activity was inducing cell cycle arrest, and enhancing apoptosis and chemo- normalized to the Renilla luciferase activity. sensitivity. Furthermore, we found that miR-506 exerts its anti- proliferative function by directly targeting the hedgehog pathway transcription factor Gli3. The newly identified miR-506/Gli3 axis Cell proliferation and chemosensitivity assays provides a further insight into the pathogenesis of cervical cancer Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8, Dojindo and indicates a novel potential therapeutic agent for the Molecular Technologies, Kumamoto, Japan). Cisplatin and paclitaxel were treatment of cervical cancer. obtained from either Chemopharmacy at the University of Chicago or Sigma. Cells for proliferation were seeded into 96-well plates at 2000 cells per well and cells for chemosensitivity at 5000 cells per well, and both incubated overnight. SiHa and Caski cells (ATCC) were stably transfected MATERIALS AND METHODS with lenti-sh-miR-506 or transiently transfected with miR-506 mimics or Cell culture siRNA-Gli3 or negative control. Cells were treated for 48h with cisplatin (0–40 μM) and paclitaxel (0–160 nM) and viability of cells were measured Caski and SiHa cell lines (ATCC, Manassas, VA, USA) were cultured in RPMI using a Cell Counting Kit-8 for chemosensitivity. In all, 10 μl of CCK-8 medium 1640 (Hyclone, Beijing, China) supplemented with 10% fetal solution was added to each well after 0, 24, 48, 72 and 96 h for bovine serum (GBICO, Beijing, China). All cells were maintained at 37 °C in proliferation measurement, respectively. In viable cells, WST-8 was an incubator with a 5% CO2 atmosphere. metabolized to produce a colorimetric dye that was detected at 450 nm using a Power Wave XS microplate reader (BIO-TEK, Neufahrn, Germany). RNA isolation from patient specimens and quantitative real-time PCR Cell cycle and apoptosis assays Fifty fresh-frozen cervical cancer clinical samples were obtained between For cell cycle analysis, transfected cells were cultured for 24 h and then April 2007 and March 2012 from the Shanghai Songjiang District Central treated with nocodazole (100 ng/ml) for 16–20 h,33 at last cells were Hospital, China Medical University Affiliated First Hospital, Shanghai Jiao collected and were fixed into 70% ethanol at À20 °C for 24 h, stained with Tong University Affiliated Sixth People's Hospital and Shanghai Fengxian 50 μg/ml propidium iodide (Kaiji, NanJing, China). District Central Hospital. The tissue specimens were frozen in liquid For apoptosis assay, cells were stained with 50 μg/ml propidium iodide nitrogen and stored at À80 °C until RNA extraction. Two gynecological and Annexin V-fluorescein isothiocyanate (Kaiji) and following the pathologists performed the pathological diagnoses of cervical cancer. manufacturer’s instructions. The results were analyzed using ModFit Informed consent was obtained from all patients, and the study was software (BD Bioscience, Franklin Lakes, NJ, USA) BD Bioscience. approved by the ethics committee of Shanghai Municipality. Total RNA The activities of caspase-3/7 were measured with the Caspase-Glo Assay extraction was performed using miRNA Extraction Kit (TAKARA, Dalian, Kit (Promega). The cells were seeded in a 96-well plate at 4 × 103 per well China). Reverse transcription of RNA and the detection of mature miR-506 and cultured overnight to ensure that the cells have enough time for proper

Oncogene (2015) 717 – 725 © 2015 Macmillan Publishers Limited miR-506 suppresses cervical cancer growth S-Y Wen et al 725 adherence to the wells. Then, the culture media were replaced with serum 8 Roy SS, Gonugunta VK, Bandyopadhyay A, Rao MK, Goodall GJ, Sun L-Z et al. deprivation medium for 24 h. Caspase-3/7 activity was measured as Significance of PELP1/HDAC2/miR-200 regulatory network in EMT and metastasis manufacturer’s protocol and calculated as follows: Caspase-3/7 activity of breast cancer. Oncogene 2014; 33: 3707–3716. = (luminescent unit of treatment well–luminescent unit of blank well)/(mean 9 Bentwich I, Avniel A, Karov Y, Aharonov R, Gilad S, Barad O et al. Identification of luminescent unit of control well). All data had been repeated for three times. hundreds of conserved and nonconserved human microRNAs. Nat Genet 2005; 37: 766–770. Tumorigenicity assay in nude mice 10 Castro RE, Rodrigues CM. Targeting miR-506 in primary biliary cirrhosis to support the HCO3- umbrella. Clin Res Hepatol Gastroenterol 2012; 36:402–404. Five to six-week-old female nude mice were purchased from the National 11 Banales JM, Saez E, Uriz M, Sarvide S, Urribarri AD, Splinter P et al. Up-regulation of Rodent Laboratory Animal Resources (Shanghai, China). After 4 days of microRNA 506 leads to decreased Cl-/HCO3- anion exchanger 2 expression in acclimatization, a total of 2 × 106 Caski cells stably transfected with either biliary epithelium of patients with primary biliary cirrhosis. Hepatology 2012; 56: sh-miR-506 or negative control were injected subcutaneously into the right 687–697. inside of the groin of each mouse. Each group contained five mice. The 12 Streicher KL, Zhu W, Lehmann KP, Georgantas RW, Morehouse CA, Brohawn P tumor weights were measured and recorded in grams. The mice were et al. A novel oncogenic role for the miRNA-506-514 cluster in initiating killed on the 28th day after injection. The mice were manipulated melanocyte transformation and promoting melanoma growth. Oncogene 2011; according to the guidelines approved by the Medical Experimental Animal 31: 1558–1570. Care Commission of Shanghai Jiaotong University. 13 Tong JL, Zhang CP, Nie F, Xu XT, Zhu MM, Xiao SD et al. MicroRNA 506 regulates expression of PPAR alpha in hydroxycamptothecin-resistant human colon Western blotting cancer cells. FEBS Lett 2011; 585: 3560–3568. Cells were harvested and lysed in 1x loading buffer (Roche Diagnostics, 14 Yang D, Sun Y, Hu L, Zheng H, Ji P, Pecot Chad V et al. Integrated Analyses Identify Indianapolis, IN, USA) added with 1:100 β-mercaptoethanol. Cell lysates were a Master MicroRNA Regulatory Network for the Mesenchymal Subtype in Serous separated by electrophoresis on a 10% sodium dodecyl sulfate—- Ovarian Cancer. Cancer Cell 2013; 23:186–199. polyacrylamide gel, transferred to nitrocellulose membranes, blocked in Tris 15 Zhao Y, Liu H, Li Y, Wu J, Greenlee AR, Yang C et al. The role of miR-506 in buffer solute with 0.1% Tween with 5% nonfat milk and incubated with transformed 16HBE cells induced by anti-benzo[a]pyrene-trans-7,8-dihydrodiol- primary antibodies against Gli3 (1:500, rabbit anti-human polyclonal 9,10-epoxide. Toxicol Lett 2011; 205: 320–326. antibody, Abcam, Cambridge, UK) and glyceraldehyde 3-phosphate dehy- 16 Kang HN, Oh SC, Kim JS, Yoo YA. Abrogation of Gli3 expression suppresses the drogenase (1:2000, rabbit anti-human polyclonal antibody, AOGMA, Chicago, growth of colon cancer cells via activation of p53. Exp Cell Res 2012; 318:539–549. IL, USA) at 4 °C overnight. The IRDye 800CW-labeled goat anti-rabbit 17 Steg A, Amm HM, Novak Z, Frost AR, Johnson MR. Gli3 mediates cell survival and secondary antibody (Li-COR Biosciences, Lincoln, NE, USA) was visualized sensitivity to cyclopamine in pancreatic cancer. Cancer Biol Ther 2010; 10: 893–902. using an Odyssey Infrared Imaging System (Li-COR) and quantified using 18 Kim KH, Kim JM, Choi Y-L, Shin YK, Lee H-c, Seong IO et al. Expression of Sonic densitometry measured by ImageJ software (NIH, Bethesda, MD, USA). hedgehog signaling molecules in normal, hyperplastic and carcinomatous endometrium. Pathol Int 2009; 59: 279–287. Statistical analysis 19 Liu K, Zhao H, Yao H, Lei S, Lei Z, Li T et al. MicroRNA-124 regulates the proliferation of colorectal cancer cells by targeting iASPP. Biomed Res Int 2013; 2013:1–10. The data are presented as percentages of control ± s.e.m. or means ± s.e.m. 20 Wang P, Chen L, Zhang J, Chen H, Fan J, Wang K et al. Methylation-mediated fi from multiple experiments. The statistical signi cance between groups was silencing of the miR-124 genes facilitates pancreatic cancer progression and ’ determined using the Student s t-test. SPSS version 11.5 (SPSS Inc., metastasis by targeting Rac1. Oncogene 2013; 33:514–524. Chicago, IL, USA) software was used for all data analyses. 21 Furuta M, Kozaki Ki, Tanaka S, Arii S, Imoto I, Inazawa J. miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carci- CONFLICT OF INTEREST noma. Carcinogenesis 2009; 31:766–776. 22 Silber J, Lim DA, Petritsch C, Persson AI, Maunakea AK, Yu M et al. miR-124 and The authors declare no conflict of interest. miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med 2008; 6:14. ACKNOWLEDGEMENTS 23 Pierson J, Hostager B, Fan R, Vibhakar R. Regulation of cyclin dependent kinase 6 by microRNA 124 in medulloblastoma. J Neurooncol 2008; 90:1–7. 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