MiR-152 and miR-185 co-contribute to ovarian cancer cells cisplatin sensitivity by targeting DNMT1 directly: a novel epigenetic therapy independent of decitabine

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Abstract

Epithelial ovarian cancer (EOC) is a highly lethal gynaecological malignancy. Cisplatin is the basal chemotherapeutic agent used to treat EOC, but resistance to cisplatin leads to chemotherapy failure. MicroRNAs are a novel class of regulators that function by controlling gene expression at the post-transcriptional level. Several recent reports have identified some microRNAs that are related to chemotherapy sensitivity. In this study, we found two microRNAs miR-152 and miR-185 that were significantly downregulated in the cisplatin-resistant ovarian cell lines SKOV3/DDP and A2780/DDP, compared with their sensitive parent line SKOV3 and A2780, respectively. Subsequently, the roles of miR-152 and miR-185 were evaluated in vitro and in vivo. The overexpression of miR-152 or miR-185 increased cisplatin sensitivity of SKOV3/DDP and A2780/DDP cells by inhibiting proliferation and promoting apoptosis, then we further confirmed that these miRNAs functioned through suppressing DNA methyltransferase 1 (DNMT1) directly. Concordantly, CD-1/CD-1 nude mice that were injected intraperitoneally with SKOV3/DDP cells transfected with miR-152 mimics exhibited upregulated cisplatin sensitivity in vivo. Interestingly, we found that there were no significant changes in the expression of these two microRNAs after treatment with decitabine (DAC), a traditional epigenetic therapeutic agent, suggesting these miRNAs represented two new regulators independent of DAC. Finally, the survival assay in A549 and HepG2 cells revealed that the two microRNAs involved in cisplatin sensitivity were related to cell types. Our results indicated that miR-152 and miR-185 were involved in ovarian cancer cisplatin resistance in vitro and in vivo by targeting DNMT1 directly. These molecules may serve as potential epigenetic therapeutic targets in other cancers.

Introduction

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer-related deaths in women in the United States and the leading cause of gynaecological cancer-related deaths.1 Owing to a lack of effective biomarkers and the absence of specific symptoms in the early stages of the disease, more than two-thirds of patients cannot be diagnosed until the disease is in an advanced stage.2 The standard therapeutic strategy is to combine surgery with a tumor residrual of not <0.5 mm diameter with chemotherapy based on the cisplatin (cis-diaminedichloroplatinum) alone or combined with other drugs.3 As EOC is associated with high implantation metastasis, chemotherapy has an important role in the 5-year survival, which rarely exceeds 30%.4 However, patients who are initially sensitive to cisplatin often develop drug resistance, leading to chemotherapy failure.5 Therefore, further investigation of the mechanism of cisplatin resistance will be clinically meaningful.

Epigenetic alterations are one mechanism of underlying acquired resistance to cisplatin. The significant upregulation of DNA methyltransferases (DNMTs) was observed in cisplatin-resistant ovarian cancer.6 Combined with platinum-based agents, DNA methyltransferase inhibitors can sensitise ovarian cancer cell lines to cisplatin,7 and the same effect can be observed in animal models and in recurrent ovarian cancer patients.8, 9 Decitabine (5-aza-2′-deoxycytidine, DAC), a FDA-approved agent, is one of the most widely used DNA methyltransferases inhibitors, not only in hematological malignancies,10, 11 but also in solid tumors.12, 13, 14 DAC treatment can upregulate the expression of some tumor repressor genes that had been silenced because of promoter hypermethylation.15 However, it is limited by its toxic activities, particularly myelosuppression.16, 17 Therefore, factors that can reverse cisplatin resistance in a more modulate way is worthy to be explored.

MicroRNAs, a class of short non-coding RNAs of 19–25 nt in length, have roles in multiple physiological and pathological functions, including growth, differentiation, proliferation and apoptosis.18 These small molecules control gene expression either by target mRNAs degradation or translation inhibition by binding to the 3′-untranslated regions (3′-UTR). Recently, some microRNAs have shown to be involved in drug resistance, acting as oncogenes or tumor suppressors. The blockage of miR-135a by a miR-135a inhibitor sensitised the MES-SA and A549 paclitaxel-resistant cell lines to paclitaxel-induced cell death.19 Knockdown or inhibition of miR-221 or miR-222 expression sensitised MDA-MB-468 cells to tamoxifen-induced cell growth arrest and apoptosis.20 The overexpression of miR-451 sensitised the breast cancer cell line MCF-7 to doxorubicin by upregulating MDR1 expression.21 Increased miR-214 and decreased let-7i expression were related to cisplatin resistance in ovarian cancer cell lines.22, 23 However, the interaction between microRNAs and cisplatin resistance is not well understood.

In the present study, we found miR-152 and miR-185 were significantly downregulated in cisplatin-resistant ovarian cell lines SKOV3/DDP and A2780/DDP compared with their sensitive parents SKOV3 and A2780 cells. Ectopic overexpression of the two microRNAs upregulated the two ovarian-resistant cell lines sensitivity to cisplatin by inhibiting their proliferation and promoting apoptosis by suppressing DNMT1 directly, the effects were independent of DAC but related to cell types.

Results

MiR-152 and miR-185 are downregulated in cisplatin-resistant ovarian cancer cells

We confirmed the cisplatin resistance of the SKOV3/DDP cell line compared with their cisplatin-sensitive parental SKOV3 cells using an MTT assay. Cells were exposed to various concentrations of cisplatin for 48 h, the 50% inhibitory concentration (IC50) of cisplatin in the SKOV3/DDP cells was two-fold than the parental SKOV3 cells (Figure 1a). To identify the microRNAs involved in cisplatin resistance, total RNA was extracted from SKOV3/DDP and SKOV3 cells. Quantitative real-time PCR revealed that miR-152 and miR-185 were significantly downregulated in SKOV3/DDP cells compared with SKOV3 cells (Figure 1b). The experiments were repeated in another pair ovarian cancer cell lines cisplatin-resistant A2780/DDP and their cisplatin-sensitive parental A2780, the IC50 of AD cells was three-fold than the parental A2780 cells (Figure 1c), miR-152 and miR-185 were also downregulated in AD cells compared with A2780 cells (Figure 1d).

Figure 1
figure1

The downregulation of miR-152 and miR-185 in cisplatin-resistant ovarian cancer cell lines. (a) SKOV3/DDP and SKOV3 cells were exposed to various concentrations of cisplatin for 48 h, and the 50% inhibitory concentration (IC50) of cisplatin was examined by the MTT assay. (b) Quantitative RT–PCR analysis of miR-152 and miR-185 expression in parental SKOV3 cells (black bars) compared with the expression in related cisplatin-resistant SKOV3/DDP cells (grey bars). (c) A2780/DDP (AD) and A2780 cells were exposed to various concentrations of cisplatin for 48 h, and the IC50 of cisplatin was examined by the MTT assay. (d) Quantitative RT–PCR analysis of miR-152 and miR-185 expression in parental A2780 cells (black bars) compared with the expression in related cisplatin-resistant AD cells (grey bars). The bars indicate the means and s.d. from triplicate experiments. *P<0.05, **P<0.01, ***P<0.001.

The overexpression of miR-152 and miR-185 increased the cisplatin sensitivity of SKOV3/DDP cells and A2780/DDP cells

To investigate the role of miR-152 and miR-185 in cisplatin-induced cytotoxicity, miR-152 and miR-185 mimic were transfected into ovarian cancer cells SKOV3/DDP cells and A2780/DDP cells, respectively. Total RNA was extracted and the transfection efficiency was evaluated at 48 h after transfection (Supplementary Figures S1A, S1B). The transfected cells were then exposed to various concentrations of cisplatin for 48 h and assessed by MTT assay. We found that the IC50 of cisplatin was downregulated in cells that had been transfected with miR-152 or miR-185 compared with the negative control (NC) (Figures 2a and c). The results revealed that the overexpression of miR-152 and miR-185 markedly increased cisplatin sensitivity of SKOV3/DDP cells and A2780/DDP cells.

Figure 2
figure2

MiR-152 and miR-185 were functionally involved in the cisplatin response of ovarian cancer cells. (a) SKOV3/DDP cells was transfected with miR-152 or miR-185 mimic or NC and incubated for 48 h then re-seeded into 96-well plates, various concentrations of cisplatin were added and cell viability was assessed by the MTT assay. The IC50 of cisplatin was shown as the means and s.d. (b) Cell survival was assayed at 48, 72 and 96 h after transfection with 20 μM cisplatin treatment for another 48 h. (c) AD cells was transfected with miR-152 or miR-185 mimic or NC and incubated for 48 h then re-seeded into 96-well plate, various concentrations of cisplatin were added and cell viability was assessed by the MTT assay. The IC50 of cisplatin was shown as the means and s.d. (d) Cell survival was assayed at 24, 48, 72 and 96 h after transfection with 10 μM cisplatin treatment for another 48 h. *P<0.05, **P<0.01.

To test whether the roles of miR-152 and miR-185 affecting cisplatin sensitivity be related to time course, cell survival assay was performed at 24, 48, 72 and 96 h after transfection. Results indicated the stable role of the two mircoRNAs after transfection (Figures 2b and d).

The overexpression of miR-152 and miR-185 increased cisplatin sensitivity by inhibiting cell growth and promoting apoptosis

To study whether the decreased expression of miR-152 and miR-185 might contribute to the malignant phenotypes of ovarian cancer cells, we evaluated the effects of miR-152 and miR-185 in SKOV3/DDP cells. Cell survival assay showed that miR-152 significantly inhibited cell growth compared with the NC group, while miR-185 had no significant inhibition (Figure 3a). This was consistent with the results of a previous study.24 To further confirm that the two microRNAs inhibited cell growth, we performed colony formation assays. We found that the numbers of colonies formed were reduced in SKOV3/DDP cells transfected with miR-152 or miR-185 compared with the NC in both the presence and absence of 10 μM cisplatin (Figure 3b). These results suggested that miR-152 and miR-185 inhibits the growth of SKOV3/DDP cells. A flow cytometry assay was performed to investigate the roles of the two microRNAs in cisplatin-induced apoptosis. The results revealed that there were more apoptotic cells in the miR-152 and miR-185 transfected groups compared with the NC group (Figures 3c and d). These experiments were repeated in A2780/DDP cells, results revealed the same trend (Supplementary Figures S2A, S2B, S2C). All the findings suggested that miR-152 and miR-185 increased cisplatin sensitivity by inhibiting cell growth and promoting apoptosis in ovarian cancer cells.

Figure 3
figure3

The overexpression of miR-152 and miR-185 increased cisplatin sensitivity by inhibiting cell growth and promoting apoptosis in SKOV3/DDP cells. (a) SKOV3/DDP cells were transfected with miR-152 or miR-185 mimic or NC and incubated for 48 h. Then the cell viability was assessed by the MTT assay. (b) SKOV3/DDP cells were transfected with miR-152 or miR-185 mimic or NC, grown for 48 h, and then treated with or without 10 μM cisplatin for another 48 h. The cells were cultured for another 7–10 days, and then colony formation was evaluated. (c) SKOV3/DDP cells transfected with miR-152 or miR-185 mimic or NC were treated with 10 μM cisplatin for 48 h, apoptotic cells were detected by flow cytometry. (d) The percentage of apoptotic cells was represented in a bar diagram as an average of three independent experiments. **P<0.01.

MiR-152 and miR-185 promoted SKOV3/DDP cell sensitivity to cisplatin through targeting DNMT1 directly

Bioinformatics analyses predicted that DNMT1 is a potential target of miR-152 and miR-185 (Figure 4a), and it has been demonstrated that DNMT1 is the true target of miR-152 and miR-185 in human endometrial cancer and gliomas, respectively.25, 26 However, microRNAs may serve different functions in different cell types by suppressing their targets, and either the abundance of the targets or the microRNAs in different cell types may contribute to the different outcome.27 We observed DNMT1 was significantly reduced at both the mRNA and protein level transfected with miR-152 or miR-185 mimic compared with the NC group in SKOV3/DDP cells (Figures 4b and c). The dual-reporter luciferase assay was performed in SKOV3/DDP cells. We proved both miR-152 and miR-185 directly targeted the 3′-UTR of DNMT1 (Figure 4d), and influenced the stability of their target mRNA.

Figure 4
figure4

DNMT1 is the direct target of miR-152 and miR-185 involved in cisplatin resistance. (a) The base-pairing interaction of miR-152 and miR-185 seed sequences and DNMT1, as predicted by bioinformatics analysis. (b) DNMT1 mRNA level measured by quantitative real-time PCR (normalized to GAPDH) and (c) DNMT1 protein level measured by western blotting (normalized to beta-actin) at 48 h after transfection with miR-152 or miR-185 mimic or NC in SKOV3/DDP cells. (d) Luciferase assay. SKOV3/DDP cells were co-transfected with a luciferase construct fused with the wild-type (WT) or truncated (MUT) 3′-UTR of DNMT1 and miR-152 or miR-185 mimic or NC. The relative luciferase activity was detected. (e, f) SKOV3/DDP cells transfected with siDNMT1 or a scramble control, DNMT1 protein level and the IC50 of cisplatin were assessed. (g) SKOV3/DDP cells co-transfected with siDNMT1 and miR-152 or miR-185 mimic or NC, the IC50 of cisplatin was assessed. *P<0.05, **P<0.01.

Further suggesting that DNMT1 was one of the functional targets involved in resistance to cisplatin in SKOV3/DDP cells, we observed decreased IC50 of cisplatin was transfected with siDNMT1 compared with the scramble control (Figure 4f), the efficiency of siDNMT1 was evaluated by decreased DNMT1 protein level (Figure 4e), the phenotype in reversal of cisplatin resistance was similar to the cells transfected with miR-152 or miR-185 mimic. We further observed that at the low expression of DNMT1 interfered with siDNMT1, overexpression of either miR-152 or miR-185 did not cause a significant decrease in cisplatin resistance in SKOV3/DDP cells (Figure 4g). These results suggested that DNMT1 was one of the functional targets of miR-152 and miR-185 in SKOV3/DDP cells.

The overexpression of miR-152 enhanced cisplatin sensitivity in vivo

As either overexpression of miR-152 or miR-185, the IC50 of cisplatin decreased significantly, and overexpression miR-152 in virto had a more significant decrease in proliferation, we chose miR-152 to evaluate its therapeutic potential in vivo. CD-1/CD-1 nude mice were intraperitoneally injected with SKOV3/DDP cells transfected with miR-152 mimic or NC. Cisplatin therapy was initiated 1 week later at a dose of 5 mg/kg twice a week. After four consecutive weeks of therapy, all of the animals were maintained normally for another 4 weeks and then killed. We found both the groups had an apparent increase in abdominal circumference and abdominal blood (Figures 5a and b). The abdominal circumference was not significantly different compared with the NC group (Figure 5e). The total numbers of nodules on the surface of organs, including the stomach, liver, intestines, diaphragm, omentum and abdominal wall, were markedly reduced compared with the NC group (Figures 5c, d and f). HE staining confirmed that the nodules were tumor tissue (Figure 5g). These data suggested that pretreatment with miR-152 mimic can promote the cisplatin sensitivity of human EOC in vivo.

Figure 5
figure5

The overexpression of miR-152 enhanced cisplatin sensitivity in vivo. (a, b) Digital photographs of the nude mice (back and obverse views). (c, d) Nodules on the abdominal walls and intestines. (e) The mean abdominal circumference. (f) The average number of nodules in each group. (g) HE staining of the nodules. Each group contained three nude mice. *P<0.05.

The role of miR-152 and miR-185 in the reversal of cisplatin resistance was independent of DAC but dependent on cell types

DAC was the most commonly used DNMT inhibitor in reversal of cisplatin resistance, we confirmed it upregulated cisplatin sensitivity in SKOV3/DDP cells and AD cells (Figures 6a and c). However, some reports had shown that DAC can cause changes of microRNA expression.28, 29 To identify whether DAC regulates the expression of miR-152 and miR-185, SKOV3/DDP cells and A2780/DDP cells were exposed to various concentrations of DAC for 48 h. The results suggested that there were no significant changes in miR-152 or miR-185 expression after DAC treatment (Figures 6b and d). Thus, the DAC-mediated reversal of sensitivity to cisplatin was not carried out through miR-152 and miR-185, and the two microRNAs were novel regulators of cisplatin resistance, independent of DAC.

Figure 6
figure6

The roles of miR-152 and miR-185 in the reversal of cisplatin resistance was independent of decitabine. (a) SKOV3/DDP cells were exposed to various concentrations of cisplatin with or without 2 μM decitabine (DAC) for 48 h. The IC50 of cisplatin was shown as the means and s.d. (b) SKOV3/DDP cells were exposed to various concentrations of DAC for 48 h, and miR-152 and miR-185 expression after treatment were measured by quantitative real-time PCR. (c) AD cells were pretreated with various concentrations of DAC for 48 h, then the cell survival was assessed after 10 μM cisplatin treatment for another 48 h. (d) AD cells were exposed to various concentrations of DAC for 48 h, and miR-152 and miR-185 expression after treatment were measured by quantitative real-time PCR. *P<0.05, **P<0.01.

To further investigate whether miR-152 and miR-185 can be widely used as a molecular therapy target, we examined the roles of miR-152 and miR-185 in cisplatin-induced cytotoxicity in other cancer cell lines. MiR-152 or miR-185 mimic were transfected into A549 and HepG2 cells, allowed to grow for 48 h, and then treated with 5 and 10 μM cisplatin. Cell survival assay revealed overexpression of miR-152 markedly upregulated cisplatin sensitivity in A549 cells but not in HepG2 cells, while overexpression of miR-185 had no significant effect on cisplatin sensitivity both in A549 cells and in HepG2 cells (Figures 7a and c), although DNMT1 protein levels were changed (Figures 7b and d). These results revealed that miR-152 and miR-185 may contribute to cisplatin sensitivity in other cancers but depend on cell types; the different mRNA level of DNMT1 and the two microRNAs may be related to the different effects (Figure 7e).

Figure 7
figure7

The roles of miR-152 and miR-185 in the reversal of cisplatin resistance was dependent on cell types. (a) A549 cells were transfected with miR-152 or miR-185 mimic or NC, respectively. Cell viability was assessed after transfection with 5 μM cisplatin for another 48 h. (b) A549 cells were transfected with miR-152 or miR-185 mimic or NC, 48 h DNMT1 protein level were measured by western blotting (normalized to GAPDH). (c) HepG2 cells were transfected with miR-152 or miR-185 mimic or NC, respectively. Cell viability was assessed after transfection with 10 μM cisplatin for another 48 h. (d) HepG2 cells were transfected with miR-152 or miR-185 mimic or NC, 48 h DNMT1 protein level were measured by western blotting (normalized to GAPDH). (e) The relative expression levels of DNMT1, miR-152, miR-185 were measures by quantitative real-time PCR in different cell lines. *P<0.05.

Discussion

Cisplatin is the first-line chemotherapy drug for many malignancies, such as testicular, ovarian, cervical, head and neck and non-small-cell lung cancers, as it was approved by the FDA in 1978 (ref. 30). Cisplatin causes intrastrand DNA crosslinks and a lower percentage of interstrand crosslinks.31 This alteration of DNA conformation not only inhibits translation but also DNA replication and makes the DNA easier to disrupt. Although cisplatin is an effective cancer treatment, drug resistance leading to chemotherapy failure is almost unavoidable.

Multiple mechanisms that mediate intrinsic or acquired resistance to cisplatin have been recognised.32 DNA repair pathways, including the mismatch repair and nucleotide excision repair pathways, are involved in cisplatin sensitivity.33 Consequently, alterations in the repair pathways have been implicated in cisplatin resistance. The NER pathway protein ERCC1 has been demonstrated to be associated with cisplatin in ovarian tumors and cancer cell lines.34 Additionally, a decreased uptake of cisplatin due to the downregulation of the copper-transporter 1 protein which increased detoxification by promoting the conjugation of cisplatin to glutathione and increasing transporter efflux, also contributing to cisplatin resistance.35

Overcoming cisplatin resistance is an urgently expected development in cancer therapy. Presently, one of the most widely accepted methods is to combine cisplatin therapy with other agents that resensitise or increase cells cisplatin toxicity. Epigenetic therapy is an area of intense research because changes in epigenetic patterns have been found in many tumors.36, 37, 38, 39 There are three DNA methyltransferases in humans, DNMT1, DNMT3a and DNMT3b. DNMT1 is the most abundant DNA methyltransferase in mammalian cells and the key enzyme for the maintenance of hemimethylated DNA during DNA replication and de novo methylation during somatic cell development and differentiation.36 DNMT1 expression is also upregulated in many malignancies.38, 40 The DNA methyltransferase inhibitor DAC is widely used in cancer therapy. The active inhibitor is the triphosphate form (5-aza-dCTP), which is activated by deoxycytidine kinase and subsequently incorporates very readily into DNA, where it inhibits DNA methyltransferase.41 Although DAC has a major role in combination with other chemotherapy drugs, its narrow safe range and effective dosage limits its clinical use: low doses can lead to insufficient demethylation, and high doses can cause excessive proliferation inhibition and cell death.42 The major toxicity produced by DAC is myelosuppression, and it is expected that safer agents could be identified.

MicroRNAs are a new class of therapeutic molecules. Compared with traditional drugs, microRNAs have a more modulatory role. Some reports have demonstrated changes in microRNA expression after DAC pretreatment. MiR-126 was strongly upregulated in HeLa, MCF-7 and T24 cell lines after treatment with the DAC and the histone deacetylase inhibitor 4-phenylbutyric acid.29 MiR-34b and miR-129 expression in gastric cancer cells increased after DAC treatment.28 We wondered whether DAC might also increase the expression of drug resistance-related microRNAs, subsequently altering the effects of these microRNAs. We measured miR-152 and miR-185 expression in SKOV3/DDP and A2780/DDP cell lines after treatment with various concentrations of DAC and found that there were no significant changes. This result implied that the reversal of cisplatin sensitivity by miR-152 and miR-185 was independent of DAC. Therefore, miR-152 and miR-185 may take place of DAC, as another epigenetic therapy targeting DNMT1 (Figure 8).

Figure 8
figure8

Comparison of two epigenetic therapeutic strategies in reversal of cisplatin resistance.

MiR-152 and miR-185 are the two major molecules that we focused on in this study. MiR-152 is located at 17q21.32 in intron 1 of the COPZ2 gene, and miR-185 is located at 22q11.21 in the C22orf25 gene. Both of these miRNAs are tumor suppressors and are expressed at low levels in many cancers,43, 44, 45 as well as in ovarian cancers.24, 46 The overexpression of miR-152 can inhibit proliferation in SKOV3 cells and endometrial cancer cell lines.24, 25 DNMT1 is the true target of miR-152 in human endometrial cancer and hepatitis B virus-related hepatocellular carcinoma and the true target of miR-185 in human glioma.25, 26, 44 However, microRNAs may serve different functions in different cell types by suppressing their targets. For example, miR-26a inhibits the cell growth and tumorigenesis of nasopharyngeal carcinoma by repressing EZH2 but promotes cholangiocarcinoma growth through the inhibition of GSK-3β and subsequent activation of β-catenin.47, 48 Moreover, either the abundance of the targets or the microRNAs may contribute to the differences.27 Here, we showed that both the level of mRNA and proteins were significant changed, and DNMT1 was the target of miR-152 and miR-185 in the ovarian cancer cells and contributes to the reversal of cisplatin resistance (Figure 4d). The different function in A549 and HepG2 cells may be related to the abundance of the target or the microRNAs (Figure 7).

Additional mechanisms for miR-152 and miR-185 in cisplatin resistance are possible and should be studied further. Recently, the AKT pathway has been shown to be activated in cisplatin-resistant ovarian cancer.49, 50 FOXO1 is the effector of PI3K/AKT, and activated AKT inhibits FOXO1 expression, subsequently inhibiting apoptosis.51 FOXO1 is a potential target of miR-152, and has been confirmed in endometrial cancer.52 The oncogenes c-myc and Six1 are potential targets of miR-185 and have been confirmed in human cancer cells, respectively.46, 53 In these cells, miR-185 expression suppresses cell proliferation and leads to G1 arrest of the cell cycle. Interestingly, a previous study also showed that miR-185 transfection caused a downregulation of cyclin E1 and cyclin-dependent kinase 6 mRNA levels.54 These other targets may also contribute to cisplatin efficacy. We also investigated the expression of some cisplatin resistance-related genes after miR-152 and miR-185 transfection, which may be downstream of DNMT1.55, 56 However, there were no significant changes of their mRNA levels (Supplementary Figure S3). That revealed the mechanism of cisplatin resistance was complicated.

In conclusion, we demonstrated that miR-152 and miR-185 were involved in cisplatin resistance and showed that miR-152 and miR-185 increased cisplatin sensitivity mainly through the direct downregulation of DNMT1. Although the present paper was focused on miR-152 and miR-185, some other microRNAs are likely to be involved in cisplatin resistance. We confirmed that miR-152 and miR-185 enhanced cisplatin sensitivity through inhibiting proliferation and promoting apoptosis and may potentially serve as therapeutic targets for overcoming cisplatin resistance in ovarian cancer. These applications are also likely to be effective in other cancers and may have beneficial implications for the development of novel cancer therapy.

Materials and methods

Cell culture

The human ovarian cancer cell line SKOV3, A2780 and its cisplatin-resistant cell line A2780/DDP, human non-small-cell lung cancer cell line A549, and human hepatic carcinoma cell line HepG2 were conserved in our laboratory. The cisplatin-resistant cell line SKOV3/DDP was purchased from the FMG-Bio company (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humid atmosphere containing 5% CO2 at 37 °C.

Transfections and luciferase assay

Cells were seeded in six-well plates and transfected with miR-152 mimics, miR-185 mimic or NC, using Lipofectamine 2000 (Invitrogen). The NC was synthetic scrambled double oligonucleotides, non-targeting against to any mRNA. For small interfering RNA-mediated DNMT1 knockdown, siDNMT1 and a non-targeting small interfering RNA scramble control (Gene Pharma Company, Shanghai, China) were transfected into SKOV3/DDP cells with Lipofectamine 2000. Protein and mRNA expression levels were analyzed 48 h post-transfection. The luciferase assay was performed in SKOV3/DDP cells. Briefly, we generated two Luc-DNMT1 3′-UTR constructs, including the potential biding sequence (DNMT1-WT-UTR) and truncated the potential biding sequence (DNMT1-MUT-UTR). Cells were seeded in 24-well plates and co-transfected with 100 ng Luc-DNMT1 3′-UTR reporter vector, 40 ng TK and 30 nM miR-152 or miR-185 mimic and incubated overnight. Then, luciferase activity was measured using the Dual-Glo Luciferase assay system (Promega, Madison, WI, USA).

Quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen). Reverse-transcribed complementary DNA was synthesized with random primers or microRNAs specific stem-loop primers. Subsequently, the cDNA was subjected to real-time PCR on a 7500 real-time PCR system (AB Applied Biosystems, Mannheim, Germany). GAPDH and U6 were used as internal controls. The primers sequences used were as follows: DNMT1 sense: 5′-IndexTermTATCCGAGGAGGGCTACCTGGC-3′, antisense: 5′-IndexTermTGGGGCTAGGTGAAGGTTCAGGC-3′; GAPDH sense: 5′-IndexTermAGCCTCCCGCTTCGCTCTCT-3′, antisense: 5′-IndexTermGCGCCCAATACGACCAAATCCGT-3′; miR-152 sense: 5′-IndexTermGGCAGTGCATGACAGAAC-3′, antisense: 5′-IndexTermCAGTGCGTGTCGTGGAGT-3′; miR-185 sense: 5′-IndexTermGGGTGGAGAGAAAGGCAG-3′, antisense: 5′-IndexTermCAGTGCGTGTCGTGGAGT-3′; U6 sense: 5′-IndexTermGCTTCGGCAGCACATATACTAAAAT-3′, antisense: 5′-IndexTermCGCTTCACGAATTTGCGTGTCAT-3′.

Western blotting

Cellular protein extracts were separated in a 12 or 8% SDS-polyacrylamide gel and electrophoretically transferred onto a PDVF membrane (Millipore, Bedford, MA, USA). Membranes were blocked overnight with 5% non-fat dried milk and incubated with antibodies to DNMT1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), GAPDH (Cell Signaling Technology, Danvers, MA, USA), or Beta-actin (Cell Signaling Technology) overnight at 4 °C. After washing with PBST, the membranes were incubated with horseradish peroxidase-linked secondary antibody. The proteins were visualized using ECL chemiluminescence and exposed to X-ray film. Bands were quantified with Image J (National Institutes of Health, Bethesda, MD, USA).

3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Non-transfected or transfected cells were re-seed into 96-well plates, 24 h later, freshly prepared cisplatin or DAC (Sigma Chemicals, St Louis, MO, USA) at various concentrations was added and the cells were incubated for a further 48 h. Cell viability was assessed using the MTT assay. The absorbance of each well at the wavelength of 492 nm was read on a spectrophotometer. At least three independent experiments were performed in quadruplicate.

Colony formation assay

Approximately 500–800 SKOV3/DDP cells transiently transfected with miR-152, miR-185 or NC were placed in 60 mm plates, treated with or without 10 μM cisplatin for 48 h, and then cells were maintained in fresh DMEM containing 10% FBS for another 7–10 days. Colonies were stained with 0.1% crystal violet in 20% methanol for 15 min. The samples were photographed and the numbers of visible colonies were counted.

Flow cytometry analysis of apoptosis

Cells transfected with miR-152, miR-185 or NC were treated with 10 μM cisplatin for 48 h and harvested. Apoptosis was evaluated using Annexin V-FITC and PI staining flow cytometry.

Nude mice model

Approximately 1 × 107 SKOV3/DDP cells transfected with miR-152 mimics or NC were injected intraperitoneally into CD-1/CD-1 nude mice (Vital Alver, Beijing, China). One week later, cisplatin therapy was initiated at a dose of 5 mg/kg twice per week. After four consecutive weeks of therapy, the animals were maintained normally for another 4 weeks and then killed. Abdominal circumference and body weight were monitored throughout the experiment. Macroscopical photographs were captured and all of the nodules on the stomach, liver, intestines, diaphragm, omentum and abdominal wall were counted. Nodule samples were fixed with 4% paraformaldehyde and submitted to HE staining. All animal studies were conducted in accordance with the guidelines established by the internal Institutional Animal Care and Use Committee.

Statistical analysis

All experimental data were shown as the mean±s.e.m. Differences between samples were analyzed using the two-tailed Student’s t-test. Statistical significance was accepted at P<0.05.

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Acknowledgements

This work was supported by the Natural Science Foundation of Heilongjiang Province for youth (QC2010002) and for outstanding youth (JC201110), the Natural Science Foundation of China (81101373/81270511/81001033), the Science Foundation of Health Department of Heilongjiang Province(2012-525) and the Science Foundation of the First Affiliated Hospital of Harbin Medical University (2009Y24).

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Correspondence to G Zhang or X Gao.

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Supplementary Information accompanies the paper on the Oncogene website

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Keywords

  • cisplatin resistance
  • microRNA
  • DNMT1
  • decitabine
  • ovarian cancer

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