MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in gene regulation. We have previously reported that activin receptor-like kinase 7 (ALK7) and its ligand, Nodal, induce apoptosis in human epithelial ovarian cancer cells. In this study, we examined the regulation of ALK7 by miRNAs and demonstrate that miR-376c targets ALK7. Ectopic expression of miR-376c significantly increased cell proliferation and survival, enhanced spheroid formation and blocked Nodal-induced apoptosis. Interestingly, overexpression of miR-376c blocked cisplatin-induced cell death, whereas anti-miR-376c enhanced the effect of cisplatin. These effects of miR-376c were partially compensated by the overexpression of ALK7. Moreover, in serous carcinoma samples taken from ovarian cancer patients who responded well to chemotherapy, strong ALK7 staining and low miR-376c expression was detected. By contrast, ALK7 expression was weak and miR-376c levels were high in samples from patients who responded poorly to chemotherapy. Finally, treatment with cisplatin led to an increase in expression of mRNA encoding Nodal and ALK7 but a decrease in miR-376c levels. Taken together, these results demonstrate that the Nodal–ALK7 pathway is involved in cisplatin-induced cell death in ovarian cancer cells and that miR-376c enhances proliferation, survival and chemoresistance by targeting, at least in part, ALK7.
MicroRNAs (miRNAs) are single-stranded, small noncoding RNAs of 18–22 nucleotides in length, which have an important regulatory role in gene expression (Chendrimada et al., 2005; Hutvagner and Zamore, 2002; Lim et al., 2003; Lund et al., 2004). A major mechanism by which miRNAs regulate gene expression is via partial base pairing with the 3′-untranslated region (3′UTR) of the target mRNAs to repress their translation (Chekulaeva and Filipowicz, 2009; Seitz et al., 2003). MicroRNAs have been shown to regulate many developmental and physiological processes and their involvement in a variety of human diseases has been increasingly recognized (Ambs et al., 2008; Aqeilan et al., 2009; Johnston and Hobert, 2003; Packer et al., 2008; Sokol and Ambros, 2005; Wienholds et al., 2005; Zhao et al., 2005). Several lines of evidence have suggested that miRNAs are involved in cancer development. About 50% of annotated human miRNAs are located in areas of the genome, known as fragile sites, which are associated with cancer (Manikandan et al., 2008). In addition, many miRNAs are known to be up- or down-regulated in a variety of cancers (Jung et al., 2009; Ventura and Jacks, 2009) and can have either tumor-promoting or tumor-suppressing effects (Selcuklu et al., 2009).
Ovarian cancer is the sixth most commonly diagnosed cancer (Permuth-Wey and Sellers, 2009) and the fourth most common cause of cancer death among women in the world (Kumaran et al., 2009). There are three major types of human ovarian cancers: epithelial, stromal and germ cell. About 90% of ovarian cancers are epithelial ovarian cancer (EOC), which has a poor prognosis mainly because of late diagnosis and acquired chemoresistance (Williams et al., 2007). Recent studies have shown that miRNAs exhibit genomic alterations at a high frequency (Zhang et al., 2006a) and that their expression is deregulated in ovarian cancer (Dahiya et al., 2008; Taylor and Gercel-Taylor, 2008), strongly suggesting that abnormal miRNA expression constitutes a crucial step in ovarian cancer development. However, the role of miRNAs in ovarian cancer is not well understood.
The transforming growth factor-β (TGF-β) superfamily includes many structurally and functionally related polypeptide growth factors (Gordon and Blobe, 2008). These factors initiate their cellular effects by binding to cell surface receptors, including type I (activin receptor-like kinases ALK1–ALK7) and type II serine/threonine receptors. Binding of ligands to type II receptors results in conformational changes that induce complex formation with type I receptors. Subsequently, type I receptors are phosphorylated by type II receptors (Attisano and Wrana, 2002) and in turn induce downstream intracellular signaling cascades (Derynck and Zhang, 2003). We have previously demonstrated that Nodal acts through ALK7 (also known as activin receptor type-1C, encoded by ACVR1C) to inhibit proliferation and to induce apoptosis of epithelial ovarian cancer cells (Xu et al., 2004; Xu et al., 2006). Interestingly, Nodal and ALK7 are more effective at inducing apoptosis in chemosensitive ovarian cancer cells than in their chemoresistant counterparts, suggesting that the Nodal–ALK7 signaling pathway is involved in the development of ovarian cancer and/or chemosensitivity.
To explore the possibility that ALK7 expression is regulated by miRNAs, FindTar (Hua et al., 2006; Wang et al., 2008) was used to analyze the interaction between miRNAs and the 3′UTR of ALK7. Several miRNAs, including miR-376c (previously known as miR-368), were found to have potential complementary binding sites on the 3′UTR of ALK7 mRNA. MiR-376c belongs to an evolutionary conserved miRNA family which also includes miR-376a, miR-376a* and miR-376b, and these genes are found in a syntenic cluster on human chromosome 14 (Seitz et al., 2004). Although miR-376c was reported to be upregulated in a subset of acute myeloid leukaemia specimens (Dixon-McIver et al., 2008; Li et al., 2008), no target genes for miR-376c have been experimentally confirmed and its functions have not yet been reported.
In this study, we examined the effect of miR-376c on ALK7 expression and cell proliferation and apoptosis using ovarian cancer cell lines. We found that miR-376c increased cell proliferation and survival, as well as spheroid formation, in part by targeting ALK7. We have also provided evidence that the Nodal–ALK7 pathway is involved in cisplatin-induced ovarian cancer cell death and that miR-376c might promote chemoresistance.
miR-376c targets ALK7 3′ UTR to suppress its expression
To determine whether ALK7 can be regulated by miRNAs, FindTar was first used to predict miRNAs that could potentially target the ALK7 3′UTR. Several miRNAs, including miR-22, miR-122a, miR-147, miR-368, miR-376a, miR-376b and miR-412 were identified to have the potential target sites at the ALK7 3′UTR (supplementary material Fig. S1A). A reporter construct containing ALK7 3′UTR downstream of the luciferase coding sequence was then generated and luciferase assays were performed to determine the effect of these miRNAs. Among all miRNAs tested, only miR-368 was found to have an inhibitory effect (supplementary material Fig. S1B). When miR-368 was renamed miR-376c in 2007 and an additional ‘A’ was added to its sequence at the 5′ end, we repeated all experiments using miR-376c. Western blot analyses revealed that miR-376c, but not miR-22, decreased ALK7 protein levels. Similarly, two siRNAs designed to knock down ALK7 also inhibited ALK7 expression (Fig. 1A). However, mRNA levels of ALK7 were not affected by miR-376c (Fig. 1A). By contrast, anti-miR-376c increased ALK7 protein levels (Fig. 1A). Luciferase reporter assays showed that miR-376c (Fig. 1B), but not related family members, miR-376a, miR-376a* and miR-376b (Fig. 1C), suppressed the luciferase activity. Mutation of the miR-376c target sites at the ALK7 3′UTR significantly reduced the inhibitory effect of miR-376c on luciferase activity (Fig. 1D). In addition, miR-376c did not alter the luciferase activity of the control p-Mir-Report vector, which does not contain the ALK7 3′UTR sequence (supplementary material Fig. S2).
miR-376c promotes ovarian cancer cell proliferation, survival and spheroid formation
To determine the potential role of miR-376c in ovarian cancer development, we first determined its effects on cell proliferation and viability. When cells were transfected with miR-376c or an siRNA to knock down ALK7, there was an increase in cell density (Fig. 2A). Further analysis using WST-1 (Fig. 2B) and BrdU (Fig. 2C) assays revealed that overexpression of miR-376c or silencing of ALK7 expression, significantly increased cell viability and proliferation in two EOC cell lines, OV2008 and A2780s. The effect of miR-376c on cell viability could be inhibited by overexpression of ALK7 (Fig. 2D). Initial studies were performed using mock transfection, negative control siRNA and scramble negative control for miR-376c and no difference was observed between these controls (data not shown). Therefore, only the negative control siRNA was used for subsequent studies.
We then used an in vitro 3D culture model to examine the potential role of miR-376c in ovarian tumor formation. Overexpression of miR-376c in SKOV-3 cells resulted in the formation of larger spheroids with higher cell density (Fig. 3A). ALK7 siRNA also increased the size and density of spheroids (Fig. 3A). As shown in Fig. 3B, transfection of miR-376c or ALK7 siRNA significantly increased the size of spheroids, whereas ectopic expression of ALK7 significantly reversed the effect of miR-376c (Fig. 3C). Furthermore, inhibition of endogenous miR-376c by anti-miR-376c phenocopied the effect of ALK7 overexpression and decreased the size of spheroids (Fig. 3D).
miR-376c suppresses Nodal-induced apoptosis
We have previously shown that Nodal acts through ALK7 to induce apoptosis in ovarian cancer cells (Xu et al., 2004; Xu et al., 2006). Since miR-376c suppresses ALK7 expression, we tested whether it would block Nodal-induced apoptosis. An OV2008 cell line stably transfected with NODAL cDNA was generated, and expression of Nodal following doxycyclin withdrawal was confirmed by RT-PCR and western blotting (Fig. 4A). Using TUNEL assays, we found that the number of apoptotic cells increased significantly after induction of Nodal expression. However, this effect was blocked by transfection with miR-376c, NODAL siRNA and ALK7 siRNA (Fig. 4B). We also tested whether Nodal signaling through Smad2 could be blocked by miR-376c. Non-transfected cells or cells transfected with negative control, miR-376c, or ALK7 siRNA were treated with recombinant human Nodal (rhNodal) for 1 hour and protein lysates were analyzed for phosphorylated and total Smad2. Activation of Smad2 by Nodal was weaker in cells transfected with miR-376c and ALK7 siRNA than that in the mock or negative control cells (Fig. 4C). Similarly, treatment of rhNodal decreased cell viability, and this action was inhibited by transfection with miR-376c and ALK7 siRNA (Fig. 4D). Finally, miR-376c, ALK7 and NODAL siRNA also reversed the effect of Nodal overexpression on caspase 3 activity (Fig. 4E).
miR-376c blocks cisplatin-induced cell death
Because we have shown that the Nodal–ALK7 pathway might be involved in chemosensitivity (Xu et al., 2004), we next tested whether miR-376c, as well as ALK7 and NODAL siRNAs, would alter the sensitivity of cells to cisplatin. Overexpression of miR-376c significantly reduced the effect of cisplatin (Fig. 5A). Dose-dependent studies using WST-1 assays revealed that miR-376c increased, whereas anti-miR-376c decreased, the IC50 of cisplatin (Fig. 5B). Similarly to miR-376c, NODAL siRNA and ALK7 siRNA also inhibited cisplatin-induced cell death (Fig. 5C). Furthermore, miR-376c and siRNAs targeting Nodal and ALK7 also blocked the effect of another chemotherapeutic agent, carboplatin (Fig. 5D). However, overexpression of ALK7 partially rescued the cell survival effect of miR-376c (Fig. 5E). To confirm that miR-376c affects the sensitivity of ovarian cancer cells to cisplatin, we transfected OV2008 cells with different doses of anti-miR-376c and treated cells with cisplatin. As shown in Fig. 6A, anti-miR-376c further decreased total cell number in a dose-dependent manner. When anti-miR-376c was cotransfected with miR-376c, the survival-enhancing effect of miR-376c was completely abolished (Fig. 6B).
Differential expression of ALK7 and miR-376c in chemosensitive and chemoresistant ovarian tumors
To further assess the role of ALK7 and miR-376c in the chemosensitivity of ovarian cancer cells, we compared the expression of ALK7 and miR-376c levels in serous ovarian tumors taken from patients who subsequently underwent platinum-based chemotherapy and showed either complete (CR) or incomplete (IR) response to the therapy. Immunohistochemistry studies revealed that CR samples displayed numerous papillary structures that were clearly outlined by the ALK staining (Fig. 7A,C). By contrast, very week staining was detected for ALK7 in IR samples (Fig. 7B,D). The levels of miR-376c in these samples were inversely related to ALK7 staining intensity; IR samples showed a significantly higher miR-376c level than the CR samples (Fig. 7E).
Cisplatin stimulates Nodal and ALK7 expression
To further examine the involvement of Nodal–ALK7 pathway and miR-376c in cisplatin-induced apoptosis, we measured NODAL and ALK7 mRNA and miR-376c levels in cells treated with different concentrations of cisplatin. In OV2008 cells, mRNA levels of NODAL (Fig. 8A) and ALK7 (Fig. 8B) increased following cisplatin treatment. Time-course studies revealed that cisplatin treatment strongly induced Nodal mRNA expression over 3–24 hours (Fig. 8C). Interestingly, miR-376c levels were significantly decreased after cisplatin treatment (Fig. 8D).
It has been reported that miR-368, which is now known as miR-376c, is overexpressed in a subset of acute myeloid leukemia samples (Dixon-McIver et al., 2008; Li et al., 2008). It has also been shown that the miR-376 cluster transcripts can undergo adenosin-to-inosine editing in a tissue-specific manner, leading to the silencing of a different set of genes (Kawahara et al., 2007). However, target genes and functions of miR-376c have not been reported. In this study, we provide the first evidence that miR-376c exerts pro-survival functions in ovarian cancer cells by suppressing the expression of ALK7. Interestingly, we found that miR-376c and the Nodal–ALK7 pathway can modulate the sensitivity of ovarian cancer cells to chemotherapeutic agents, such as cisplatin and carboplatin.
Using bioinformatic tools, we first identified a number of miRNAs that potentially target the ALK7 3′UTR. However, only one out of the seven miRNAs tested was able to inhibit ALK7 expression in luciferase reporter assays and in western blot analyses. The effect of miR-376c on ALK7 is highly specific, because closely related family members, such as miR-376a and miR-376b, which differ from miR-376c by two or three base pairs, were unable to suppress ALK7 expression in the reporter assays. Furthermore, mutation of a single nucleotide in the targeting region of the ALK7 3′UTR significantly reduced the activity of miR-376c, whereas mutation of several nucleotides completely rendered miR-376c inactive. These findings confirm that ALK7 is a target of miR-376c.
In this study, we found that ectopic expression of miR-376c promotes cell proliferation, survival and spheroid formation. Consistent with our previous findings that Nodal inhibits proliferation and induces apoptosis via ALK7 (Munir et al., 2004; Xu et al., 2004; Xu et al., 2006), knockdown of ALK7 using siRNA also resulted in an increase in cell proliferation, viability and spheroid formation. Furthermore, we observed that the effect of miR-376c could be reversed by overexpression of ALK7. These findings further support the role of ALK7 in regulating cell proliferation and apoptosis and suggest that miR-376c promotes cell proliferation and survival, in part by inhibiting ALK7 expression.
Our studies provide evidence that the Nodal–ALK7 pathway is involved in cisplatin-induced cell death. We found that silencing of Nodal and ALK7 expression using siRNAs significantly reduced the effect of cisplatin on cell death. Furthermore, treatment with cisplatin increased NODAL and ALK7 mRNA levels. These findings strongly suggest that the Nodal–ALK7 pathway has an important role in determining sensitivity of ovarian cancer cells to cisplatin. The TGFβ signaling pathway has been previously implicated in drug resistance in cancer cells. For example, breast cancer cells that are resistant to the growth inhibiting effect of TGFβ1 were also resistant to cisplatin (Stoika et al., 2008). In addition, cisplatin and other anti-cancer drugs induce the production of TGFβ1 in breast cancer cells (Stoika et al., 2003). Moreover, TGFβ1 sensitizes oral cancer cells to cisplatin treatment (Thavaraj et al., 2005). Finally, Mullerian-inhibiting substance enhances the effect of chemotherapeutic agents on ovarian cancer cells (Pieretti-Vanmarcke et al., 2006). The mechanism by which the Nodal–ALK7 pathway enhances chemosensitivity is not known. However, it is possible that this might involve a downregulation of survival pathways. Nodal and ALK7 have been reported to inhibit Akt activity (Wang et al., 2006; Zhang et al., 2006b), as well as the expression of X-linked inhibitor of the apoptotic protein (Xiap) (Xu et al., 2006). Since it has been well documented that Akt and Xiap promote chemoresistance in ovarian cancer cells (Asselin et al., 2001; Fraser et al., 2008; Sasaki et al., 2000; Yang et al., 2006), an inhibition of Xiap and Akt by the Nodal–ALK7 pathway would enhance the effect of cisplatin.
Several studies have suggested that miRNAs are novel players in the development of chemoresistance. MicroRNAs are differentially expressed in chemosensitive and chemoresistant cells (Boren et al., 2009; Xia et al., 2008). MicroRNA-98 and miR-21 have been shown to potentiate chemoresistance (Hebert et al., 2007; Moriyama et al., 2009). In this study, we found that forced overexpression of miR-376c inhibited the effects of cisplatin, whereas inhibition of endogenous miR-376c by anti-miR-376c sensitized the cells to cisplatin. Importantly, analysis of miR-376c and ALK7 levels in tumor samples revealed that ALK7 levels were higher in chemosensitive tumors than in their chemoresistant counterparts, whereas miR-376c exhibited the opposite expression pattern. Moreover, cisplatin significantly reduced miR-376c levels. These findings suggest that the miR-376c level is a determinant of the responsiveness of ovarian cancer cells to chemotherapeutic agents. Future studies using a larger set of tumor samples might reveal whether or not ALK7 and miR-376c could be used as prognostic markers to predict responsiveness to chemotherapy. The cisplatin-blocking effect of miR-376c is mimicked by siRNA targeting ALK7 and partially rescued by overexpression of ALK7, indicating that miR-376c promotes chemoresistance in part by downregulating ALK7.
In summary, we have demonstrated that miR-376c targets ALK7 expression to promote proliferation and survival of ovarian cancer cells. We have also provided evidence that the Nodal–ALK7 pathway and miR-376c are involved in cisplatin-induced cell death. Future in vivo studies will further clarify the role of miR-376c in ovarian cancer development and chemoresistance.
Materials and Methods
Cell lines, expression plasmids and transient transfection
Human ovarian cancer cells, OV2008 and A2780s were cultured as described previously (Xu et al., 2004; Xu et al., 2006). Generation of Nodal and ALK7 expression plasmids was also as described (Xu et al., 2004). A cDNA fragment representing nucleotides 87–756 of the human ALK7 3′UTR, which contains the predicted ALK7 targeting site was obtained by RT-PCR and cloned into pMIR-Report (Ambion, Austin, TX) downstream of the luciferase coding sequence. Three mutants, each containing a point mutation or several mutations at the predicted miR-376c binding region, were created by site-directed mutagenesis using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Transient transfection of plasmids and siRNA or miRNA duplexes was carried out using Lipofectamine 2000 (Invitrogen, Burlington, ON) following the manufacturer's suggested procedure.
miRNAs and siRNAs
The FindTar algorithm (Hua et al., 2006) was first used to predict miRNAs that have potential binding sites at the ALK7 3′UTR. MicroRNA duplexes, non-targeting negative control duplexes, scramble miR-376c negative control, and siRNAs were purchased from GenePharma (Shanghai, China) (supplementary material Table S1). Anti-miR-376c and its negative control were purchased from Ambion (Applied Biosystems, Foster City, CA).
Luciferase reporter assays
Cells were seeded in 12-well plates at a density of 1×105 cells/well and transfected with 0.2 μg pMIR-Report-ALK7 and 0.02 μg pRL-TK internal control (Promega, Madison, WI) plasmids. Twenty-four hours after transfection, luciferase activities were measured using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
ALK7 antibody production and western blot analysis
Polyclonal antiserum against a peptide (SYRKKKRPNVEEPLSEC) corresponding to amino acids 142–158 of the human ALK7 sequence was synthesized by Alpha Diagnostic (San Antonio, TX) and four New Zealand white rabbits were inoculated for antibody production. The primary injection was performed using antigen mixed with complete Freund adjuvant. The same amount of antigen mixed with incomplete Freund adjuvant was injected for boosts. Following the second boost, the antiserum activities were examined by ELISA. After primary immunization and four additional injections, the antisera from all of the rabbits were screened, and the one with the highest titer was used for western blot detection of ALK7. For western blotting, protein lysates were separated in 12% SDS polyacrylamide gels, transferred to Immobilon PVDF Membranes (Millipore, Toronto, ON), and probed with anti-ALK7 (1:2000 dilution), anti-Myc (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), anti-total or phosphorylated Smad2 (1:1000, Invitrogen Canada, Burlington, ON), or anti-cleaved caspase3 (1:1000, Cell Signaling, Pickering, ON) antibodies. ECL kit (GE, Baie d'Urfe, Quebec) was used for chemiluminescent detection of immobilized proteins. Protein loading was evaluated using mouse anti-β-actin (Sigma, Oakville, ON). In some cases, the intensity of protein fragments was quantified using AlphaEaseFC imaging software.
Cell proliferation and viability assays
Cell proliferation and viability were determined using BrdU and WST-1 assays according to the manufacturer's protocols (Roche, Mississauga, ON). Briefly, cells were seeded on 96-well culture plates at a density of 10,000 cells per well and cultured for 24 hours before transfection. Transfection was performed with 200 nM miR-376C or its negative control and ALK7 siRNAs. Seventy-two hours after transfection, WST-1 or BrdU reagent was added to the cells and absorbance was measured at 450 nm using an ELISA plate reader. The viability and cytotoxicity of cells treated with rhNodal (500 ng/ml, R&D Systems, Minneapolis, MN) were also evaluated with the MultiTox-Fluor Multiplex Cytotoxicity Assay kit (Promega, Madison, WI). Live-cell and dead-cell protease activities were detected at 400Ex/505Em and 485Ex/520Em, respectively. Cell viability was assessed as the ratio of live and dead signals. Manual cell counting using DAPI staining was used to determine the effect of cisplatin. Briefly, cells were fixed with methanol containing 1 μg/ml DAPI. The DAPI stained cells were viewed at 100× magnification under fluorescent microscopy (Nikon Eclipse TE 2000-U). Five randomly selected fields in each sample were pictured, scored and averaged for cell number.
Hanging-drop culture for spheroid formation
SKOV-3 cells were cultured in M199 and MCDB105 (1:1) with 5% FBS. Cells were then transfected with miR-376c, ALK7 siRNA or negative control. At 24 hours after transfection, cells were trypsinized and resuspended in the same culture medium. Hanging-drop culture was performed using a method described for ovarian cancer cells (Zietarska et al., 2007) with slight modifications. Briefly, 15 μl droplets, each containing 10,000 cells, were plated on the inner surface of a Petri dish cover. Approximately 20 droplets from control and miRNA or siRNA transfected group were plated into one cover. The covers were then inverted and placed on a dish containing 15 ml PBS. After 4 days of culture, spheroids were photographed and their sizes were measured using the ImageJ software,
Generation of inducible Nodal-expressing stable cell line
An inducible stable cell line expressing Nodal containing a Myc tag was generated using the Tet-off advanced inducible gene expression system (Clontech, Mountain View, CA). OV2008 cells were first transfected with the pTet-off-advanced plasmid, and G418-resistant clones were selected. A luciferase reporter plasmid was used to screen clones that had low background and high doxycycline-dependent induction. These clones were then transfected with pTRE-Tight-Nodal and stable clones were selected with hygromycin. Cells were cultured in doxycycline-containing medium and at various time points following doxycycline withdrawal, RT-PCR and western blot analysis were performed to confirm induction of Nodal expression. A forward primer corresponding to the vector sequence and a Nodal specific reverse primer were used to detect transgene expression. Exogenous Nodal was detected using an anti-Myc antibody.
The inducible stable cell line expressing Nodal was seeded on 96-well plates at a density of 10,000 cells per well and cultured overnight. Doxycycline was then removed from the medium to induce Nodal expression. At 24 hours following the withdrawal of doxycycline, cells were transfected with 200 nM negative control, miR-376c or ALK7 and NODAL siRNAs. At 48 hours after transfection, apoptosis was determined by TUNEL method using the TMR Red kit (Roche, Mississauga, ON) according to the manufacturer's suggested protocol. Briefly, cells were rinsed with PBS, fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. They were then incubated in TUNEL reaction mixture at 37°C for 60 minutes in the dark. After three rinses with PBS, the samples were analyzed under a fluorescence microscope (NIKON ECLIPSE TE 2000-U) using an excitation wavelength of 540 nm and emission wavelength of 580 nm. Ten randomly selected fields in each sample were pictured, scored and averaged for cell numbers.
Treatment with cisplatin and carboplatin
Cells cultured in 24-well or 96-well plates were transiently transfected with negative controls, siRNA, miR-376c or anti-miR-376c as indicated in each experiment for 5 hours. Cells were then treated with different concentrations of cisplatin or carboplatin for 48 hours. At the end of the experiments, the number of live cells was determined by DAPI staining and manual cell counting or WST-1 assays. Dose-response curves were fitted and IC50 values were calculated using the Graphpad Prism program.
Human ovarian tumor samples were obtained from patients undergoing primary excision surgery at the Hospital of Obstetrics and Gynecology, Dalian, China, who then received platinum-based primary chemotherapy. The use of tumor samples was approved by the ethics committee at the hospital and all patients provided informed consent. Stage III serous carcinoma samples were selected from patients who had a complete response (CR) or incomplete response (IR) to chemotherapy. A CR was defined as a complete disappearance of all measurable and assessable disease or a normalization of the serum CA-125 level (< 30 U/ml) after chemotherapy. Patients were considered to have an IR if they demonstrated only a partial response, progressive disease during primary therapy and a higher level of the serum CA-125 (> 30 U/ml) after 6 months of chemotherapy. Seven CR and six IR samples were used in the immunohistochemistry staining to detect ALK7 expression and real-time PCR to determine miR-376c levels.
The paraffin-embedded tumor samples were incubated in primary antibody (anti-ALK7; 1:100 dilution) at 4°C overnight. After washing three times in PBS for 1 hour, the samples were incubated with the secondary biotin-related anti-rabbit antibody for 1 hour (ZSGB-BIO, China). The tissue sections were then incubated in ABC solution (Vectastain Kit) and further developed with DAB (ZSGB-BIO) color staining. Finally, the samples were counterstained with hematoxylin, dehydrated, mounted and photographed.
RNA extraction and real-time PCR
Trizol reagent (Invitrogen) was used to extract total RNA, including miRNAs, from culture cells following published protocols (Davis et al., 2008; Martello et al., 2007) with slight modifications. Specifically, one volume of isopropyl alcohol was added to the extracted aqueous phase and precipitation of RNA was carried out at −20°C overnight to enrich the small RNA population (Lin et al., 2009). RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion) was used to extract total RNA from paraffin-embedded ovarian tumor sections. Briefly, up to four 20 μm sections were deparaffinized using a series of xylene and ethanol washes. The tissue pellets were subjected to a protease digestion at 60°C for 2 hours. Nucleic acids were isolated by rapidly passing the mixture through a filter cartridge provided. Following removal of DNA by DNase digestion, RNA was eluted into nuclease-free water. One microgram of total RNA was polyadenylated and then reverse-transcribed using the QuantiMir RT Kit (System Biosciences). Forward primers specific for mature microRNAs (supplementary material Table S1) were used to detect the corresponding microRNA transcripts in real-time PCR using SYBR Green and Chromo4™ Real-time detection system (BioRad). The miR-376c expression levels were normalized to U6 snRNA transcript levels. For quantification of Nodal and ALK7 mRNA levels, cDNA was synthesized from 2.5 μg total RNA using oligo(T)23VN primer (Sigma). Quantitative RT-PCR was carried out using gene specific primers for Nodal and ALK7 (Table S1) and SYBR® Green Master Mix (Bio-Rad). The PCRs were carried out at 95°C for 4 minutes followed by 40 cycles of 20 seconds at 95°C, 30 seconds at 60°C and 45 seconds at 72°C. Expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were also determined and used as endogenous controls to normalize the expression levels of mRNA.
Unless indicated otherwise, data were converted to fold of control value, combined from several experiments and expressed as means ± s.e.m. Statistical significance was determined using one-way analysis of variance and Student–Newman–Keuls test for multiple group comparisons. Student's t-test was used for comparison between two groups. Statistical significance was defined as P<0.05.
G.Y. was supported in part by a fellowship from the Toronto Ovarian Cancer Network with funds raised by the Toronto Fashion Show and S.Z. was supported by a visiting scholarship from Hebei Province Government. C.P. is a recipient of a Mid-Career Award from OWHC/CIHR and B.B.Y. is a recipient of a Career Investigator Award (CI 5958) from the Heart and Stroke Foundation of Ontario. This work was supported by a CIHR grant (MOP-89931) to C.P.
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.072223/-/DC1
- Accepted September 23, 2010.
- © 2011.