The introduction of the Simian virus 40 (SV40) early region, the telomerase catalytic subunit (hTERT) and an oncogenic allele of H-Ras directly transforms primary human cells. SV40 small T antigen (ST), which forms a complex with protein phosphatase 2A (PP2A) and inhibits PP2A activity, is believed to have a critical role in the malignant transformation of human cells. Recent evidence has shown that aberrant microRNA (miRNA) expression patterns are correlated with cancer development. Here, we identified miR-27a as a differentially expressed miRNA in SV40 ST-expressing cells. miR-27a is upregulated in SV40 ST-transformed human bronchial epithelial cells (HBERST). Suppression of miR-27a expression in HBERST cells or lung cancer cell lines (NCI-H226 and SK-MES-1) that exhibited high levels of miR-27a expression lead to cell growth arrested in the G0–G1 phase. In addition, suppression of miR-27a in HBERST cells attenuated the capacity of such cells to grow in an anchorage-independent manner. We also found that suppression of the PP2A B56γ expression resulted in upregulation of miR-27a similar to that achieved by the introduction of ST, indicating that dysregulation of miR-27a expression in ST-expressing cells was mediated by the ST–PP2A interaction. Moreover, we discovered that Fbxw7 gene encoding F-box/WD repeat-containing protein 7 was a potential miR-27a target validated by dual-luciferase reporter system analysis. The inverse correlation between miR-27a expression levels and Fbxw7 protein expression was further confirmed in both cell models and human tumor samples. Fbxw7 regulates cell-cycle progression through the ubiquitin-dependent proteolysis of a set of substrates, including c-Myc, c-Jun, cyclin E1 and Notch 1. Thus, promotion of cell growth arising from the suppression of Fbxw7 by miR-27a overexpression might be responsible for the viral oncoprotein ST-induced malignant transformation. These observations demonstrate that miR-27a functions as an oncogene in human tumorigenesis.
Simian virus 40 (SV40) is a well-characterized member of the polyomavirus family, with a 5-kb small double-stranded, circular DNA genome. The SV40 early region encodes three proteins through alternative splicing, the large T (LT), small T (ST) and 17 kT antigens (Shenk et al., 1976; Sleigh et al., 1978; Khalili et al., 1987; Zerrahn et al., 1993). Among these three early proteins, LT and ST have been intensively studied because they not only have important roles in viral transcription and replication but also participate in cell immortalization and transformation (Ali and DeCaprio, 2001; Rundell and Parakati, 2001). Coexpression of LT and the human telomerase catalytic subunit (hTERT) allows mammalian cells to evade cellular senescence and become immortal (Ali and DeCaprio, 2001). In addition, the coexpression of LT and hTERT with an oncogenic allele of the H-Ras gene can successfully induce cell transformation in rodent cell models (Michalovitz et al., 1987; Hirakawa and Ruley, 1988). However, human cells expressing LT, hTERT and H-Ras cannot grow in an anchorage-independent manner or form tumors in animals without the additional introduction of ST (Hahn et al., 1999, 2002; Yu et al., 2001). Several lines of evidence suggest that ST perturbs cellular targets that participate in human tumor development (Shenk et al., 1976; Crawford et al., 1978; Choi et al., 1988). The ability of ST to transform human cells requires binding to the abundant serine–threonine protein phosphatase 2A (PP2A) (Yang et al., 1991; Sontag et al., 1993). PP2A is a heterotrimer composed of a conserved catalytic C subunit, a structural A subunit and a regulatory B subunit. The A and C subunits each exist as two isoforms, whereas the B subunits fall into four families (Cohen, 1989). Previously, we demonstrated that the SV40 ST antigen, which formed a complex with PP2A and inhibits PP2A activity by replacing specific PP2A B subunits, has a critical role in human cell transformation (Chen et al., 2004). However, the precise mechanism by which ST induces human cell transformation remains unclear.
MicroRNAs (miRNAs) are non-coding small RNAs of ∼22 nt that regulate gene expression by targeting mRNAs in a sequence-specific manner, inducing translational repression or mRNA degradation (Bartel, 2004; Esquela-Kerscher and Slack, 2006). Recent report has revealed that mRNA degradation is predominant for reduced protein levels in mammalian cells (Guo et al., 2010). miRNAs have been estimated to regulate at least 20% to 30% of all human genes (Lewis et al., 2005). Each miRNA may target >200 transcripts directly or indirectly, and multiple miRNAs can act on a given gene (Lai, 2004). An emerging body of evidence suggests that miRNAs are often overexpressed or downregulated in a number of human malignancies, and these genes are thought to function as tumor suppressors or oncogenes (Ambros, 2004). Recent studies demonstrate that various viruses, including human T-cell leukemia virus type 1 and Epstein–Barr virus, disturb the physiological functions of host cells by altering cellular miRNAs expression, leading to changes in cell proliferation or to a malignant phenotype (Connolly et al., 2008; Godshalk et al., 2008; Pichler et al., 2008; Tomita et al., 2009). Based on these observations, we speculate that particular genes targeted by miRNA might be involved in the process of SV40 ST-induced cell transformation. Although human cancers rarely have a viral etiology, we believe that investigating of how tumor oncoproteins induce the transformation of human cells will lead to a greater understanding of the molecular events that constitute the malignant state. To identify the critical miRNAs that mediate the SV40 ST-induced cell transformation, we used miRNA microarray chips to investigate miRNA expression in human bronchial epithelial cells (HBE cells) expressing SV40 ST. Here, we found that elevated miR-27a expression played an important role in the perturbation of cellular functions induced by ST in a PP2A-dependent manner. Our findings contribute to the understanding of the epigenetic mechanisms of SV40 ST in the progression of cell transformation.
miRNA expression profiles at different stages of HBE cell transformation
Previously, we created HBE cells at different stages of the cell transformation (Pang et al., 2008). In brief, primary HBE cells were immortalized by expressing SV40 LT and hTERT (named as HBELH cells). HBER cells were created by introduction of an oncogenic version of H-Ras into HBELH cells. In agreement with previous studies, the expression of the oncogene H-Ras led to an increase in cell proliferation; however, these cells (HBER) were unable to grow in soft agar or form tumors in immunodeficient mice. Subsequently, HBERST cells generated by the additional introduction of SV40 ST into HBER cells grew in an anchorage-independent manner and formed tumors in immunodeficient mice. Therefore, these cell lines represent distinct stages of immortalized cells (HBELH), pretransformed cells (HBER) and transformed cells (HBERST).
To identify miRNAs involved in ST-induced cell transformation, we used microarray chips containing 856 miRNA probes to define miRNA expression profiles in HBELH, HBER and HBERST cells. The results revealed that the expression of 76 miRNAs was at least two-fold different in HBERST cells compared with HBELH cells (P<0.05) (Figure 1). These 76 miRNAs were subjected to a stepwise selection. To exclude the influence of oncogenic H-Ras expression, 61 miRNAs that were also significantly altered in HBER cells compared with HBELH cells were excluded from the list of candidate miRNAs. The remaining 15 miRNAs were reduced to six miRNAs after removing miRNAs with hybridization intensities (fluorescence signal on the microarray chip) lower than 500 in all three cell lines. Thus, six miRNAs (miR-20a, miR-27a, miR-374b, miR-1246, let-7d and let-7f) were identified as the candidates for mediating ST-induced malignant transformation (Supplementary Table S1). Among these six miRNAs, miR-20a and miR-27a were upregulated, whereas the remaining four miRNAs, miR-374b, miR-1246, let-7d and let-7f were downregulated. Next, we validated the differential expression of miRNAs observed in the microarray results by performing reverse transcription and quantitative real-time PCR (qRT–PCR) assays on these six miRNAs from three types of HBE cell lines. Consistent with the results of the miRNA microarray analysis, we confirmed that the levels of miR-20a and miR-27a were overexpressed in HBERST cells, whereas miR-374b, miR-1246, let-7d and let-7f were suppressed in HBERST cells compared with both HBELH and HBER cells (Figure 2a).
To exclude the possibility that alteration of miRNA expression was a consequence of malignancy rather than the cause, we used qRT–PCR to examine miR-20a, miR-27a, miR-374b, miR-1246, let-7d and let-7f expression in immortalized HBE cells expressing ST. We found that the introduction of ST in immortal HBE cells did not cause cell transformation and resulted in a 1.8-fold increase in miR-27a expression, a 1.9-fold increase in miR-20a and a 30% decrease in miR-1246 expression (P<0.05) (Figure 2b). However, we observed no changes in the expression of miR-374b, let-7d or let-7f, indicating that the expression of these three miRNAs was not directly affected by the expression of ST. These findings suggest that the aberrant expression of miR-20a, miR-27a and miR-1246 might be involved in the regulation of malignant cell transformation. Thus, miR-20a, miR-27a and miR-1246 were subjected to further functional tests (Figure 1).
Inhibition of miR-27a on cell proliferation
Since ST-induced cell transformation largely depends on its ability to stimulate cell proliferation (Rundell and Parakati, 2001), we examined whether changes in miR-20a, miR-27a or miR-1246 expression affected cell proliferation.
To this end, miR-20a antisense oligonucleotide (ASO), miR-27a ASO or miR-1246 mimic were transfected into the HBERST cell line. The effects of miRNA mimic and ASO on the expression of miRNAs were detected by qRT–PCR after transfection. The miRNA mimic negative control (NC) and the miRNA ASO NC were used to exclude off-target effect. As expected, 200 nM miR-27a ASO or 200 nM miR-20a ASO resulted in a 70% decrease of miR-27a or a 55% decrease of miR-20a expression 24 h after transfection. Meanwhile, treatment with 200 nM miR-1246 mimic induced a 22-fold increase in miR-1246 expression (Supplementary Figure S1). For functional test, we found that transfection of 200 nM miR-20a ASO or miR-1246 mimic into HBERST cells had no impact on cell proliferation compared with the control within 144 h. Interestingly, transfection of 200 nM miR-27a ASO inhibited cell proliferation by 41% compared with ASO NC (P<0.01) (Figure 3a). In addition, treatment of HBER cells with 200 nM miR-27a mimic increased cell proliferation by 60% compared with mimic NC (P<0.01) (Figure 3b). We also examined the effect of miR-27a ASO on the proliferation of cancer cells. The lung cancer cell lines, NCI-H226 and SK-MES-1 were selected as they exhibited high levels of miR-27a compared with immortal HBE cells (Supplementary Figure S2a). As shown in Figures 3c and d, miR-27a ASO inhibited cell growth by 34% in NCI-H226 cells and 32% in SK-MES-1 cells at 144 h, respectively, compared with ASO NC (P<0.01), suggesting that miR-27a acts as a negative regulator of cell proliferation. In concert with growth inhibition, cell-cycle analysis revealed that transfection of HBERST cells with miR-27a ASO arrested cells at G0–G1 phase (Figure 3e). These observations indicate that upregulation of miR-27a is responsible for SV40 ST-induced cell growth stimulation.
Suppression of miR-27a inhibits clonogenicity in vitro
We further assessed the impact of miR-27a suppression on ST-induced cell transformation. HBERST cells were transfected with miR-27a ASO or ASO NC and allowed to grow at very low density (100 cells/well). Notably, transfection with miR-27a ASO decreased the number of colonies by 33% compared with ASO NC treatment (Figure 4a). In addition to the colony formation assay, we found that miR-27a ASO dramatically repressed the growth of HBERST cells and the ability to grow in an anchorage-independent manner, as observed by a significant reduction in the number of colonies (Figure 4b). To further clarify the oncogenic effect of miR-27a, we investigated whether the transfection of miR-27a ASO is antagonistic to ST antigen-induced transformation of HBER cells. The result showed that pretreatment of HBER cells with 200 nM miR-27a ASO led to a decrease of 56% of colony number in soft agar induced by SV40 ST (Figure 4c). In contrast, overexpression of miR-27a in HBER cells resulted in a 1.8-fold increase of colony number compared with cells transfected by mimic NC (Figure 4d). Taken together, these results demonstrate that upregulation of miR-27a contributes directly to ST-induced malignant cell transformation.
miR-27a induction by ST antigen is PP2A dependent
Given the results that ST forms stable complexes with PP2A Aα subunit and this interaction leads to partial inhibition of PP2A in vitro (Pallas et al., 1990) and PP2A appears to be the relevant cellular target of ST necessary for cell transformation (Hahn et al., 2002), we investigated whether PP2A mediated ST-induced upregulation of miR-27a expression.
To address this issue, we first examined the role of PP2A enzymatic activity in the control of miR-27a expression. We treated cells with the PP2A inhibitor, okadaic acid to specifically inhibit the total PP2A activity. As shown in Figure 5a, inhibition of PP2A activity resulted in a 48% increase in miR-27a expression, suggesting that PP2A activity is a critical event in the control of miR-27a expression. Previously, we demonstrated that ST targets PP2A enzymatic complexes containing the B56γ subunit and that alteration in the expression of this subunit contributed to cancer development (Chen et al., 2004). We reasoned that particular B subunits might be involved in the regulation of miR-27a expression. To address this issue, we used retroviral vectors that drive the expression of a short-hairpin RNA (shRNA) targeting the PP2A B subunit (shB55α and shB56γ) or a control vector encoding a short-hairpin RNA specific for GFP (shGFP). The efficiency and specificity of these siRNAs had been demonstrated previously (Chen et al., 2004). We then infected HBELH cells with the shB55α, shB56γ or shGFP vector and generated the stable cell lines HBELH-shB55α, HBELH-shB56γ and HBELH-shGFP, respectively. We examined whether the silencing of the B55α or B56γ subunit had an impact on the expression of miR-27a. As a result, we found that depletion of PP2A B56γ subunit (HBELH-shB56γ cells) induced a 1.9-fold increase in the expression of miR-27a, similar to that achieved by the introduction of ST. In parallel, we detected a slight decrease in the level of miR-27a expression in HBELH-shB55α cells compared with the control (Figure 5a). These findings indicate that suppression of PP2A activity or dysfunction of PP2A B56γ subunit lead to stimulation of miR-27a expression.
To further demonstrate that PP2A B56γ subunit is functionally mediated ST-associated upregulation of miR-27a expression, we introduced a version of wild-type PP2A B56γ into HBERST cells and found that overexpression of PP2A B56γ induced a 31% decrease of miR-27a expression in HBERST cells (Figure 5c). Taken together, these observations reinforce the notion that the interaction between ST and PP2A leading to a displacement of specific regulatory subunit is critical for human cell transformation.
The Fbxw7 gene is a direct target of miR-27a
To determine the target gene controlled by miR-27a, we first searched three algorithm programs (PicTar, TargetScan Release 5.0 and miRBase Targets) to predict the targets of miR-27a. These programs suggested a great number of genes as possible targets of miR-27a. Among the predicted targets, several genes including FOXO1 (forkhead box O1), Myt-1 (myelin transcription factor 1), prohibitin, sprouty2 and ZBTB10 (zinc finger and BTB domain containing 10) have been previously identified as the targets of miR-27a (Mertens-Talcott et al., 2007; Guttilla and White, 2009; Liu et al., 2009; Ma et al., 2010). We focused on genes meeting the following criteria: (1) cancer suppressor genes; (2) ability to bind miR-27a (the free energy (Mfe) of binding of the target gene’s mRNA with miR-27a <−13.4 kcal/mol (Zhao et al., 2007)); and (3) genes that function in the regulation of cell proliferation. As a result, three genes, FOXO1, prohibitin and ZBTB10 mentioned above were found to meet these criteria. Besides these three targets, the cell-cycle regulatory protein F-box and WD repeat domain containing 7 (Fbxw7; also known as Cdc4 in Saccharomyces cerevisiae, Sel-10 in Caenorhabditis elegans or Ago in Drosophila melanogaster) was also selected because Fbxw7 has been reported as a tumor suppressor, the expression of which is generally suppressed in many tumors (Rhodes et al., 2007). In addition, all three programs predicted two high affinity binding sites of miR-27a (site 1: Mfe=−21.90 kcal/mol; site 2: Mfe=−20.30 kcal/mol) in the 3′-UTR of Fbxw7 mRNA. To compare the binding affinity of miR-27a with these four genes’ mRNAs including FOXO1, prohibitin, ZBTB10 and Fbxw7, we used a dual-luciferase reporter system to examine their luciferase activity. As shown in Supplementary Figure S3, all of 3′-UTR of four genes displayed significant affinity with miR-27a. However, the affinity of Fbxw7 mRNA with miR-27a is of the highest with 50% luciferase activity suppression compared with NC treatment, while other three showed 30–37% activity suppression. Therefore, we chose Fbxw7 for further analysis. To ensure the specificity of this interaction, three mutants of the Fbxw7 mRNA in 3′-UTR (named Mut-1, Mut-2 or Mut-1+Mut-2) were constructed that contained a 4-nt change in seed region of the target sequence (Figure 6a). As we expected, mutated both miR-27a-binding sites (Mut-1+Mut-2) at the 3′-UTR of Fbxw7 mRNA completely abrogated the effects on inhibition of luciferase activity (Figure 6b). Notably, each of the miR-27a-binding sites tested in the 3′-UTR of Fbxw7 mRNA appeared to be effective, as demonstrated by a decreased in luciferase activity of 30% in Mut-1 and 40% in Mut-2. These observations indicate that Fbxw7 is also an important target of miR-27a.
Since miRNAs suppress the expression of target genes through mRNA translational repression or degradation of mRNA, we examined the levels of Fbxw7 mRNA and protein expression upon treatment with an miR-27a mimic. The results showed that the level of Fbxw7 mRNA in HBELH cells transfected with 200 nM miR-27a mimic was decreased by 60% compared with mimic NC measured by quantitative RT–PCR, indicating that miR-27a regulates Fbxw7 expression by mRNA degradation (Figure 6c). Consistent with the reduction at the mRNA level, treatment of HBELH cells with 200 nM miR-27a mimic led to a 40% decrease in Fbxw7 protein expression (Figure 6c). In addition, decreases in Fbxw7 mRNA and protein expression of Fbxw7 were also observed in HBERST cells and lung tumor cells (Supplementary Figure S2b).
Fbxw7 has been defined as a tumor suppressor that regulates cell-cycle progression through the ubiquitin-dependent proteolysis of a set of substrates whose function is frequently perturbed in cancer cells. The loss and mutation of this gene is frequently detected in various types of cancers (Mao et al., 2004; Perez-Losada et al., 2005; Tan et al., 2008; Crusio et al., 2010). To address whether downregulation of Fbxw7 mimicked the effects from increase of miR-27a expression, we introduced specific siRNAs duplex targeting Fbxw7 into HBER cells. As shown in Figure 7a, three siRNAs successfully suppressed the expression of Fbxw7. To exclude the possibility of off-target effects, we selected two siRNAs (siFbxw7-1 and siFbxw7-2) that target the different region of the Fbxw7 to investigate the effect of suppressing Fbxw7 on cell proliferation. As a result, we found that suppression of Fbxw7 by both siFbxw7-1 and siFbxw7-2 in HBERST cells led to an increase (53% or 41%) in cell proliferation, indicating that maintenance of Fbxw7 expression is critical in acceleration of cell growth in ST-expressing cells (Figure 7b).
In addition, we noted that the targets of Fbxw7 (cyclin E1 and Notch 1) were also upregulated in ST-expressing cells (Figure 7c). The elevated expression of these Fbxw7 targets induced by ST expression was suppressed when miR-27a ASO was applied (Figure 7d). These observations demonstrate that suppression of Fbxw7 due to upregulation of miR-27a mediates ST-induced malignant cell transformation.
To further explore whether miR-27a expression is inversely correlated with Fbxw7 expression, we examined the levels of miR-27a and Fbxw7 protein expression in 10 paired lung tumors and adjacent normal lung tissues (the clinical pathological features of these samples are shown in Supplementary Table S2). We found that the levels of miR-27a were upregulated in 6 out of 10 pairs of lung tumors (Figure 8). Meanwhile, the levels of miR-27a and Fbxw7 expression were inversely correlated in 8 out of 10 pairs of lung tumors (Figure 8). Additionally, we found that the levels of PP2A B56γ were downregulated in six pairs of lung tumors. The inverse correlation of PP2A B56γ and miR-27a expression was also observed in 8 out of 10 pairs of lung tumors (Figure 8). Notably, the corresponsive expression of PP2A B56γ-miR-27a-Fbxw7 regulatory axis was found in 7 out of 10 pairs of lung tumors (Figure 8). Thus, we further confirm that Fbxw7 is one of the targets of miR-27a. ST antigen stimulates cell proliferation and induces cell transformation, at least in part by upregulation of miR-27a and suppression of Fbxw7 expression, and this action depends on regulation of PP2A pathway.
The viral oncoproteins SV40 ST has been demonstrated to perturb critical host cell pathways, facilitating the experimental transformation of mammalian cells (Sontag and Sontag, 2006; Sablina and Hahn, 2008). The study of SV40-induced transformation in human cell culture models has promoted the identification of pathways related to cancer development. The current study performed miRNA array testing on ST-expressing cells and identified miR-27a as a differentially expressed miRNA that contributes to ST-induced malignant cell transformation in a PP2A-dependent manner. Further functional studies revealed that the upregulation of miR-27a cooperates with hTERT, LT and H-Ras to make human cells transformed as measured by an increase in colony formation in soft agar. We also identify that the Fbxw7 is one of the targets of miR-27a. These observations support the notion that miR-27a functions as a tumor oncogene.
Previously, several groups have demonstrated that the interaction of ST with PP2A is necessary for the malignant transformation of human cells (Mumby, 1995; Yu et al., 2001; Hahn et al., 2002). For example, ST targeting of PP2A enzymatic complexes containing the B56γ subunit contributes to cancer development (Chen et al., 2004). In this study, we used miRNA microarray analysis to identify critical miRNAs mediating ST-induced cell transformation. miRNA expression and the effects on cell proliferation confirmed that miR-27a is an important epigenetic regulator involved in the promotion of the G1/S transition in ST-transformed cells. Upregulation of miR-27a mimicked the effect of ST expression on increase of cell proliferation and growing in an anchorage-independent manner. On the contrary, suppression of miR-27a resulted in inhibition of cell proliferation and a reduction in anchorage-independent growth, demonstrating that the effects of miR-27a on the regulation of cell proliferation underlie the mechanism of ST-associated tumorigenicity.
miR-27a is differentially upregulated in ST-transformed HBE cells. Suppression of miR-27a expression in transformed cells leads to inhibition of cell proliferation and anchorage-independent cell growth. These findings strongly support the conclusion that miR-27a is involved in the regulation of cancer development. Indeed, it has been reported that miR-27a dysregulated in many human cancers. For example, miR-27a accelerates breast cancer cell growth by targeting mRNAs of the cell-cycle-associated protein ZBTB10 and myelin transcription factor 1 (Mertens-Talcott et al., 2007). Recent studies have shown that miR-27a contributes to the transformation or maintenance of a malignant state in breast cancer cells or gastric adenocarcinoma by targeting mRNAs of the transcription factor FOXO1 (Guttilla and White, 2009) and the membrane protein prohibitin (Liu et al., 2009). In hepatocellular carcinoma cells, upregulation of miR-27a confers the cells resistant to apoptosis induced by transforming growth factor-β (Huang et al., 2008). Moreover, miR-27a modulates the malignant biological behavior of pancreatic cancer cells by targeting sprouty2 mRNA, a crucial molecule involved in the Ras/MAPK signaling pathway (Ma et al., 2010). In light of these findings, miR-27a is emerging as a potent tumor promoter that contributes to the development of human cancer.
ST stimulates cell proliferation and transforms many types of human cells. These biological functions are believed to be mediated by the interaction between ST and PP2A (reviewed in Arroyo and Hahn, 2005; Eichhorn et al., 2009). The alteration of the phosphorylation status of signaling molecules has been demonstrated to be the primary action of the ST antigen in cell transformation (Janssens and Goris, 2001; Moreno et al., 2004; Sablina and Hahn, 2008). Indeed, PP2A complexes formed by various B subunits confer specificity of substrates dephosphorylation (Ferrigno et al., 1993; Cegielska et al., 1994). Among them, several members of the B56 family have been reported to have important roles in control of PP2A potential tumor-suppressive activity by dephosphorylation of its target oncogene (Arnold and Sears, 2006; Margolis et al., 2006; Grochola et al., 2009; Shouse et al., 2010). However, the precise mechanisms by which inactivation of PP2A B subunits functions in neoplastic transformation is not yet clear (Chen and Hahn, 2003). In this study, we identify the novel epigenetic molecule miR-27a that acts as a mediator in SV40 ST-induced cell transformation. Given the result that the specific silencing of the PP2A B56γ subunit also triggered the elevation of miR-27a, we extrapolate that miR-27a expression is the downstream effector controlled by the interaction of ST and PP2A. To date, the functional loss of PP2A B56γ is believed to be the key factor in SV40 ST-induced tumorigenesis (reviewed in Eichhorn et al., 2009). PP2A B56γ subunit appears to be a negative upstream regulator controlling miR-27a expression. The disruption of the function of B56γ due to displacement by ST could account for the enhancement of miR-27a expression. The existence of corresponsive PP2A B56γ-miR-27a-Fbxw7 regulatory axis in human lung tumors further confirms that B56γ subunit is indispensable in ST-induced upregulation of miR-27a. Recently, we have demonstrated that the dysfunction of a novel GSK-3β-C/EBPα-miR-122-IGF-1R regulatory circuitry contributes to the development of hepatocellular carcinoma (Zeng et al., 2010). We identified that C/EBPα, the activity of which is controlled by the status of phosphorylation, directly binds to the promoter of the miR-122 gene and initiates transcription. Thus, we assume that perturbation of the dephosphorylation of crucial downstream factors by ST expression or B56γ dysfunction lead to the alteration of transcriptional activity by regulation of miR-27a expression. Taken together, these findings demonstrate that epigenetic alteration is involved in SV40-mediated transformation. The PP2A pathway is involved in the induction of miR-27a by ST.
The biological function of miRNAs is to regulate their targets by inducing degradation of the mRNA or by inhibiting mRNA translation, according to the degree of complementarity with the 3′-UTR of the target (Bartel, 2004; Engels and Hutvagner, 2006). To explore functional mechanism of miRNAs, it is important to identify their control targets. Here, we identified Fbxw7 as a novel target of miR-27a, confirmed by both cell models and human lung tissue samples. Fbxw7 mutations occur in various human cancer types with an overall mutation frequency of ∼6% (Akhoondi et al., 2007). In addition, the Fbxw7 locus is located at chromosome region 4q31.3, which is deleted in ∼30% of human cancers (Knuutila et al., 1999). Meanwhile, Fbxw7 expression has been found to be suppressed in a number of tumors (Rhodes et al., 2007). Indeed, Fbxw7 is an evolutionarily conserved E3 ubiquitin ligase that is thought to be involved in regulating cell growth and apoptosis by ubiquitination of cyclin E (Koepp et al., 2001), Notch 1 (Tsunematsu et al., 2004), c-Myc (Welcker et al., 2004), c-Jun (Wei et al., 2005) and other downstream target genes. Mutation of a single Fbxw7 allele was found to cooperate with p53 in tumor development, suggesting that it is a haploinsufficient tumor suppressor (Mao et al., 2004). Although the precise mechanism by which the suppression or functional defect of Fbxw7 leads to cancer needs further investigation, we discovered in this study a novel signaling pathway involving epigenetic modulation of Fbxw7 in human tumorigenesis.
Activation of miR-27a appears to be essential in mediating SV40 ST-induced malignant cell transformation. Although further study is required to explore the targets of PP2A B56γ complexes and their correlation with the activation of miR-27a, these studies demonstrate that epigenetic alteration and genetic dysfunction might work together to regulate critical pathways that contribute to the cell transformation.
Materials and methods
The HBE cell line was a gift from Dr DC Gruenert (University of California, San Francisco, CA, USA). Stable HBELH, HBER and HBERST cell lines were generated from HBE cells by infection with amphotropic retroviruses carrying hTERT, G12V H-Ras and SV40 ST as previously described (Pang et al., 2008). The human lung carcinoma cell lines, NCI-H226 and SK-MES-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).
Human cancer tissue specimens
Fresh human lung cancer tissues and matched adjacent non-tumor tissues were obtained from the Affiliated Cancer Hospital of Guangzhou Medical School and the Affiliated Cancer Hospital of Sun Yat-sen University. All tissue samples were confirmed by the pathologic examination. The human specimens were obtained according to consent regulations and were used in accordance with the policies of the Ethical Review Committee of Sun Yat-sen University.
miRNA microarray expression analysis
miRNA microarray analysis was performed by the LC Sciences Corporation (Houston, TX, USA). In brief, RNA samples were isolated, size fractioned and labeled with Cy3 or Cy5. The labeled samples were then hybridized to a dual channel microarray using μParaflo microfluidics chips (miR human 13.0) (LC Sciences). Hybridization images were collected using a GenePix 4000B laser scanner (Molecular Devices, Sunnyvale, CA, USA) and digitized using Array-Pro image analysis software (Media Cybernetics, Silver Spring, MD, USA). Raw data were normalized by the locally weighted scatterplot smoothing (LOWESS) method using the background-subtracted data. The microarray data have been deposited in the Gene Expression Omnibus database, http://www.ncbi.nlm.nih.gov/geo (accession no. GSE26166). A one-way analysis of variance test was performed to analyze the statistical significance of signal differences among the three cell lines, HBELH, HBER and HBERST.
Cell proliferation and cell-cycle analysis
For cell proliferation studies, cells were seeded in a 24-well plate and transiently transfected with miRNA ASO, miRNA mimic, mimic NC or ASO NC at the indicated final concentrations. Cells were transfected with miRNA mimic, miRNA ASO or siRNA twice at 0 and 48 h to sustain the transfection efficiency. The cell number was counted at 48, 96 and 144 h after transfection using a Z2 Particle Counter and Size Analyzer (Beckman-Coulter, Miami, FL, USA). Three independent experiments were performed. For cell-cycle analysis, 2 × 106 cells were seeded in 6 cm plates and transfected with miR-27a ASO or ASO NC. Twenty-four hours after transfection, cells were trypsinized, rinsed and collected by centrifugation. Cells were resuspended in staining solution and incubated at 37 °C for 15 min. Stained cells were analyzed on a FACS Calibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using Cell Quest software.
Cell line establishment
The plasmids (pMKO.1-shB55α, pMKO.1-shB56γ, pMIG-ST) were generously provided by Dr WC Hahn (Dana Farber Cancer Institute, Boston, MA, USA). To generate stable HBE-shB55α or HBE-shB56γ cells, each plasmid was transfected into 293T cells along with pCL-Ampho using calcium phosphate precipitation to generate retroviruses, and the viral supernatants were applied to infect HBE cells separately and followed by selection with puromycin (1 μg/ml).
Clonogenecity and soft agar assay
After transfection with miR-27a ASO or ASO NC, HBERST cells were plated at a density of 100 cells/well in a six-well plate and maintained in culture for 14 days. The medium was changed twice weekly during this period. At the end of the incubation, all dishes were fixed with methanol, stained with 10% aqueous Giemsa and scored for colony formation. For soft agar assay, 5 × 104 cells were plated in DMEM supplemented with 10% FBS in 0.4% agar (Sigma, St Louis, MO, USA) above a layer of 0.6% agar. Colonies were counted after 4 weeks of culture.
3′-UTR of Fbxw7 vector construction and luciferase reporter assay
The 3′-UTR of Fbxw7 (2387–3801 nt, NM_033632) containing two putative miR-27a-binding sites (2776–2783 nt; 3652–3659 nt) was cloned into the EcoRV and Xba1 sites of pGL3cm, generating the pGL3-Fbxw7-WT construct. Three types of pGL3-Fbxw7-Mut constructs containing the mutations located at miR-27a-binding sites were generated by site-specific mutagenesis as shown in Figure 6a.
For the luciferase reporter assay, HBELH cells were seeded at 1 × 104 cells/well in a 96-well plate 24 h before transfection. Cells were cotransfected with either 50 nM of miR-27a mimics or a NC, 50 ng of either pGL3-Fbxw7-WT or pGL3-Fbxw7-Mut, and 50 ng of pRL-TK (Promega, Madison, WI, USA) by lipofectamine 2000 (Invitrogen, Branford, CT, USA) for 48 h. Then, luciferase activity was measured using the dual-luciferase reporter system (Promega). pRL-TK was used as an internal control.
Data are expressed as the mean±s.e. of at least three separate experiments. The differences between groups were analyzed using SPSS 13.0. Unless otherwise indicated, the differences between two groups were analyzed using an unpaired Student's t-test and the differences among more than two groups were assessed using a one-way analysis of variance followed by a Mann–Whitney test. Differences were considered statistically significant at P<0.05.
F-box/WD repeat-containing protein 7
- HBE cells:
human bronchial epithelial cells
- hTERT :
human telomerase catalytic subunit
protein phosphatase 2A
quantitative real-time PCR
small-hairpin RNA interference against the B56γ subunit
SV40 small T antigen
- HBER cells:
cells expressing hTERT, the SV40 large T antigen (LT) and an oncogenic allele of H-Ras
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This work was supported by program of Distinguished Young Scholar of NSFC (30925029, 30925036), a Key NSFC Program (30630055) and NSFC (30800930, 30771832, 30901211), National Key Basic Research and Development Program (2010CB912803), National High Technology Research and Development Key Program of China (2008AA062504), Ministry of Health of China (200902006), the Fundamental Research Funds for the Central Universities (10ykjc05 and 10lgzd10), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme GDUPS (2010).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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