mir-23b and mir-130b expression is downregulated in pituitary

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Molecular and Cellular Endocrinology xxx (2014) xxx–xxx

MCE 8778 No. of Pages 7, Model 5G

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Contents lists available at ScienceDirect

Molecular and Cellular Endocrinology

journal homepage: www.elsevier .com/locate /mce

miR-23b and miR-130b expression is downregulated in pituitaryadenomas

http://dx.doi.org/10.1016/j.mce.2014.03.0020303-7207/� 2014 Elsevier Ireland Ltd. All rights reserved.

⇑ Corresponding author. Address: Istituto di Endocrinologia ed OncologiaSperimentale del CNR, via Pansini 5, 80131 Naples, Italy. Tel.: +39 081 7463602;fax: +39 081 2296674.

E-mail address: [email protected] (A. Fusco).

Please cite this article in press as: Leone, V., et al. miR-23b and miR-130b expression is downregulated in pituitary adenomas. Molecular and CEndocrinology (2014), http://dx.doi.org/10.1016/j.mce.2014.03.002

Vincenza Leone a, Concetta Langella a, Daniela D’Angelo a, Paula Mussnich a, Anne Wierinckx b,e,Luigi Terracciano c, Gerald Raverot d, Joel Lachuer e, Sandra Rotondi f, Marie-Lise Jaffrain-Rea f,g,Jacqueline Trouillas b,d, Alfredo Fusco a,⇑a Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Scuola di Medicina e Chirurgia di Napoli, Universitàdegli Studi di Napoli ‘‘Federico II’’, Naples, Italyb INSERM U1052, Centre de Recherche en Cancerologie de Lyon, F-69000 Lyon, France and University Lyon, F-69000 Lyon, Francec Molecular Pathology Division Institute of Pathology, University Hospital Schönbeinstrasse, 40CH-4003 Basel, Switzerlandd INSERM U1028, CNRS UMR 5292, Lyon Neuroscience Research Center, Neuro-oncology and Neuroinflammation Team, F-69372 Lyon Cedex 08, France and University Lyon1,F-69000 Lyon, Francee Profilexpert UNIV-US7 INSERM-UMS 3453 CNRS Lyon, Francef Department of Clinical Applied Sciences and Biotechnology, University of L’Aquila Via Vetoio, Coppito 2, 67100 L’Aquila, Italyg Neuromed, IRCCS, Pozzilli, Italy

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a r t i c l e i n f o

Article history:Received 21 October 2013Received in revised form 5 March 2014Accepted 5 March 2014Available online xxxx

Keywords:microRNAPituitary adenomaHMGA2CCNA2

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a b s t r a c t

MicroRNA (miRNA) deregulation plays a critical role in tumorigenesis. miR-23b and miR-130b areinduced by thyrotropin in thyroid cells in a cAMP-dependent manner.

The aim of our work has been to investigate the possible role of miR-23b and miR-130b in pituitarytumorigenesis. We have analyzed their expression in a panel of pituitary adenomas (PAs) including GHand NFPA adenomas.

We report that miR-23b and miR-130b are drastically reduced in GH, gonadotroph and NFPA adenomasin comparison with normal pituitary gland. Interestingly, the overexpression of miR-23b and miR-130binhibits cell proliferation arresting the cells in the G1 and G2 phase of the cell cycle, respectively. More-over, we demonstrate that miR-23b and miR-130b target HMGA2 and cyclin A2 (CCNA2) genes, respec-tively. Finally, downregulation of miR-23b and miR-130b expression is associated with increased levelsof their respective targets in human PAs.

These findings suggest that miR-23b and miR-130b downregulation may contribute to pituitarytumorigenesis.

� 2014 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

Pituitary adenomas (PAs) are one of the most frequentintracranial tumors with a prevalence of clinically apparent tumorsclose to one in 1000 of the general population and are the thirdmost common intracranial tumor type after meningiomas andgliomas (Scheithauer et al., 2006). GH- or ACTH-secreting PAsrepresent 20–25% and 10% of pituitary tumors, respectively, pro-lactinomas account to about 50% of PAs and TSH-secreting PAsare rare (1%) (Arafah and Nasrallah, 2001). About one-third ofPAs are clinically non-functioning pituitary adenomas (NFPAs),

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which do not exhibit signs of hormone hypersecretion. These aremostly composed by the so called ‘‘null cell’’ PAs (devoided ofpituitary hormone immunoreactivity) and gonadotroph adenomas(defined by FSH and/or LH immunoreactivity) (Trouillas et al.,1986). NFPA are usually large tumors diagnosed following localcompressive effects on brain structures and cranial nerves.30–50% of the GH adenomas are histologically mixed GH/PRL celladenomas co-secreting PRL and GH (Lopes, 2010). Pituitary tumor-igenesis is generally considered a model of the multi-step processof carcinogenesis, in which molecular genetic alterations representthe initializing event that transforms cells, and hormones and/orgrowth factors promote cell proliferation (Melmed, 2003; Asaand Ezzat, 2004). However, the molecular events leading topituitary tumor development are still unclear. Epigenetic events,such as hypermethylation and/or microRNA (miRNA)-dependent

ellular

http://dx.doi.org/10.1016/j.mce.2014.03.002

mailto:[email protected]


http://www.sciencedirect.com/science/journal/03037207

http://www.elsevier.com/locate/mce


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impairment of protein translation, are likely to be responsible forthe downregulation of gene and/or protein expression associatedwith pituitary tumorigenesis (Amaral et al., 2009; Tateno et al.,2010; Yacqub-Usman et al., 2012).

MiRNAs are a class of small non-coding RNAs which regulategene expression at post-transcriptional level. They bind to 30-untranslated (30-UTR) regions of target mRNAs, causing block oftranslation or mRNA degradation (Bartel, 2004). They play impor-tant roles in essential cellular processes such as differentiation, cellgrowth and cell death (Miska, 2005). Several studies have demon-strated altered expression of specific miRNAs in different types ofhuman neoplasias suggesting that they play a key role in tumori-genesis (Fabbri et al., 2008). Many studies have shown alterationsof miRNA expression in pituitary adenomas. Several of these dereg-ulated miRNAs may be involved in cell proliferation, apoptosis,cancer development and progression (Bottoni et al., 2007; Amaralet al., 2009; Butz et al., 2011; D’Angelo et al., 2012; Palmieriet al., 2012).

We have recently reported the identification of specific miRNAs,miR-23b, miR-130a and miR-130b, whose upregulation by TSH isrequired for thyroid cell growth and is protein kinase A-CREBdependent (Leone et al., 2012; Leone, unpublished results formiR-130b). We also identified SMAD3, a member of TGF-b path-way that has an inhibitor role in thyroid follicular cell proliferation,as target of miR-23b. Functional studies demonstrate that the over-expression of miR-23b promotes thyroid cell growth (Leone et al.,2012).

The aim of our work was to verify whether miR-23b and miR-miR-130b misexpression occurs in non-thyroid cell system, suchas pituitary adenomas, where alterations of the cAMP pathwayare frequent. Indeed, cAMP signaling is hyperactivated in GH-secreting adenomas, with an increased phosphorylation of thecAMP response element-binding protein (p-CREB) (Nishizawaet al., 2013; Gadelha et al., 2013). Moreover, it has been demon-strated that the arylhydrocarbon receptor–interacting protein(AIP), whose mutations have been linked o predisposition to pitu-itary adenomas, acts as a tumor suppressor by maintaining a lowcAMP signaling and concentration (Formosa et al., 2013). An in-creased TGF-b1 activity has also been reported in some PAs, whereTGF-b1 may represent a usefull serum marker for invasiveness(Elenkova et al., 2013; Katsuno et al., 2013). Futhermore, thesemiRNAs have been reported as downregulated in several humanneoplasias (Tong et al., 2009; Salvi et al., 2009; Kovaleva et al.,2012).

Here, we report that miR-23b and miR-130b are drastically re-duced in somatotroph, gonadotroph and null cell PAs in compari-son with normal pituitary gland. Moreover, we demonstrate thatmiR-23b and miR-130b target HMGA2 and cyclin A2 (CCNA2)genes, respectively, which are already known to play a critical rolein pituitary tumorigenesis.

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2. Materials and methods

2.1. Tissue collection and RNA isolation

PAs were obtained from patients operated on for medical rea-sons. For each tumor, some fragments were either snap-frozen inliquid nitrogen or collected in RNA Later (Ambion) at surgery andstored at �80 �C until RNA extraction. Diagnostic immunohisto-chemistry for pituitary hormones was performed in all cases onparaffin-embedded sections and tumors were classified into soma-totroph (n = 15) and NFPA (n = 21) including gonadotroph (n = 16)and null cell (n = 5) PA (Lloyd et al., 2004). We declare that in-formed consent for the scientific use of biological material was ob-tained from all patients.

Please cite this article in press as: Leone, V., et al. miR-23b and miR-130b exEndocrinology (2014), http://dx.doi.org/10.1016/j.mce.2014.03.002

Total RNA isolation from PAs was performed with TRIzol re-agent (Invitrogen) according to manufacturer’s instructions. RNAsfrom human normal pituitary glands were used as control. Normalpituitaries were obtained from autopsies of two females and threemales, aged between 50 and 60 years, devoid of endocrinediseases.

2.2. Bioinformatic prediction of miRNA target genes

Genes potentially targeted by the selected miRNAs were identi-fied by using different on-line available tools such as TargetScan(www.targetscan.org), miRanda (www.microrna.org) or miRwalk(www.umm.uni-heidelberg.de/apps/zmf/mirwalk). For more de-tails see Supplemental Information.

2.3. Cell lines and transfection

GH3 rat pituitary tumor cells secreting PRL and GH and HumanEmbryonic Kidney HEK-293 cells were cultured in DMEM contain-ing 10% FBS.

For transfection of miRNA oligonucleotides, cells were transfec-ted with 50 nmol/ml pre-miRNA precursors or control no-targetingscrambled oligonucleotides (Ambion) using siPORT neoFX Trans-fection Agent (Ambion).

2.4. Reverse Transcription and quantitative Real Time (qRT)-PCR

Reverse Transcription and qRT-PCR for mature miRNA wereperformed according to manufacturer’s instructions of miScriptSystem Kits (Qiagen) (for more details see Supplemental Informa-tion). qRT-PCR analyses for HMGA2 and CCNA2 expression wereperformed as previously described (De Martino et al., 2009). To cal-culate the relative expression levels we used the 2�DDCT method(Livak and Schmittgen, 2001). Primers for glucose-6-phosphatedehydrogenase (G6PD) were used for mRNA normalization andprimers for RNU6 (Qiagen) were used for miRNA normalization.The primers used to amplify the above mentioned genes are re-ported in Supplemental Information.

2.5. Western blotting and antibodies

Western blot analysis was performed as previously described(Lloyd et al., 2004) and the membranes were incubated with anti-bodies against CCNA2 (sc-751, Santa Cruz), a-tubulin (SC-8035,Santa Cruz), a-actin (sc-1616, Santa Cruz), Vinculin (sc-7649, SantaCruz), anti-HMGA2 antibody previously described (Finelli et al.,2002).

2.6. Luciferase target assays

Cells were co-transfected with the modified Firefly luciferasevectors described in Supplemental Information, along with theRenilla luciferase reporter plasmid and the miRNA oligonucleo-tides. Firefly and Renilla luciferase activities were measured 36 hafter transfection with the Dual-Luciferase Reporter Assay System(Promega). Firefly activity was normalized to Renilla activity ascontrol of transfection efficiency.

2.7. Growth curve assay

Exponentially growing GH3 and HEK-293 cells were plated in 6-well plates and transfected with 50 nmol/ml of pre-miR miRNAprecursor or scrambled oligonucleotide using siPORT neoFX. Cellswere counted in triplicate at 24, 48, 72 and 96 h after transfectionwith a Burker hemocytometer chamber.

pression is downregulated in pituitary adenomas. Molecular and Cellular

http://www.targetscan.org

http://www.microrna.org

http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk


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2.8. Flow cytometry

HEK-293 cells were plated and synchronized by serum depriva-tion for 48 h. Then, the cells were transfected with 50 nmol/ml ofpre-miR miRNA precursor or scrambled oligonucleotide using si-PORT neoFX and collected after 72 h.

After trypsinization, cells were washed in phosphate-bufferedsaline and fixed in 70% ethanol. Staining for DNA content was per-formed with 2 lg/ml propidium iodide and 20 lg/ml RNase A for30 min. For each measure 20,000 events have been analyzed. Weused a FACScan flow cytometer (Becton Dickinson) that was inter-faced with a Hewlett–Packard computer (Palo Alto, CA). Cell cycledata were analyzed with the CELL-FIT program (Becton Dickinson).

2.9. Statistical analysis

Student’s t-test was used to determine the differences betweentwo population of samples. Data are presented in the text asmean ± SEM and p < 0.05 was accepted as statistically significant.

To compare the differences between groups, one-way analysisof variance was used. The significance of differences was deter-mined by Analysis of Variances (ANOVA) followed by Dunnett’stest as the post hoc test using Graph Pad Prism 5.0. Data are pre-sented in the text as mean ± SEM and p < 0.05 was accepted as sta-tistically significant.

3. Results

3.1. miR-23b and miR-130b are downregulated in pituitary adenomas

To evaluate the possible role of miR-23b and miR-130b in pitu-itary tumorigenesis, we analyzed the expression of these miRNAs ina panel of 15 somatotroph PA and 21 NFPA in comparison with 5normal pituitary glands. As shown in Fig. 1, miR-23b was downreg-ulated in all tumors, except one GH (Panel A). MiR-130b was alsodownregulated in all tumors, apart from one NFPA (Panel B).

Fig. 1. Analysis of miR-23b and miR-130b expression in pituitary adenomas by qRT–PCRbetween somatotroph PA and NFPA (including gonadotroph (n = 16) and null cell (n = 5)samples was equal to 1. The range of variability of the expression of these miRNAs in northree independent experiments performed in triplicate. �p < 0.05 compared to normal p


3.2. Identification of the target genes for the miR-23b and miR-130b

In order to understand the mechanism by which the downreg-ulation of miR-23b and miR-130b might be involved in pituitarytumorigenesis, we identified, using bioinformatic tools (miRanda,TargetScan and MiRwalk), several potential target genes. Interest-ingly, we found HMGA2 and CCNA2 genes as predicted targets formiR-23b and miR-130b, respectively. Therefore, we focused ourattention on these target genes since HMGA proteins are involvedin cell cycle dysregulation, which plays an important role in thedevelopment of PA (Fedele et al., 2006). Moreover, we have previ-ously demonstrated that HMGA2 plays a critical role in pituitarytumorigenesis. Indeed, the HMGA2 gene was found overexpressedin most human PAs (De Martino et al., 2009; Qian et al., 2009)and transgenic mice overexpressing HMGA2 developed mixedPRL/GH PAs (Fedele et al., 2002). A-type cyclins also have a criticalrole on the cell cycle regulation. In fact, by coupling successively toCDK2 and CDK1, they participate in the S/G2 transition and pro-gression through G2 (Yam et al., 2002).

In Fig. 2A we report the miRNA-targeting sites of miR-23b onHMGA2 30-UTR. To validate its influence on the expression of theHMGA2 gene, we have performed a time course, collecting HEK-293 cells transfected with miR-23b and relative scrambled, at 24,48, 72 and 96 h. As shown in Fig. 2B, transfection of miR-23b de-creased HMGA2 protein levels at 72 h as compared to the scram-bled oligonucleotide-transfected cells. Interestingly, as shown inFig. 2C, this was accompanied by a decrease in HMGA2 transcripts,even though there is not a perfect complementarity between themiR-23b and its target sequences. This result strongly suggeststhat miR-23b reduces HMGA2 protein level also by affectingHMGA2 mRNA stability. In Fig. 3A we report the miRNA-targetingsites of miR-130b on CCNA2 30-UTR. Similarly, we looked at timecourse of CCNA2 protein levels in cells transfected withmiR-130b. As shown in Fig. 3B, transfection of miR-130b decreasedCCNA2 protein levels at 48 and 72 h as compared to the scrambledoligonucleotide-transfected cells. In contrast, no significant

. The relative expression values indicate the relative change in the expression levelsPA) versus five normal pituitary glands, assuming that the mean value of the normalmal pituitary tissues is less than 10%. Each bar represents the mean value ± s.e. fromituitary.



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Fig. 2. miR-23b targets HMGA2. (A) Schematic representation of human HMGA230UTR and the relative position of the predicted miRNA-binding site. (B) Westernblot analysis of HMGA2 protein levels in HEK-293 cells transfected with indicatedmiRNA precursors or scrambled oligonucleotide and collected at 24, 48, 72 and96 h. a-tubulin was analyzed as loading control. A representative experiment isshown. The bars below represent the densitometric analysis performed usingImageJ software and normalized with tubulin. (C) qRT–PCR analysis of HMGA2mRNA in HEK-293 cells transfected and collected at 72 h. Relative expression valuesindicate the relative change in HMGA2 mRNA expression levels between miR-treated and scrambled oligonucleotide-treated cells, normalized with G6PD. Theerror bars represent the mean ± s.e of three independent experiments performed intriplicate. (D) Relative luciferase activity in HEK-293 cells transiently transfectedwith 30UTR-HMGA2 and 30UTR-HMGA2MUT along with the indicated miRNAoligonucleotides or with a no-targeting scrambled oligonucleotide. The relativeactivity of firefly luciferase expression was standardized to a transfection control,using Renilla luciferase. The scale bars represent the mean ± s.e. of three indepen-dent experiments performed in triplicate. p < 0.05 compared to scrambled oligo-nucleotide-transfected cells.

Fig. 3. miR-130b targets CCNA2. (A) Schematic representation of the 30-UTR sites ofthe CCNA2 gene targeted by miR-130b and the relative position of the predictedmiRNA-binding site. (B) Western blots of the CCNA2 protein expression in HEK-293cells transfected with miR-130b and a scrambled oligonucleotide and collected at24, 48, 72 and 96 h. a-actin was analyzed as loading control. A representativeexperiment is shown. The bars below represent the densitometric analysisperformed using ImageJ software and normalized with actin. (C) qRT–PCR analysisof CCNA2 mRNA in HEK-293 cells transfected and collected at 72 h. Relativeexpression values indicate the relative change in CCNA2 mRNA expression levelsbetween miRNA-treated and scrambled oligonucleotide-treated cells, normalizedwith G6PD. The error bars represent the mean ± s.e. p < 0.05 compared to scrambledoligonucleotide-transfected cells. (D) Relative luciferase activity in HEK-293 cellstransiently transfected with 30UTR-CCNA2- and 30UTR-CCNA2MUT along with miR-130b oligonucleotide. A no-targeting scrambled oligonucleotide was used as acontrol. The relative activity of firefly luciferase expression was standardized to atransfection control, using Renilla luciferase. The scale bars represent themean ± s.e. of three independent experiments performed in triplicate. �Significancevalues of p < 0.05 compared to scrambled oligonucleotide-transfected cells.



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changes in the CCNA2 transcripts were observed (Fig. 3C). Thesedata are consistent with post-transcriptional regulation of theCCNA2 protein by miR-130b without effects on CCNA2 mRNAdegradation.


In order to verify that a direct interaction between miRNAs andthe CCNA2 and HMGA2 mRNAs was responsible for protein leveldecrease, the 30-UTRs of CCNA2 and HMGA2 mRNA were inserteddownstream of the luciferase ORF, either in sense (30UTR-CCNA2and 30UTR-HMGA2) or in antisense (30UTR-CCNA2 MUT and



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30UTR-HMGA2 MUT) orientation. These reporter vectors weretransfected in HEK-293 cells together with miRNA oligonucleotideprecursors or a scrambled oligonucleotide. As shown in Fig. 2 and3D, the luciferase activity of the Luc-sense-30UTR constructs wasmarkedly decreased after transfection of both miRNAs, comparedwith the scrambled oligonucleotide, while the luciferase activityof Luc-antisense-30UTR constructs did not vary. This indicates thatthese miRNAs interfere with CCNA2 and HMGA2 translationthrough direct interaction with their 30-UTR.

3.3. The miR-23b and miR-130b inhibit cell proliferation

To understand the role of miRNA downregulation in pituitarytumorigenesis we analyzed their effects on cell proliferation. Theeffect of miR-23b expression on cell growth was studied on humanHEK-293 cells, we did not use the rat GH3 cells for this experimentsince HMGA2 is not expressed in these cells. As shown in Fig. 4A(left panel), a significant reduction of cell number was observed96 h after transient transfection with miR-23b compared withscrambled oligonucleotide-transfected cells. Even a more drasticreduction of the proliferation was obtained when the HMGA2 pro-tein expression was silenced by shRNA interference (data notshown). Conversely, the role of miR-130b was studied on GH3 cellstransiently transfected with miR-130b or the scrambled oligonu-cleotide. As shown in Fig. 4A (right panel), a significant reductionof cell number was also observed 96 h after transfection withmiR-130b compared with scrambled-oligonucleotide transfectedcells. We have also performed the same experiments on the HEK-293 cell line achieving almost identical results (SupplementaryFig. 1A).

Finally, to better characterize the effects of the analyzed miR-NAs on cell cycle progression, miRNA precursors or scrambled oli-gonucleotide were transfected in HEK-293 (Fig. 4B, left panel) andGH3 cells (Fig. 4B, right panel), and analyzed by flow cytometry.Interestingly, miR-23b-transfected cells displayed an increase in

Fig. 4. miR-23b and miR-130b inhibit cell proliferation. (A). Cell growth curves of HEK-2with miR-130b and counted each 24 h for 96 h after plating. Y-axis represents the mean o�Significance values of p < 0.05 compared to scrambled oligonucleotide-transfected cellspanel) cells transfected with the indicated miRNA precursors or scrambled oligonucleotidcytometry after propidium iodide staining. Each bar represents the mean ± s.e. from threcompared to scrambled oligonucleotide-transfected cells.


the G1 phase population and a decrease in the S-phase, comparedwith scrambled-transfected cells. These results indicate that theoverexpression of this miRNA affects the G1-S transition of the cellcycle progression, as expected by its target (HMGA2). In contrast,miR-130b-transfected cells displayed a decreased number of cellsin the G1-phase, whereas the cell population in the G2-phasewas increased, compared with scrambled-oligonucleotide trans-fected cells. These findings are consistent with downregulation ofits target gene (CCNA2), which is involved in the regulation of S/G2 transition and progression through G2 phase of the cell cycle(Yam et al., 2002). We have also performed the same experimentson the HEK-293 cell line achieving almost identical results (Supple-mentary Fig. 1B).

3.4. miR-23b and miR-130b downregulation is associated with anincrease in HMGA2 and CCNA2, respectively, in human PAs

In order to further analyse the potential role of miRNA down-regulation in human pituitary tumorigenesis, the expression ofCCNA2 was studied by western blot in a subset of GH-PAs showinga marked downregulation of miR-130b. As shown in Fig. 5A, an in-creased CCNA2 protein expression was observed in all cases, incomparison with normal pituitary. Similarly, we previously re-ported increased HMGA2 protein levels in GH-PAs, in comparisonwith normal pituitary (D’Angelo et al., 2012). In this study, wefound that miR-23b downregulation was associated with overex-pression of HMGA2 mRNA evaluated by qRT-PCR in GH, gonadotro-phin and null cell PAs (Fig. 5 B).

4. Discussion

In this study, we have analyzed the expression of miR-23b andmiR-130b in pituitary adenomas. Both were found to be downreg-ulated in GH, gonadotroph and null cell PA in comparison with

93 (left panel) cells transfected with miR-23b or GH3 (right panel) cells transfectedf viable cell count ± s.e. from three independent experiments performed in triplicate.. (B) Flow cytometric analysis of synchronized HEK-293 (left panel) and GH3 (righte. After transfection, the DNA of the transfected cells was analyzed 72 h later by flowe independent experiments performed in triplicate. �Significance values of p < 0.05



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Fig. 5. miR-23b and miR-130b downregulation is associated with an increase in HMGA2 and CCNA2 in human PAs compared with normal pituitary (NP). (A) Western blotanalysis of CCNA2 protein expression in a subset of somatotroph PA. a-tubulin has been used as loading control. (B) qRT–PCR analysis of HMGA2 mRNA levels in somatotrophPA and NFPA (including gonadotroph (n = 16) and null cell (n = 5) PA) and in normal pituitary tissue (5 samples). The relative expression values indicate the relative change inthe expression levels between adenomas and normal pituitary assuming that the value of normal pituitary tissue is equal to 1.



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normal pituitary gland, in contrast with the results obtained inthyroid cells stimulated by TSH (Leone et al., 2012) and thyroidadenomas (Leone, manuscript in preparation) showing upregula-tion of these miRNAs. We also identified the HMGA2 gene as targetof miR-23b. Overexpression of HMGA2 is a frequent event in PAsand high levels of HMGA2 protein have been correlated with tumorsize, invasiveness and higher Ki-67 index in PRL, silent ACTH- andgonadotroph adenomas (Qian et al., 2009). We have previouslyshown that HMGA2 overexpression in prolactinomas was fre-quently associated with alterations of the HMGA2 gene, locatedin chromosome 12, including amplification, rearrangements andtrisomy of chromosome 12 (Finelli et al., 2002). In this study, wedemonstrate that miR-23b interaction with HMGA2 mRNA 30 UTRin vitro is directly responsible for HMGA2 protein decrease. In addi-tion, miR-23b was found to affect HMGA2 mRNA stability in vitroand an opposite behavior between HMGA2 transcripts and theexpression of miR-23b was observed in human PAs. Therefore,miR-23b downregulation can, at least partially, account for HMGA2overexpression in NFPAs where genomic HMGA2 alterations havebeen rarely observed (Pierantoni et al., 2005). Then, downregula-tion of miRNAs targeting HMGA2 represents an alternative mecha-nism of HMGA2 overexpression in PA and may potentiallysynergize with HMGA2 gene alterations to induce high HMGA2protein levels. Moreover, we identified the CCNA2 gene as targetof miR-130b. A-type cyclins exert a critical role on the cell cycleregulation. In fact, Cyclin A is the only cyclin able to regulate multi-ple steps of the cell cycle, since it participates in the G1/S transitionand progression through G2 phase of the cell cycle (Yam et al.,2002).

In this study, we demonstrate that miR-130b interaction withCCNA2 mRNA 30 UTR in vitro is directly responsible for CCNA2 pro-tein decrease, with no effect on CCNA2 mRNA stability. Accordingly,CCNA2 was found to be overexpressed in a subset of GH-secretingPAs showing a marked downregulation of miR-130b. Functionalstudies suggest a role for the downregulation of these miRNAs in


tumorigenesis. Indeed, overexpression of both miR-23b and miR-130b was found to inhibit cell growth. Moreover, FACS analysisdemonstrated that, consistently with their identified target genes,miR-23b retains the cells in the G1 phase of the cell cycle, whereasmiR-130b retains the cells in the G2 phase. These findings furthersupport the hypothesis that downregulation of miR-23b andmiR-130b may contribute to development of human PAs throughan overexpression of HMGA2 and CCNA2, respectively. However,we have to consider that downregulation of these miRNAs maycontribute to pituitary tumorigenesis also by targeting other genescoding for proteins involved in cell proliferation as SMAD3, a mem-ber of TGF-b pathway that we identified as target of miR-23b.Indeed, it is well known that each miRNA regulates the expressionof several genes.

Interestingly, recent studies have demonstrated that miR-23band miR-130b are also downregulated in prolactinomas (Chenet al., 2012a,b), suggesting this might represent a general eventin pituitary tumorigenesis. It is noteworthy, to observe that bothmiRNAs were not reported in our previous study where the miRNAexpression profile of 12 GH-adenomas was analyzed. Unfortu-nately, these techniques are not completely precise yet, and thisalso accounts for different results obtained by different groupsanalyzing the same tumor histotypes. Moreover, we did not ana-lyze miR-23b in the study by Palmieri et al., where we focused juston a limited number of miRNAs able to target the genes coding forthe HMGA proteins.

Notably, both miR-23b and miR-130b have been found deregu-lated in several human neoplasias. Indeed, miR-23b is overexpres-sed in bladder (Gottardo et al., 2007) and oral squamous cellcarcinoma (Scapoli et al., 2010) in breast cancer (Paris et al.,2012) where it acts as a potential oncomir. Conversely, it is down-regulated in prostate cancer (Tong et al., 2009), hepatocellular car-cinoma (Salvi et al., 2009) where it acts as a tumor suppressor bytargeting the urokinase-type plasminogen activator (uPA) and c-met (Salvi et al., 2009). Also miR-130 is upregulated in several



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types of cancer, such as nonsmall cell lung cancer (Yang et al.,2010), pancreatic (Chen et al., 2012a,b) and breast cancer (Shiet al., 2011), whereas is down-regulated in chronic lymphocyticleukemia (Kovaleva et al., 2012).

In conclusion, we report the identification of miRNAs drasticallyand constantly downregulated in PAs. Since these miRNAs targetgenes, such as HMGA2 and CCNA2, whose overexpression plays acritical role in pituitary tumorigenesis, it is reasonable to retainthat their downregulation might contribute to this process sug-gesting an approach to the therapy of PAs based on the restorationof the downregulated miRNAs.

Acknowledgments

This work was supported by grants from Associazione Italianaper la Ricerca sul Cancro (AIRC) (IG 5346), the Project ‘‘Invec-chiamemto’’ of the National Research Council (CNR) and from theItalian Ministry of Economy and Finance to the CNR for the ProjectFaReBio di Qualità. D. D’Angelo is a recipient of a fellowship fromItalian Foundation for Cancer research (FIRC).

We are grateful to E. Jouhanneau, V. Esposito e F. Giangasperofor adenomas collection and analysis and Mario Berardone forartwork.

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Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.mce.2014.03.002.

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mir-23b and mir-130b expression is downregulated in pituitary

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