Research Article

IntroductionMicroRNAs (miRNAs) are a new class of small non-codingendogenous RNA molecules approximately 22 nucleotides inlength. They are transcribed from genomic DNA as long primarytranscripts (pri-miRNAs), which are then modified by RNase-III-type enzymes Drosha and Dicer to produce pre-miRNAs andsubsequently mature miRNAs (Lee et al., 2004; Mansfield et al.,2004). miRNAs can function as a guide molecule in post-transcriptional repression of protein synthesis by partially pairingwith the 3�-untranslated region (UTR) of target mRNAs (Seitz etal., 2003). miRNAs have key roles in diverse regulatory pathways,including control of development (Shan et al., 2009; Wienholds etal., 2005; Zhao et al., 2005), cell proliferation (Corney et al., 2007;Fang et al., 2011; Johnson et al., 2007), differentiation (Kahai etal., 2009; Li and Carthew, 2005; Naguibneva et al., 2006; Wang etal., 2008), apoptosis (Chan et al., 2005; Chen and Stallings, 2007),cell cycle progression (Brennecke et al., 2003; O’Donnell et al.,2005), immune response (Stern-Ginossar et al., 2007; Wu et al.,2007), protein secretion (Mello and Czech, 2004; Poy et al., 2004),and many other physiological and pathological processes (Hansenet al., 2007; Wu et al., 2007).

A recent study demonstrated that more than 50% of miRNAgenes are located in cancer-associated genomic regions or in fragile sites, suggesting that miRNAs have an important role in the pathogenesis of cancers (Calin et al., 2004). Some studieshave also found that miRNAs might function as oncogenes or tumor suppressor genes (Zhang et al., 2007). Normally,oncogenic miRNAs are overexpressed in cancers and they cantarget tumor suppressor genes, whereas tumor-suppressor-like miRNAs are underexpressed in cancers and they might targetoncogenes.

MiRNA-199 was determined by computer analysis of mouseand Fugu rubripes sequences by Lim and colleagues in 2003 (Limet al., 2003). In the same year, it was cloned from the humanosteoblast sarcoma cell line Saos-2 and identified from mouse skincells (Lagos-Quintana et al., 2003). The two putative hairpinprecursors reside in chromosome 19 and chromosome 1 in thehuman genome. More recent microarray analysis revealed thatmiR-199a-3p is highly expressed in hair follicles (Yi et al., 2006)and in some tumor cells (such as ovarian and breast cancer cells)(Chen et al., 2008), suggesting its possible involvement in tumorprogression, but it was also significantly underexpressed inhepatocellular carcinoma (Murakami et al., 2006) and in bladdercancer (Ichimi et al., 2009). The published work suggests thatmiR-199a is involved in cancer development directly or indirectlyby targeting cancer oncogenes or tumor suppressors, or both. Thisstudy was designed to investigate the functional role of miR-199ain normal cells, as well as in neoplastic cells. Although miR-199aprecursor produces mature miRNAs miR-199a-5p and miR-199a-3p, here we focused on miR-199a-3p because it potentially targetscaveolin-2, a protein that is involved in cell cycle progression.

Caveolin-2 is a scaffolding protein of the caveolae family thatcoats plasma membrane invaginations. As the key structural proteinthat organizes caveolae, caveolins have been shown to have animportant role in regulating endocytosis and other aspects ofcellular signaling (Krajewska and Maslowska, 2004; Williams andLisanti, 2004). Caveolins interact directly with a number ofcaveolae-associated signaling molecules, such as H-Ras, hetero-trimeric G-proteins, epidermal growth factor receptor, proteinkinase C, Src-family tyrosine kinases and nitric oxide synthaseisoforms. It has been documented that caveolin-binding caneffectively inhibit the enzymatic activity of these signaling

SummaryRecent advances in the study of microRNAs indicate that they have an important role in regulating cellular activities such asproliferation, morphogenesis, apoptosis and differentiation by regulating the expression of various genes. MiR-199a-3p is highlyexpressed in hair follicles and in some tumor cells, suggesting its participation in tumor progression, but it is significantly underexpressedin hepatocellular carcinoma and in bladder cancer. The mechanism underlying these effects is not yet known. Here, we dissect theeffects of miR-199a-3p on YPEN-1 endothelial cells, and MDA-MB-231 and MT-1 breast cancer cell lines. We found that expressionof miR-199a-3p promotes proliferation and survival of endothelial cells as well as breast cancer cells. Remarkably, miR-199a-3pinhibited both endogenous caveolin-2 activity and exogenous caveolin-2 activity, which was confirmed by a reporter construct bearingthe 3�-untranslated region of caveolin-2. However, overexpression of caveolin-2 completely counteracted the enhancement of miR-199a-3p-mediated activities on cell proliferation, survival and sensitivity of tumor cells to anticancer drugs. Our findings suggest thatMiR-199a-3p targeting of caveolin-2 might have an important role in breast cancer tumor progression, making it a potential candidatefor intervention in cancer.

Key words: MicroRNA, miR-199a-3p, siRNA, 3�UTR, Caveolin, Cell cycle

Accepted 3 May 2011Journal of Cell Science 124, 2826-2836 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.077529

MicroRNA miR-199a-3p regulates cell proliferation andsurvival by targeting caveolin-2Tatiana Shatseva1,2, Daniel Y. Lee1,2, Zhaoqun Deng1,2 and Burton B. Yang1,2,*1Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada2Department of Laboratory Medicine and Pathobiology, University of Toronto, ON M5S 1A8, Canada*Author for correspondence (byang@sri.utoronto.ca)

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molecules. Compared with the other two members of the caveolinfamily, caveolin-1 and caveolin-3, the role of caveolin-2 is lesswell defined. Recent studies have alluded to cell- and tissue-specific roles of caveolin-2. There is also evidence that caveolin-2can regulate proliferation of lung endothelial cells and rat fibroblastcell line Hirc-B (Kim and Pak, 2005; Kwon et al., 2009a; Kwon etal., 2009b). Caveolin-2 was shown to be expressed at high levelsin normal breast-like cancer and in the majority of basal-like cancer(Perou et al., 2000), but at low levels in non-microdissected breastcancer. In fact, levels of caveolin-2 were found to be inverselycorrelated with tumor size (Sagara et al., 2004).

ResultsThe effect of miR-199a-3p on cell proliferationWe examined miR-199a-3p expression in human breast carcinomatissue and found that miR-199a-3p was expressed at higher levelsin carcinoma tissue than in normal breast tissue or tissue adjacentto the tumor, using RNAs purchased from Ambion (AppliedBiosystems/Ambion, Austin, TX) (supplementary material Fig.S1A). To study the role of miR-199a-3p, we generated a constructexpressing the miR-199a precursor, which can produce both miR-199a-3p and miR-199a-5p (Fig. 1A, supplementary material Fig.S1B). The construct was transfected into a number of cell linesincluding YPEN-1 cells and MT-1 and MDA-MB-231 breastcarcinoma cell lines. We have successfully used this plasmid toexpress different miRNAs in vitro (Lee et al., 2007; Wang et al.,2008). Analysis of miR-199a-3p expression was performed by RT-PCR and real-time PCR. We detected increased expression of miR-199a-3p in both cell types to different levels, perhaps because ofvarying transfection and expression efficiency (Fig. 1B,C).

We tested the roles of miR-199a-3p in regulating cell growth.Cells stably transfected with miR-199a were subjected toproliferation assays along with cells transfected with an emptyvector, which thus acted as a control. Cell proliferation wasdetermined by MTT and cell number counting on the second andthird days after cell inoculation. The experiments revealed thatexpression of miR-199a-3p increased proliferative activity ofendothelial cells and breast cancer cells (Fig. 2A–C).

We generated an anti-miR-199a-3p expression construct to studythe activity of endogenous miR-199a-3p in cell proliferation. Stabletransfection of anti-miR-199a-3p plasmid into breast cancer cellsMB-231 and MT1 originally expressing moderate and high levelsof miR-199a-3p, respectively, led to suppression of proliferativeactivity (Fig. 2D). Anti-miR-199a-3p exerted a similar effect oncell survival, and this was observed in transient transfection of theanti-miR-199a-3p plasmid into YPEN-1 cells, and MB-231 andMT-1 breast cancer cells (Fig. 2E, supplementary material Fig.S2).

In wound healing assays there was no difference observed in themigration of YPEN-1 cells between vector-transfected and miR-199a-3p-transfected cells. However, the miR-199a-3p-transfectedMB-231 cells migrated into the wound areas much faster than thevector-transfected cells (supplementary material Fig. S3).

Effect of miR-199a-3p on expression of Cav-2To identify the potential targets of miR-199a-3p that would facilitatecell growth, we used bioinformatic search tools (PicTar, Berlin),and found that a great number of genes could be potential targetsof miR-199a-3p. Caveolin-2 was selected for analysis.Computational screening showed that there were two potentialtarget sites of miR-199a-3p in the 3�UTR of rat (GenBank accession

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number NM_131914) and human (GenBank accession numberNM_001233) caveolin-2. Caveolin-2 is a member of a family ofscaffolding proteins that coats plasma membrane invaginations(caveolae). The two potential target sites for mir-199a-3p interactionare located at the nucleotides 627–649 and 664–684 of ratcaveoloin-2 3�UTR (Fig. 3A) and at the nucleotides 887–918 and1476–1500 of human caveolin-2 (Fig. 3B). Because expression ofmiR-199a-3p had similar roles in rat and human cell lines, theconserved target sites in rat and human suggests that caveolin-2can potentially mediate miR-199a-3p functions in cell proliferation.

To test targeting of caveolin-2 by miR-199a-3p, we examinedcaveolin-2 expression in cells stably transfected with miR-199ausing an anti-caveolin-2 monoclonal antibody. Cell lysates frommiR-199a- and vector-transfected YPEN-1 and MB-231 cells wereanalyzed by western blotting. We observed that caveolin-2expression was repressed in cells transfected with miR-199acompared with vector-transfected cells (Fig. 3C). When caveolin-2 expression was assayed by immunocytochemistry staining, lowerlevels of caveolin-2 were observed in the miR-199a-transfectedcells compared with the controls (Fig. 3D). However, transfection

Fig. 1. Construction and expression of miR-199a-3p. (A) Diagram of aconstruct expressing GFP, a neomycin-resistance gene, and two hairpinstructures of pre-miR-199a-3p. Two restriction sites (underlined), two pre-miR-199a-3p (capital letters), a linker (lowercase), and the terminationsequence (tttttt) are shown. (B) RT-PCR of pre-miR-199a-3p using RNAprepared from YPEN-1 endothelial cells stably transfected with miR-199a anda control vector, confirming expression of pre-miR-199a-3p. GAPDH wasamplified as a control. (C) Graph of results from real-time PCR of maturemiR-199a-3p from breast cancer cell line MB-231 confirming properprocessing of miR-199a-3p. Error bars represent s.d.; *P<0.05.

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of MT1 cells with anti-miR-199a expression construct promotedcaveolin-2 expression.

Interestingly, the levels of Cdc42, which, according tocomputational analysis, is a potential target of miR-199a-3p, wereupregulated (Fig. 3E), suggesting that Cdc42 is not a true target ofmiR-199a-3p. A number of reports indicate that Cdc42 is involvedin the molecular control of diverse cellular functions, includinggene transcription, cytoskeletal organization, cell proliferation,migration and transformation (Bishop and Hall, 2000). It has beenreported that caveolin-1 can function as a Cdc42 guanine nucleotidedissociation inhibitor in pancreatic b-cells, maintaining Cdc42 inan inactive state (Nevins and Thurmond, 2006). Because caveolin-1 and caveolin-2 share structural similarities and are commonlycoexpressed, caveolin-2 and Cdc-42 might share a similarrelationship to that observed for caveolin-1 and Cdc-42. We alsoanalyzed caveolin-1 expression in different cell lines stablytransfected with miR-199a or the control vector, but detected littledifference in caveolin-1 expression (supplementary material Fig.S4). There appears to be a possible link between miR-199a-3p,caveolin-2 and Cdc42, which is that miR-199a-3p might be

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involved in regulation of cell proliferation, survival and cellmigration by directly targeting caveolin-2, which then upregulatesCdc42 expression to exert the biological functions of miR-199a-3p.

We analyzed expression of miR-199a-3p, caveolin-2 and Cdc42in a number of cell lines including A431, A549, C8186, HepG2,OV2008, HT1080, Jurkat, MT1 and A2058. We observed thatMT1 cells expressed extremely high levels of miR-199a-3p (Fig.4A). Interestingly, the level of caveolin-2 was the lowest, whereasthe level of CDC42 was the highest in this cell line (Fig. 4B). Wealso observed that C8186 and A2058 expressed higher levels ofmiR-199a-3p, whereas the levels of caveolin-2 were lower in thesecell lines than the other cell lines. This reversed co-relationshipsuggests that miR-199a-3p has roles in repressing caveolin-2expression in these cell lines. To confirm the function of miR-199a-3p in repressing caveolin-2 expression, we transfected anantisense construct anti-miR-199a-3p in MT1 cells, which expressextremely high levels of miR-199a-3p and detected a very highincrease in caveolin-2 expression (Fig. 4C). However, the effect ofanti-miR-199a-3p was not evident in MB-231 cells, possibly

Fig. 2. Effects of miR-199a-3p on endothelial cellproliferation. (A) YPEN-1 endothelial cells stablytransfected with miR-199a or a control vector GFPwere seeded on tissue culture plates and culturedfor 2–3 days. Cell numbers were counted.Expression of miR-199a-3p enhanced cellproliferation. (B) Cell proliferation was alsomeasured using MTT assay kits. Cells wereincubated with MTT for either 0.5 or 1 hour. Cellsexpressing miR-199a-3p exhibited an increase ingrowth rate. Error bars represent s.d. (n=8);*P<0.01. (C) MDA-MB231 breast cancer cellsstably transfected with miR-199a or a controlvector GFP were seeded on tissue culture platesand cultured for 2–3 days. Cell numbers werecounted. Expression of miR-199a-3p enhanced cellproliferation. Error bars represent s.d. (n=6);*P<0.01. (D) MT1and MDA-MB231 cells stablytransfected with miR-199a, empty vector or anti-miR-199a-3p were seeded at a density of 1–4�105

cells per well in six-well plates, with 2 ml ofculture medium containing serum. Transfection ofanti-miR-199a-3p led to suppression ofproliferation. Error bars represent s.d. (n=9);*P<0.05 (E) Transient transfection of anti-miR-199a-3p plasmid into endothelial and breast cancercells for 48 hours led to suppression of cellsurvival. Error bars represent s.d. (n=9); *P<0.01.

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because these cells express low levels of miR-199a-3p to startwith.

To directly test whether or not miR-199a-3p was able to inhibitgene expression by binding to the 3�UTR of the target mRNA in asequence-specific manner, a luciferase construct, harboring twopotential binding sites for miR-199a-3p, was generated(supplementary material Fig. S5). In addition, a mutant constructwas produced wherein both potential target sites were mutated(Fig. 5A). Luciferase activity decreased significantly in the Luc-caveolin-transfected cells when co-transfected with miR-199a-3p.However, there was no significant difference in luciferase activitywhen cells were co-transfected with the mutated construct andmiR-199a-3p (Fig. 5B).

Effect of docetaxel on MT1 breast cancer cellsIt has been shown that caveolin-2 expression is associated withhighly aggressive tumors such as inflammatory breast carcinoma

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(Van den Eynden et al., 2006), basal-like (Savage et al., 2008) andtriple-negative breast carcinoma (Savage et al., 2008; Tan et al.,2008). Docetaxel has been shown to exert strong cytotoxic effectson advanced breast cancer cells (Fumoleau et al., 1996). Therefore,we investigated whether overexpression of miR-199a-3p andsilencing of caveolin-2 could modulate the response of tumor cellsto anticancer drug treatment.

Sensitivity to docetaxel was assessed in MT1 cells stablytransfected with vector and antisense miR-199a-3p. Lowconcentrations of docetaxel were able to induce significant growthinhibition in cells transfected with either the empty vector orantisense miR-199a-3p. A high proportion of detached cells wereobserved after exposure to 1 M and 2 M docetaxel. The vector-transfected cells showed higher sensitivity to the drug than cells-transfected with the antisense construct (Fig. 6A). Theanti-miR-199a-3p- and vector-transfected cells were also treatedwith 2 M docetaxel for 0, 24, 48 and 72 hours. This time-course

Fig. 3. Role of miR-199a-3p in the expression ofcaveolin-2. (A) Computational analysis indicatedthat miR-199a-3p potentially targets rat caveolin-2at nucleotides 627–649 and nucleotides 664–684.(B) miR-199a-3p was also found to target humancaveolin-2 at nucleotides 887–918 and nucleotides1476–1500. (C) Cell lysate was prepared fromvector (GFP)- and miR-199a-transfected YPEN-1and MB-231 cells, followed by western blotanalysis probed with anti-caveolin-2 antibody.Expression of miR-199a repressed caveolin levels.(D) YPEN-1 and MB-231 cells were transfectedwith miR-199a and an empty vector, whereas MT1cells were transfected with anti-miR-199a and anempty vector. Cells were stained for caveolin-2expression (arrows). The miR-199a-transfectedcells expressed lower levels of caveolin-2 than thevector-transfected cells did, whereas expression ofthe anti-miR-199a construct promoted caveolin-2expression. (E) The cell lysate from C was alsoanalyzed by western blot probed with anti-Cdc42antibody. Expression of miR-199a increasedCdc42 levels.

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experiment showed that the cytotoxic effect of docetaxel (2 M)was highly pronounced in the vector-transfected cells comparedwith the cells transfected with the antisense construct. Expressionof the anti-miR-199a construct enhanced cell survival(supplementary material Fig. S6A).

Next, we analyzed apoptosis and cell cycle profiles of cellstreated with docetaxel. Annexin-V staining demonstrated that mostof the apoptotic cells in the detached fraction (Annexin-V positive,PI negative and Annexin-V positive, PI positive) were confined tothe vector-transfected cells and not in the antisense-transfectedcells (Fig. 6B). 14.4±1.02% of the vector-transfected cells weredetected in the G2–M phase. This was accompanied by a prominentincrease in sub-G1 apoptotic cells to 58±1.02%. The sub-G1population was represented by cells displaying internucleaosomalDNA fragmentation, as well as chromatin condensation anddisintegration, which are all typical features of apoptotic cells. Bycontrast, 27.8±0.9% of the antisense-transfected cells were foundin the G2–M phase and the sub-G1 population was lower (34.4± 0.8%). These results revealed that docetaxel at aconcentration of 2 M could effectively arrest both vector-transfected and anti-miR-199a-3p-transfected MT1 cells at the G1and G2–M phases. The dramatic increase in sub-G1 accumulationseen in vector-transfected cells that expressed high levels of miR-199a-3p and low levels of caveolin-2 demonstrates that they aremuch more sensitive to docetaxel treatment compared withantisense-transfected cells with reverse expression of miR-199a-3p

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and caveolin-2. As a result, docetaxel treatment produced moreapoptotic cells in the vector-transfected cells than in the anti-miR-199a-3p-transfected cells (Fig. 6C).

Effect of caveolin-2 suppression on cell proliferativeactivity and cell survivalTo demonstrate that high proliferative activity and survival in cellsare caused by suppression of caveolin-2, four different siRNAscomplementary to caveolin-2 (siRNA-Cav2) were synthesized andtransfected in YPEN-1 cells (supplementary material Fig. S6B).We observed that all siRNAs were able to suppress expression ofcaveolin-2 (Fig. 7A) and enhance cell proliferation (Fig. 7B).These results suggest that caveolin-2 has an important role inmediating the effect of miR-199a-3p on cell activities.

We then performed rescue experiments by cloning and generatingan expression construct of caveolin-2 in the expressing vectorpcDNA3, producing the construct pcDNA3-Cav-2. Expression ofpcDNA3-Cav-2 plasmid was confirmed by western blotting ofcells transfected with the plasmid (supplementary material Fig.S6C). Analysis of cell proliferation and survival in serum-freemedium indicated that exogenous expression of caveolin-2 in themiR-199a-transfected cells decreased cell growth (Fig. 7C) andsurvival (Fig. 7D). However, transfection with caveolin-2 or anti-miR-199a-3p construct increased caveolin-2 expression, whichresulted in decreased sensitivities of the cells to docetaxel treatment.Cells expressing increased levels of caveolin-2 had a smaller

Fig. 4. Relationship of miR-199a-3p and caveolin-2 expression.(A) Expression of miR-199a-3p was analyzed by real-time PCR in anumber of cancer cell lines as indicated. Error bars represent s.d. (B) Expression of caveolin-2 and Cdc42 were analyzed in cancer celllines by western blot probed with anti-caveolin-2 and anti-Cdc42antibodies. (C) Cell lysates prepared from the vector (GFP)-, miR-199a- and anti-miR-199a-transfected YPEN-1 and MT-1 cells weresubjected to western blot analysis probed with anti-caveolin-2antibody. Whereas expression of miR-199a repressed caveolin-2levels, expression of the anti-miR-199a construct increased caveolin-2 levels.

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population of apoptotic cells (Fig. 7E) and sub-G1 cells (Fig. 7F).These results suggest that the slower growing cells are moreresistant to docetaxel-induced cell death.

DiscussionMicroRNAs have an important role in regulating cellular activitiessuch as proliferation, morphogenesis, apoptosis and differentiation.They can profoundly affect the expression of a large number ofgenes that encode proteins either by degradation of mRNA throughthe RNA interference pathway or by inhibiting protein translationby partially pairing with the 3�UTR of target mRNAs. Recentstudies have shown alterations in miR-199a-3p expression invarious human cancers. MiR-199a-3p has been found to be highlyexpressed in ovarian and breast cancer (Chen et al., 2008), butsignificantly under expressed in hepatocellular carcinoma(Murakami et al., 2006) and in bladder cancer (Ichimi et al., 2009).Moreover, it was reported that miR-199a-3p is involved in tumorprogression and chemoresistance in ovarian cancer by regulatingIKKb expression (Chen et al., 2008). Overexpression of miR-199a-3p was found to inhibit the invasiveness of tumor cells (Kimet al., 2008) and to modulate their sensitivity to doxorubicin-induced apoptosis in hepatocellular carcinoma (Fornari et al., 2010)by targeting the Met proto-oncogene.

In this study, we found that expression of miR-199a-3p promotedproliferation and survival of endothelial cells as well as breastcancer cells. To identify the potential targets of miR-199a-3p thatwould facilitate cell growth, we used bioinformatic search tools.Computational screening showed that there were two potential

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target sites of miR-199a-3p in the 3�UTR of rat and humancaveolin-2. Caveolin-2 is a member of a family of scaffoldingproteins that coat plasma membrane invaginations (caveolae).There are three members within the caveolin protein family:caveolin-1, caveolin-2 and caveolin-3 (Williams and Lisanti, 2004).Caveolin-2 is usually co-expressed with caveolin-1 in most celltypes and tissues, whereas caveolin-3 expression is restricted tomuscle cell types (Williams and Lisanti, 2004). Being key structuralproteins that organize caveolae, caveolins have been shown tohave an important role in regulating endocytosis and various aspectsof cellular signaling (Krajewska and Maslowska, 2004; Williamsand Lisanti, 2004). Caveolins interact directly with a number ofcaveolae-associated signaling molecules. In many of these cases,it has been documented that caveolin-binding can effectively inhibitthe enzymatic activity of these signaling molecules in vitro. Relativeto caveolin-1 and caveolin-3, the functional role of caveolin-2 isless defined. Recent studies have demonstrated cell- and tissue-specific roles of caveolin-2. For example, obvious pulmonarydefects and cell hyperproliferation in caveolin-2-knockout micesuggest that caveolin-2 is involved in regulation of lung endothelialcell proliferation and differentiation. There is also evidence thatcaveolin-2 regulates proliferation of rat fibroblast cell line Hirc-B(Kim and Pak, 2005; Kwon et al., 2009a; Kwon et al., 2009b).Furthermore, there has been increasingly more coherent datasuggesting that caveolin-2 has oncogenic effects in cancers of thebreast (Elsheikh et al., 2008), prostate (Yang et al., 1998), bladder(Fong et al., 2003), oesophagus (Ando et al., 2007), thyroid,pancreas, and lung (Wikman et al., 2004). Caveolin-2 has beenshown to be expressed at high levels in normal breast-like cancerand in the majority of basal-like cancer (Perou et al., 2000), but atlow levels in non-microdissected breast cancer. The caveolin-2levels were inversely correlated with tumor size (Sagara et al.,2004). These findings indicate that expression of caveolin-2 mighthave an important role in breast cancer tumor progression andmight be correlated with tumor grade and aggressiveness. Itsuggests that repression of caveolin-2 expression by miR-199a-3pcould have a crucial role in cancer development.

Consistent with this, it is interesting that we observed noticeablepromotion of cell proliferation and survival accompanied byinhibition of caveolin-2 expression in cells transfected with miR-199a-3p compared with vector-transfected cells. The potentialinteraction between caveolin-2 and miR-199a-3p was confirmedby reversed expression of miR-199a-3p and caveolin-2 in a numberof cell lines including A431, A549, C8186, HepG2, OV2008,HT1080, Jurkat, MT1 and A2058. Also, when the antisenseconstruct anti-miR-199a-3p was transfected into MT1 cells, whichexpress extremely high levels of miR-199a-3p, immenseupregulation of caveolin-2 expression was detected followed bycell growth inhibition. However, anti-miR-199a-3p was not detectedto affect MB-231 cells, possibly because MB-231 cells expresslow levels of miR-199a-3p. Finally, MiR-199a-3p was confirmedto inhibit both endogenous caveolin-2 activity and exogenouscaveolin-2 activity by using a reporter construct bearing the 3�UTRof caveolin-2.

In addition, we found that inhibition of caveolin-2 expression bymiR-199a-3p can modulate sensitivity of breast cancer cells todocetaxel-induced apoptosis. This is consistent with reported resultsshowing that caveolin-2 expression is associated with highlyaggressive tumors such as inflammatory (Van den Eynden et al.,2006), basal-like (Savage et al., 2008) and triple-negative breastcarcinoma (Savage et al., 2008; Tan et al., 2008).

Fig. 5. Luciferase activity assays. (A) The seed regions of the miR-199a-3ppotentially target sites in rat caveolin-2. Mutated regions are shown in bold.(B) Luciferase activities decreased significantly in the Cav2-luc-transfectedcells when co-transfected with miR-199a-3p in three independent experiments(Assay 1, 2 and 3). The inhibition of luciferase activities by miR-199a-3p wasabolished when the potential target sites were mutated. Error bars represents.d. (n=3); **P<0.01.

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It has been shown that the level of caveolin-2 is high in MCF7drug-resistant breast cancer cells (Lavie et al., 1998). However,knockdown of caveolin-2 hinders the development of drug resistance,which was demonstrated in adenocarcinoma A549 cells. Currently,one of the most effective treatments for advanced breast cancertargets microtubules. Microtubules are polymerized tubulin structuresthat make up the cytoskeleton, and are involved in cell locomotion,movement of organelles and mitosis. Both actin filaments andmicrotubules are implicated in membrane trafficking in mitotic cells(Mundy et al., 2002). Docetaxel has been shown to exert strongcytotoxic effects on advanced breast cancer cells (Fumoleau et al.,1996). At the molecular level, docetaxel acts by stabilizing tubulinheterodimers, and impairing mitosis and cell proliferation in tumors

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(Jordan et al., 1996). The drug also has been implicated in theinduction of apoptosis through activation of components in signaltransduction cascades. Recent data indicates a connection betweencytoskeletal components and caveolae-regulated cellular events suchas endocytosis (Parton and Richards, 2003), calcium homeostasis(Isshiki and Anderson, 2003), skeletal muscle transverse tubuleformation (Parton et al., 1997), and compartmentalization of receptor-mediated signaling components (Steinberg and Brunton, 2001).According to our results, an increased sensitivity of breast cancercells to docetaxel was observed upon both overexpression of miR-199a-3p and inhibition of caveolin-2 expression.

In summary, our data demonstrate that miR-199a-3p can enhanceproliferation and survival in endothelial cells, as well as in breast

Fig. 6. Effect of docetaxel onmiR-199a-3p-mediated cellactivity. (A) Sensitivity todocetaxel (0–20 M) wasassessed in MT1 cells transfectedwith the vector or antisense miR-199a-3p. Low concentrations ofdocetaxel were able to inducesignificant growth inhibition inboth vector- and antisense-transfected cells after 24 hours ofexposure. The vector-transfectedcells, originally expressed highlevels of miR-199a-3p butshowed greater sensitivity to thedrug than cells transfected withthe antisense. Error bars indicates.d. (n=9); *P<0.01. (B) Apoptosis was evaluatedusing flow cytometry aftertreating MT1 cells with 2 Mdocetaxel for 48 hours. MoreAnnexin-V positive apoptoticcells were detected in the vector-transfected cells than in the anti-miR-199-3p-transfected cells. (C)DNA content was assessed ingrowing cells by PI staining andFACS analysis at 48 hours afterdocetaxel treatment. Percentageof MT1 cells arrested in G1 andG2–M phases as well as cell cycleprofile are shown. A prominentincrease in subG1 accumulationwas detected in the vector-transfected cells compared withcells transfected with anti-miR-199a-3p. Shown is arepresentation of threeindependent experiments.

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cancer cells. Expression of miR-199a-3p produced remarkableinhibition of endogenous caveolin-2 expression and exogenousexpression introduced by a reporter construct bearing mRNAencoding the 3�UTR region of caveolin-2. The finding of caveolin-2 as a target of miR-199a-3p is very important for a betterunderstanding of how this miRNA contributes to cancer development.

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Overexpression of caveolin-2 completely counteracted miR-199a-3p-mediated enhancement of cell proliferative activity, survival andsensitivity of tumor cells to anticancer drugs. In addition, our resultssuggest that targeting caveolin-2 by MiR-199a-3p could have animportant role in breast cancer tumor progression, which could bean interesting concept to apply to cancer intervention.

Fig. 7. Confirmation of miR-199a-3p function by targeting caveolin-2. (A,B) YPEN-1 cells transiently transfected with the siRNA or the control oligo were grownon six-well tissue culture dishes. Downregulation of caveolin-2 was confirmed by western blotting (A). Cell proliferation was assayed (B). Transfection with siRNAenhanced cell proliferation. Error bars represent s.d. (n=6). (C,D) YPEN-1 and MD-MB-231 cells stably transfected with miR-199a were transiently transfected withcaveolin-2 or a control vector, whereas MT1 cells stably transfected with GFP were transiently transfected with caveolin-2 or a control vector. Expression of caveolin-2was confirmed by western blot probed with anti-caveolin-2 antibody (C, top). The cells were grown on six-well tissue culture dishes, followed by proliferation (C,bottom) and survival (D) assays. Transfection with caveolin-2 reversed the effect of miR-199a resulting in reduced cell proliferation and survival. *P<0.01. Error barsrepresent s.d. (n=3). (E) MT1 cells stably transfected with GFP were transiently transfected with caveolin-2 or a control vector, whereas MT1 cells stably transfectedwith anti-miR-199a-3p construct were transiently transfected with the same vector. Apoptosis was evaluated using flow cytometry after the cells were subjected to 2M docetaxel for 48 hours. Cells transfected with caveolin-2 and anti-miR-199a-3p were less sensitive to docetaxel-induced cell death than cells transfected with theGFP control. (F) DNA content was assessed by FACS analysis on the same cells as in E with propidium iodide staining 48 hours after docetaxel treatment. There weresmaller sub-G1 populations in the cells transfected with caveolin-2 (59.5%) and anti-miR-199a-3p (50.6%) than in the cells transfected with the GFP control (78.9%).

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Materials and MethodsGeneration and expression of miR-199a-3p plasmidTo study the effect of miR-199a-3p on cell function, the miR-199a-3p plasmid wasgenerated by insertion of duplicate miR-199a-3p precursors into a mammalianexpression vector, BluGFP, which contained a Bluescript backbone, a CMV promoterdriving expression of green fluorescent protein (GFP), and a H1 promoter drivingmiR-199a-3p between the BglII and HindIII restrictions sites. In brief, the miR-199aprecursor, generated by PCR was ligated into the mammalian expression vectorBluGFP, as previously described (Lee et al., 2007). The sequence of the construct isgiven in Fig. 1A.

To define the role of endogenous miR-199a-3p in mediating cell activities, theanti-miR-199a-3p plasmid was engineered. It contained a short RNA sequencecomplementary to the hairpin sequence of miR-199a-3p molecule. By binding to themiR-199a-3p, the products of this plasmid inhibited miR-199a-3p functionality. Inbrief, two primers, antihsa-miR-199a* (5�-ccgggagatctaaccaatgtgcagactactgtattt -tggaaaagctttag; underlined is the anti-miR-199a-3p sequence) and EGFP-700R (5�-ctcctcgcccttgctcaccat), were synthesized. PCR was performed using these two primersand the plasmid BluGFP indicated above. The PCR product was digested with BglIIand NheI to get the insert, followed by ligation with the BglII- and NheI-digestedBluGFP plasmid. The resulting plasmid anti-miR-199a-3p expressed an anti-miR-199a-3p sequence and GFP unit.

Rat prostate endothelial cells (YPEN-1) and human breast carcinoma cells (MDA-MB-231) were obtained from the American Tissue Culture Collection (ATCC,Manassas, VA). MT1 cells were originally isolated from a breast cancer patient(Naundorf et al., 1992). YPEN-1 cells were grown in Iscove’s modified Dulbecco’smedium (IMDM), containing 5% fetal bovine serum (FBS), and antibiotics; MB-231and MT1 cells were cultured in DMEM medium supplemented with 10% FBS, andantibiotics in a 5% CO2 atmosphere at 37°C. 3�105 cells/well in six-well cultureplates were transfected with miR-199a or a control vector using LipofectamineTM

2000 (Invitrogen). Stable transfected cells were selected by G418 antibiotic(Calbiochem, San Diego, CA) at a final concentration of 400 g/ml. On day 15 aftertransfection, cells were assayed for reporter gene activity. To confirm overexpressionof both pre- and mature miR-199a-3p, RT-PCR was performed using RNA isolatedfrom cells stably transfected with miR-199a-3p and an empty vector.

Cell proliferation assaysMiR-199a- and GFP-transfected cells were seeded in six-well tissue culture platesat 1�105 cells per well. Cell numbers were counted on second and third days todetermine cell number. Cell proliferation was also determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma). MiR-199a- and GFP-transfected cells were grown in 96-well cell culture plates in 5%FCS medium and then the medium was replaced with 50 l of MTT reagent (2mg/ml) and incubated in 5% CO2 at 37°C for 0.5 and 1 hours. Afterwards, themedium was aspirated and 50 l of dimethyl sulfoxide (DMSO, Sigma) was addedto each well. The optical density was measured using a microplate reader.

For cell cycle analysis, the miR-199a- and GFP-transfected MT1 cells (3�105

cells/six-well tissue plate) were treated with 2 M docetaxel for 48 hours. The cellswere harvested, washed with ice-cold PBS, and resuspended in ice-cold 70% ethanolfor 30 minutes. The cells were treated with 10 g/ml of RNase at 37°C, then spundown and stained with 10 g/ml of PI for 30 minutes. The DNA content wasmeasured by flow cytometry with FlowJo software (FACS Calibur; Becton Dickinson,CA).

Cell survival assaysMiR-199a- and GFP-transfected YPEN-1, MD-MB231 and MT1 cells were seededat a density of 2–4�105 cells per well in six-well plates respectively. After 24 hours,cells were washed twice with serum-free culture medium, and then incubated inserum-free conditions. After 3 or 5 days, cells were harvested, and the number ofviable cells was determined with Trypan Blue staining.

MT1 cells stably transfected with GFP or miR-199a-3p antisense were cultured inDMEM medium supplemented with 10% FBS, and antibiotics. Docetaxel wasobtained from Sanofi-Aventis Canada at a concentration of 10 mg/ml (12.6 mM) andit was used to test the sensitivity of cells to the drug at scalar concentrations from 0to 20 M.

After treatment with 2 M docetaxel for 48 hours, the MT1 cells transfected withGFP and miR-199a-3p antisense were separately washed twice with cold PBS andstained with binding buffer containing fluorescein-labeled Annexin-V and withPropidium Iodide (PI) (APC Annexin V Apoptosis Detection Kit I, BDPharmingenTM) following the manufacturer’s instructions. The cells that were positivefor apoptosis (APC Annexin-V positive, PI negative or APC Annexin-V positive, PIpositive) were analyzed by flow cytometry.

Wound-healing assayCells were plated at 3�105/dish in 60-mm-diameter dishes. A plastic pipette tip wasdrawn across the center of the plates to produce clean 1-mm-wide wound lines afterthe cells had reached confluence. Cell movement into the wound area was examinedand photographed at different time points (3 and 24 hours) using a phase-contrastmicroscope. The distance between the leading edge of the migrating cells and theedge of the wound was measured to estimate cell mobility.

Putative target analysisTo examine expression of Cdc42 and caveolin-2 in miR-199a- and GFP-transfectedcells, 5�105 cells were plated in 100 mm cell culture plates and incubated for 48and 72 hours. After incubation, cells were collected and resuspended in Tris-HClbuffer, lysed using an equal volume of 4% SDS, and sonicated. Protein concentrationswere measured using protein assay reagents (Bio-Rad). Cell extracts were boiled for5 minutes and subjected to 12% SDS-PAGE, and electrophoretically transferred toa nitrocellulose membrane. Each membrane was incubated with monoclonal Cdc42and caveolin-2 (1:500, BD) and b-actin (1:10,000, Sigma) antibodies, washed withTTBS and incubated with secondary antibody conjugated with peroxidase. Thesignal was then detected using chemiluminescent detection system (Pierce).

To visualize caveolin-2 expression, immunocytochemistry was performed. YPEN-1, MD-MB231 and MT1 cells were seeded at a density of 2–4�105 cells per wellon glass coverslips placed in six-well plates. After 4 hours, cells were washed twicewith serum-free culture medium, and then were fixed with 2% paraformaldehyde.Cells were permeabilized with 0.5% Triton X-100, washed three times and incubatedfor 10 minutes with 0.6% H2O2 in TBS to quench endogenous peroxidase activity.To suppress non-specific binding of IgG, cells were incubated for 20 minutes in 10%goat serum in TBS and then with antibodies against caveolin-2 (1:200, BD) overnightat 4°C. After washing in TBS, cells were incubated with biotin-conjugated secondaryantibody for 45 minutes at 37°C, then with streptavadin–HRP and visualized withfresh DAB solution for 5 minutes. Counterstaining was performed with hematoxylin.Then slides were dehydrated with alcohols followed by processing in xylenes andpermanent coverslip mounting. Images of ten randomly selected fields were acquired.

Luciferase assayTo determine whether miR-199a-3p is able to repress gene expression by binding tothe 3�UTR of the target mRNA in a sequence-specific manner, two luciferaseconstructs were generated. One (Luc-Cav-2) contained the 3�UTR of caveolin-2obtained by PCR using two primers, ratCav2-N3�SpeI (5�-gggactagtacactcagacc-ccaggtc-3¢) and ratCav2-C3�XbaI (5�-gggaagctttctagattcttacgttgagtgttgcc-3¢). ThePCR products were digested with SacI and MluI and the fragment was inserted into aSacI- and MluI-digested pMir-Report Luciferase plasmid (Ambion). The other (Luc-Cav-mut) contained mutations at the putative binding site, which was generated withtwo primers, ratCav-2-N3�XbaI and ratCav-2-C3�SpeI-Mut (5�-gggactagtacactcagac-cccaggtccaaatatttggaatgacatcaactctgttgatgtgacacaaaagctgatgacatctgttc-3¢) with asimilar approach.

U343 cells, a cell line that is of high efficiency for gene transfection and resistanceto transfection-induced cell death, were used for luciferase activity assays as described(Jeyapalan et al., 2011; Lee et al., 2009). In brief, the cells were seeded onto 12-welltissue culture plates at a density of 1�105 cells/well and co-transfected with theluciferase report constructs and miR-199a-3p construct. The cells were then collectedby trypsin and lysed with lysis buffer from Luciferase Assay Kit (Promega). Cellswere centrifuged at 3000 r.p.m. for 5 minutes. Supernatant was transferred into ablack 96-well plate (3�10 l) for luciferase activity measurement and into atransparent 96-well plate (3�50 l) for b-gal activity determination. For the luciferaseactivity measurement, 70 l of luciferase assay reagent was added to each well andthe luciferase activity was detected by using microplate scintillation and luminescencecounter (Packard, Perkin Elmer). For the internal control of b-gal activities, 90 l ofassay reagent (4 mg/ml ONPG, 0.5 M MgSO4, b-mercaptoethanol and 0.4 M sodiumphosphate buffer) was added into each well. The plate was then incubated at 37°Cfor 30 minutes, or until a color change was detectable. The absorbance at 410 nmwas measured by using a microplate reader (Bio-Tek Instruments).

Rescue assaysTo confirm that the effects of miR-199a-3p on proliferative activity and survival arecaused by suppressing caveolin-2, rescue experiments were performed by usingpcDNA3-caveolin-2 plasmid construct. The coding sequence of caveolin-2 was clonedby PCR using two primers, ratCave2Kozak-EcoRI (5�-ggggaattcgccgccaccatggggctg-gagactgagaaggcc-3¢) and ratCaveC-XbaI (5�-gggtctagatcagtcatggctcagttg-3¢). Afterdigestion with restriction enzymes XbaI and EcoRI, the PCR product was inserted intoXbaI- and EcoRI-digested pcDNA3.1. YPEN-1, MD-MB231 and MT1 cells stablytransfected with miR-199a-3p and empty vector were seeded at a density of 2–4�105

cells per well in six-well plates. After 24 hours, cells were washed twice with serum-free culture medium, transfected with pcDNA3-Cav-2 using LipofectamineTM 2000,and subjected to proliferation and survival assays. Cell number was determined 48hours after transfection.

To confirm that miR199a-3p sensitizes cells subjected to docetaxel to apoptosisby suppressing caveolin-2, MT1 cells stably transfected with an empty vector andan antisense miR construct were transfected with pcDNA3-Cav-2 plasmid constructand pcDNA3 plasmid only using LipofectamineTM 2000 followed by treatment with2 M docetaxel. 48 hours after treatment, cells were analyzed for apoptosis and cellcycle profile. A western blot assay was performed to check for the presence of thedesired construct.

siRNA assaysTo confirm that caveolin-2 has a role in mediating cell proliferation, migration andsurvival, four different siRNAs complementary to caveolin-2 sequences weredesigned and synthesized by GenePharma (Shanghai, China). YPEN-1 cells were

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seeded at a density of 2�105 cells per well in six-well plates, washed twice withserum-free culture medium after 24 hours after cell inoculation, and incubated withdifferent siRNAs. The cells were harvested 48 hours after the treatment and subjectedto western blotting and proliferation assays. The viable cell number was determined.

The authors thank Gisele Knowles for assistance in flow cytometryand Jennifer Yang for editing the manuscript. This work was supportedby grants from Heart and Stroke Foundation of Ontario (NA6732) toB.B.Y., who is the recipient of a Career Investigator Award (CI 5958)from the Heart and Stroke Foundation of Ontario.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/16/2826/DC1

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