MiR-200c and HuR in ovarian cancer
© Prislei et al.; licensee BioMed Central Ltd. 2013
Received: 27 September 2012
Accepted: 30 January 2013
Published: 8 February 2013
MicroRNAs in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors. This study assessed the role of miR-200c as regulator of class III β-tubulin (TUBB3), a factor associated with drug-resistance and poor prognosis in ovarian cancer.
Expression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Stable overexpression of miR-200c was obtained in A2780 and Hey cell lines. Crosslinking-coupled affinity purification method and ribonucleic-immunoprecipitation assay were used to characterise the complexes between miR-200c, HuR and 3′UTR region of TUBB3 mRNA. Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients.
In a panel of ovarian adenocarcinoma cell lines, we observed a direct correlation between miR-200c expression and chemoresistance. In A2780 cells miR-200c targeted TUBB3 3′UTR, while a positive correlation was observed between miR-200c and TUBB3 expression in most of the other cell lines. Through the analysis of 3′UTR-associated complexes, we found that the miR-200c can increase the association of the RNA binding protein HuR with TUBB3 mRNA, whereas HuR binding enhanced TUBB3 mRNA translation. Most importantly, in our analysis on 220 ovarian cancer patients we observed that overexpression of miR-200c correlated with poor or good outcome depending on the cellular localization of HuR.
This study suggests a model for the combined regulatory activity of miR-200c and HuR on TUBB3 expression in ovarian cancer. When HuR is nuclear, high expression of miR-200c inhibits TUBB3 expression and results in a good prognosis, whereas when HuR occurs in cytoplasm, the same miRNA enhances TUBB3 expression and produces a poor outcome. These findings reveal the usefulness of multidimensional analysis in the investigation of the prognostic role of miRNA expression.
KeywordsOvarian cancer miR-200c Class III beta-tubulin HuR Predictive biomarkers
About 80% of patients with ovarian cancer respond to first-line platinum/taxane chemotherapy, but the majority who experience a relapse will be refractory to further treatment . Understanding the molecular events underlying relapse is essential for identifying patients at high risk of poor outcome. Several studies reported that miRNAs can act either as oncogenes or tumor suppressors . In this context, expression of the miR-200 family in particular (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) was linked with ovarian cancer in multiple reports with contradictory findings. In some studies, high expression levels of the miR-200 family were associated with early relapse and decreased overall survival [3–5], while in others the opposite effect of high miR-200 expression was reported [6–9]. The precise reasons underlying such contradictory findings remain unknown. The miR-200 family is known as the main suppressor of the epithelial-to-mesenchymal transition (EMT), a reversible embryonic program aberrantly activated in tumor progression and metastasis. While most carcinomas have a differentiated phenotype, ovarian cancer cells that have undergone EMT are more invasive and more aggressive [10, 11]. For miR-200s to function as inhibitors of EMT seems difficult to reconcile with observations that the miR-200 family of miRNAs is overexpressed in aggressive ovarian cancers [3–5], so the designation of this family of miRNAs as drivers of biological aggressiveness and drug-resistance is a puzzle.
One of the validated targets of the miR-200 family is TUBB3 (class III β-tubulin) , whose overexpression has been reported in several malignancies, including ovarian cancer . Posttranscriptional inhibition of TUBB3 gene expression has been reported for miR-200c in Hey ovarian cancer cells and in HeC50 endometrial cancer cells [12, 14]. Forced expression of miR-200c in HeC50 cells and in a xenograft model reversed resistance to chemotherapy [12, 14, 15] suggesting that miR-200c is a factor protective against tumor aggressiveness and chemoresistance. We have shown previously that TUBB3 gene is conditionally expressed as an adaptive mechanism of resistance to low oxygen and glucose levels [16, 17], conditions correlated with aggressiveness in cancer . Interestingly, in hypoglycemic conditions, TUBB3 is induced by posttranscriptional regulation and enhanced by association with the RNA-binding protein (RBP) HuR to the 3′UTR region of the mRNA, but the role of miR-200c in this context is unknown.
The molecular mechanisms whereby HuR modulates translation are not fully clarified, although it is clear that HuR regulates numerous genes encoding proteins implicated in carcinogenesis . Nevertheless, an increasing number of studies have revealed that HuR can modulate gene expression through its interplay with miRNAs. Binding of HuR may suppress the inhibitory effect of miRNAs, although HuR can also synergize miRNAs to repress gene expression [20, 21].
This study aimed to clarify the role of the miR-200c as driver of biological aggressiveness in ovarian cancer. We noticed in vitro a direct correlation between expression of miR-200 family members and chemoresistance in most of the ovarian adenocarcinoma cell lines analyzed, and in particular we noted a clear correlation between miR-200c and TUBB3 expression. Using a multidimensional approach, we analyzed TUBB3 (gene and protein), HuR and miR-200c. The results suggested that the same miRNA, miR-200c, can act either as a suppressor or enhancer of the aggressive phenotype, depending upon the localization of HuR. This result offered a possible explanation for the discrepancies among the clinical reports describing miR-200c as a suppressor or enhancer of aggressiveness in solid malignancies.
Cell cultures and reagents
A2780, OVCAR-3, A2780-CIS, and A2780-ADR cells were purchased from the European Collection of Cell Cultures. TC1 is a clone derived from A2780 cells chronically exposed to paclitaxel . OVCAR-EPO cells correspond to OVCAR-EPO10 cell line obtained from OVCAR-3 cells as patupilone-resistant, while Hey-EPO are Hey-derived patupilone resistant cells. Culture media were selected according to the suggestions of European Collection of Cell Cultures. Growth experiments and transient transfection with siHuR and siC oligonucleotide duplex were performed and analyzed as previously described .
A 301-bp DNA fragment including the sequence of the pre-miR-200c (NT_009759) was amplified with the primers forward 5′-ACAAGCTTAGGAAGTGTCCCCAGGGACTCG-3′ and reverse 5′-AACTCGAGACGCTCTCAGCTCAAGACGAGG-3′ and cloned in pUSE(+) expression vector (Upstate Biotechnology), obtaining the pUSE-200c plasmid. The empty pUSE vector served as control. After electroporation, cells were selected in the presence of G418 (1.5 μg/mL) and when colonies appeared cloned at limiting dilution. Twelve clones were screened and those with the highest expression chosen for further analysis.
Real-time quantitative PCR, Western blotting and Immunohistochemistry
MiRNAs reverse transcription and PCR reactions were performed on Trizol (Invitrogen, Carlsbad, CA, USA) isolated total RNAs using TaqMan MicroRNA Assays kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR on mRNAs was performed as previously described . Western blots were done on total lysates or on nuclear/cytoplasmic fractions as previously described , with the following antibodies: anti-human TUBB3 polyclonal (1:1000, Covance, Princeton Township, NJ), anti-HuR (1:500, Santa Cruz, Santa Cruz, CA), anti-β-actin (1:5000, Sigma, Saint Louis, MO), anti-SNRP70 (1:1000, Abcam, Cambridge, UK), anti-GAPDH (1:5000, Abcam, Cambridge, UK). Blots were visualized by enhanced chemiluminescence procedures (Amersham, GE-Healthcare, Buckinghamshire, UK) as described by the manufacturers. The expression of HuR and TUBB3 was immunohistochemically assessed in a series of 220 ovarian cancers. Immunostaining for HuR was performed as previously described . For the analysis of the expression of TUBB3, antigen retrieval procedure was performed by microwave oven heating in 10mM citric acid, pH 6.0 (2 times for 4 min.). TUBB3 protein was identified after overnight incubation at 4°C by using the monoclonal antihuman antibody (clone TUJ1;1:300; Covance) in 20% normal goat serum. The En Vision-mouse+ System-HRP (DAKO, Carpinteria, CA, USA) was used. Diaminobenzidine was used as a chromogen (DAB substrate System, DAKO). Sections were counterstained with haematoxylin.
The 292-bp 3′UTR sequence of the TUBB3 gene (NM_006086) was cloned in the XbaI site of pGL3-Promoter Vector (Promega, Madison, WI), downstream of the firefly luciferase coding region, obtaining the pGL3-TUBB3-UTR construct. pGL3-TUBB3-UTRm vector was derived by inverse PCR on pGL3-TUBB3-UTR plasmid, resulting in a 10-bp deletion (GCAGTATTTA) which includes the seed sequence for miR-200c. The control vector pGL3-V was obtained cloning a 445-bp sequence from the MCS of pBluescript SK (nt 977–532) in the XbaI site of pGL3-Promoter Vector. A2780 cells were transfected with the described reporter vectors together with the renilla luciferase normalization plasmid (pRL-TK), using Transfectin (Bio-Rad, Hercules, CA). Cells were harvested 48 hours later for analysis using Dual Luciferase Reporter assay system (Promega, Madison, WI).
Purification of S1-tagged mRNPs and RIP assay
The UTR-S1 vector was obtained inserting in pUSE(+) expression vector (Upstate Biotechnology, Lake Placid, NY) the following sequences: 1) the coding sequence of the firefly luciferase and the TUBB3 3′UTR region, amplified from the described pGL3-UTR construct; 2) the linker sequence L1 amplified from pBluescript SK vector (nt 701–825); 3) the S1 aptamer sequence, obtained by annealing the primers tag-S1-F 5′-AATTCACCGACCAGAATCATGCAAGTGCGTAAGATAGTCGCGGGCC-3′ and tag-S1-R 5′-GGCCGCCCCGGCCCGCGACTATCTTACGCACTTGCATGATTCTGGT-3′. The UTRm-S1 vector differs from UTR-S1 for the mutation already described in pGL3-UTRm construct. The S1-tagged reporter constructs were stably transfected in A2780 cell line as described above and the expression of the exogenous mRNA was quantified by Q-PCR with primers LUC-F1 5′-CTTACTGGGACGAAGACGAACAC-3′ and LUC-R1 5′-GGGAAGACCTGCGACACCTG-3′. The purification of the S1-tagged mRNA/RBP was performed on cells treated with glucose-free medium for 48 hours, then incubated with 0.2% formaldehyde for the cross-linking and processed as described-by Vasudevan & Steitz . Western blot were performed with the antibody anti-Ago2 (1:250, Abcam, Cambridge, UK) and anti-HuR (1:500, Santa Cruz, Santa Cruz, CA). RIP was performed as described , with the modification of treating the cells in 0.2% formaldehyde before the harvesting, as above described for purification of S1-tagged mRNA/RBPs.
Nanofluidic analysis of micro-RNA and gene expression
FFPE samples were obtained from ovarian cancer that had been preserved between 2000 and 2008 following the approved Danbury Hospital Internal Review Board protocol. FFPE samples were cut to 10 μm thickness and two tissue slices were put into a 1.5 ml tube. One milliliter of xylene was added for deparaffinization followed by mixing twice with a high speed vortex for 3 min at room temperature. Total RNA was then automatically extracted with the QIAcube using the Qiagen miRNeasy FFPE kit (Valencia, CA) following manufacturers’ protocols. The RNA from the cell line A2780 was automatically extracted with the QIAcube using the Qiagen miRNeasy kit (Valencia, CA) following manufacturer’s protocols. RNA quantity and the quality were assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Analysis was carried out using the 48.48 dynamic array (Fluidigm Corporation, CA, USA) and a Biomark platform following the manufacturer’s protocol.
Overall survival (OS) and progression free survival (PFS) were calculated from the date of diagnosis to the date of progression/death or date last seen. Medians and life tables were computed using the product-limit estimate by the Kaplan-Meier method and the Wilcoxon test was employed only to assess statistical significance. Multivariate analysis assessed the clinical role of TUBB3, miR-200c, HuR pattern of staining in a model including additional significant variables in univariate analysis such as (age, stage and histotype) using the Cox proportional hazards model and nonparametric testing with the Kruskal Wallis test. T-test served to test differences of expression among different cells/conditions. A P value<0.05 was considered significant. Statistical analysis was carried out using JMP9 (SAS).
Expression of the miR-200c in drug-resistant ovarian adenocarcinoma cell lines
Q-PCR analysis of the miR-200c in the aforementioned cell lines revealed that this miRNA exhibited a higher expression with respect to the drug-sensitive isogenic cells, regardless of whether resistance to paclitaxel and cisplatin was inherent or acquired (Figure 1B). The only exception was the expression in the Hey cell line, which was approximately the same as in A2780 cells, while miR-200c expression was fourfold higher in Hey-EPO cells.
Correlation between miR-200c and TUBB3 expression in drug-resistant cell lines
Because TUBB3 is among the factors associated with drug-resistance in ovarian cancer, we set out to investigate the correlation between TUBB3 expression and expression of miR-200c in the same panel of cell lines. The highest TUBB3 mRNA and protein expression is observed in cell lines with native chemoresistance (OVCAR-3, Hey, SKOV-6, and OV2774 cells), but a marked increase in expression is also evident in A2780-derived cell lines with acquired drug-resistance (Figure 1C-D). OVCAR-EPO and Hey-EPO cells showed decreased TUBB3 expression compared with parental cells, as we reported previously, since epothilones seemed capable of selectively killing off those cells with high levels of TUBB3. Comparisons between TUBB3 and miR-200c expression revealed a direct correlation (R=0.49, p=0.03) in the majority of the cells analyzed. The only exception was in Hey and Hey-EPO cells, where an inverse correlation was observed between TUBB3 and miR-200c expression.
Since HuR is involved in the control of TUBB3 translation , HuR expression was analyzed in the same panel of cells. The total amount of HuR protein did not show evident correlation with TUBB3 expression in normoglycemia (Figure 1E). Nevertheless, the nuclear/cytoplasmic ratio of HuR in the different cell lines was different in normoglycemia, with A2780 exhibiting a prevalent nuclear HuR localization, while in OVCAR-3 the localization was mostly in the cytoplasm (Figure 1F).
Hypoglycemia and miR-200c control of TUBB3 expression
TUBB3 protein expression was also analyzed in the OVCAR-3 cell line, featured with high levels of TUBB3 and miR-200c (see Figure 1B-D). In this model, hypoglycemia induced a faint not significant increase (Figure 3A-B). This finding suggested that TUBB3 expression cannot be increased over a certain level in this cell line, at least in the conditions analyzed.
Endogenous miR-200c expression was assayed in A2780 and OVCAR-3 cells cultured in hypoglycemic conditions (Figure 3C). In A2780 cells miR-200c expression decreased, while in the same conditions it increased in OVCAR-3. Overall, these results suggested that A2780 and OVCAR-3 cell lines represent two different models, and prompted us to investigate the mechanism of miR-200c in regulation of TUBB3 expression, analyzing the involvement of additional factors.
Analysis of the interaction of miRNA-200c and HuR in modulation of TUBB3 expression
We reported previously that HuR mediates increased translation of TUBB3 through the 3′UTR region of TUBB3 mRNA . Since specific mRNAs can be relieved of miRNA-mediated suppression by HuR [20, 21, 28], we explored the possible interactions between HuR and miR-200c in modulating TUBB3 mRNA. The study of mRNA/protein complexes in vivo is often hampered by the rapidity of the interactions and by nonspecific binding of abundant RBPs. To overcome these limitations we set up an in vivo cross-linking–coupled RNA/RBP purification assay. We obtained a vector in which the TUBB3 3′UTR region was followed by an S1 sequence, an RNA aptamer which binds the streptavidin (UTR-S1, Figure 4A), along with the UTRm-S1 vector, in which the tagged 3′UTR sequence featured a mutation at the miR-200c interaction site (Figure 4A). After the stable transfection in A2780 cells and the treatment of the cells in hypoglycemic conditions, we checked that the expression of exogenous mRNAs was similar in A2780-UTR-S1 and A2780-UTRm-S1 cell lines in normoglycemia, as well as upon hypoglycemic treatment (Figure 4B). The analysis of specific RNAs and RNA-binding proteins associated in the S1-complexes was performed. We verified that the recovery of the exogenous mRNAs was similar in A2780-UTR-S1 and A2780-UTRm-S1 cell lines in normoglycemia, as well as upon hypoglycemic treatment (Figure 4C). The RNA polymerase II mRNA, analyzed as a negative control, was indeed detected at a much lower rate in the complexes (Figure 4C).
Western blot analysis revealed that HuR binding on UTR-S1 mRNA increased with hypoglycemic conditions, whereas binding on UTRm-S1 mRNA decreased under these conditions (Figure 4D). Ago2 binding was detectable in hypoglycemic conditions on UTR-S1 mRNA, but not on UTRm-S1 mRNA (Figure 4D). The S1-unbound samples were utilized as controls. MiR-200c was needed for the interaction of Ago2 with the TUBB3 3′UTR, since the deletion of its seed sequence prevented the association of any RISC containing Ago2, suggesting that miR-200c is the main miRNA interacting with TUBB3 3′UTR. What is more, these data support the notion that miR-200c interacted with the TUBB3 3′UTR in hypoglycemia and that such an interaction was important for HuR binding and increased expression of TUBB3 under stressing conditions. In this context, and at variance previous reports , miR-200c seemed to be capable of acting as a positive regulator of TUBB3 expression and as a factor determining biological aggressiveness.
Analysis of HuR, TUBB3, and miR-200c expression in 220 ovarian cancer patients
Clinical features of the analyzed setting of ovarian cancer patients
No. pts (%)
Adenocarcinoma (not specified)
625 U/mL (12.5-10000)
Median follow up (Alive)
Survival rates of different groups of patients
HuR C- TUBB3+ miR-200c-
HuR C- TUBB3+ miR-200c+
HuR C- TUBB3- miR-200c-
HuR C- TUBB3- miR-200c+
HuR N- TUBB3+ miR-200c-
HuR N- TUBB3+ miR-200c +
HuR N- TUBB3- miR-200c-
HuR N- TUBB3- miR-200c +
Survival rates at 5 years and Odds Ratio (OR) for different groups of patients obtained through COX regression multivariate model
HuR C- TUBB3+ miR-200c +
HuR C- TUBB3- miR-200c +
HuR N- TUBB3+ miR-200c +
HuR C- TUBB3+ miR-200c -
HuR N- TUBB3+ miR-200c -
HuR N- TUBB3- miR-200c -
HuR C- TUBB3- miR-200c -
HuR N- TUBB3- miR-200c +
In summary, the results of our study revealed that HuR plays a pivotal role in driving TUBB3 expression and the aggressive phenotype in ovarian cancer, through its interaction with miR-200c. We are confident that this discovery will foster the development of protocols specific to ovarian cancer patients. These findings suggest the complex HuR/miR-200c/TUBB3 as a new potential target for the development of therapies aimed at overcoming aggressive disease in ovarian cancer patients.
Quantitative real-time reverse transcription polymerase chain reaction
Hypoxanthine phosphoribosyltransferase 1
Epithelial to Mesenchymal Transition.
This work was partially supported by a Grant from AIRC (Associazione Italiana Ricerca sul Cancro IG11975), by the Ruth C. Donovan Cancer Research Program, and by a liberal donation from Mr. and Mrs. Ruggles. We thank Cristiana Gaggini and Flavia Filippetti for excellent technical assistance.
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