Epigenetic silencing of microRNA-199b-5p is associated with acquired chemoresistance via activation of JAG1-Notch1 signaling in ovarian cancer - PubMed (original) (raw)

Epigenetic silencing of microRNA-199b-5p is associated with acquired chemoresistance via activation of JAG1-Notch1 signaling in ovarian cancer

Michelle X Liu et al. Oncotarget. 2014.

Abstract

Epithelial ovarian cancer is a highly lethal and aggressive gynecological malignancy. The high mortality rate is due in part to the fact that many advanced cancer patients become refractory to current chemotherapeutic agents, leading to tumor recurrence and death. However, the underlying mechanisms leading to chemoresistance remain obscure. Here, we report that the loss of miR-199b-5p due to progressive epigenetic silencing leads to the activation of the JAG1-mediated Notch1 signaling cascade, thereby leading to the development of acquired chemoresistance in ovarian cancer. Using miRCURY LNA™ microRNA array and Q-PCR analyses of two pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines, we identified miR-199b-5p as significantly down-regulated in cisplatin-resistant ovarian cancer cells and confirmed that miR-199b-5p is clinically associated with advanced and poor survival ovarian cancers. Interestingly, the loss of miR-199b-5p could be restored by 5-Aza-dC-mediated demethylation, and methylated specific PCR (MS-PCR), bisulfite-sequencing and pyrosequencing revealed that the promoter region of miR-199b-5p was hypermethylated. Computational and mechanistic analyses identified JAG1 as a primary target of miR-199b-5p. Notably, the reduced expression of miR-199b-5p was found to be inversely correlated with the increased expression of JAG1 using an ovarian cancer tissue array. Enforced expression of miR-199b-5p sensitized ovarian cancer cells to cisplatin-induced cytotoxicity both in vitro and in vivo. Conversely, re-expression of miR-199b-5p and siRNA-mediated JAG1 knockdown or treatment with Notch specific inhibitor γ-secretase (GSI) attenuated JAG1-Notch1 signaling activity, thereby enhancing cisplatin-mediated cell cytotoxicity. Taken together, our study suggests that the epigenetic silencing of miR-199b-5p during tumor progression is significantly associated with acquired chemoresistance in ovarian cancer through the activation of JAG1-Notch1 signaling.

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Figures

Fig 1

Fig 1. Loss of miR199b-5p is involved in acquired chemoresistance and advanced-stage ovarian cancer

(A) miR-199b-5p expression was measured by quantitative RT-PCR in a panel of ovarian cancer cell lines: A2780s, A2780cp, OV2008, C13*, SKOV3, OVCA429, OVCA433, and ES-2. The results are reported as the mean ± SD of three independent experiments performed in triplicate, ** P<0.01. (B) Comparison of miR-199b-5p expression in early-stage (Stage I, N=28) vs. advanced-stage (Stage II, III and IV, N =51) ovarian cancer (P=0.047) and in low-grade vs. high-grade disease as determined by Q-PCR. (C) Comparison of the survival curve of ovarian cancer patients with high (n=12) and low levels (n=51) of miR-199b-5p. The relative expression level of miR-199b-5p (0.024) was used as a cutoff point. * P = 0.047

Fig 2

Fig 2. Hypermethylation silences miR-199b-5p expression in ovarian cancer

(A) The expression of miR-199b-5p could be restored in cisplatin-resistant ovarian cell lines upon 5-Aza-dC mediated demethylation treatment (*P<0.05, ** P<0.01). (B) Schematic illustration of CpG islands in the promoter region of miR-199b-5p. Vertical lines indicate CpG sites. The arrows indicate the MS-PCR, BGS and pyrosequencing (-6318 to -6228) regions. (C) Promoter methylation status of miR-199b-5p was evaluated by MS-PCR in ovarian cancer cells upon treatment of 5-Aza-dC-mediated demethylation. (D) BGS evaluation of the promoter methylation status of miR-199b-5p in ovarian cancer cells A2780s, A2780cp and SKOV3 with/without 5-Aza-dC mediated demethylation. Each row of circles indicates a single clone. Each circle indicates a CpG site. Filled, methylated CpG site. Unfilled, unmethylated CpG site. (E) Pyrosequencing analysis revealed that methylation density of ovarian cancer cell lines. (F) Pyrosequencing results of ovarian cancer samples indicated that there was a progressive increase in the methylation status with tumor stage (stage 3 vs. stage 2, P=0.0482; stage 4 vs. stage 2, P=0.0412), whereas there was no significance relationship between the methylation status and tumor grade. Each dot represents the methylation percentage of each clinical sample.

Fig 3

Fig 3. MiR-199b-5p sensitizes chemoresistant ovarian cancer cells to cisplatin-induced cytotoxicity in vitro and in vivo

(A) XTT cell proliferation assay revealed that ectopic expression of miR-199b-5p significantly reduce cell viability from 95-99% to 80-85% in A2780cp, C13*, SKOV3 and ES-2 cells upon cisplatin treatment (3 μg/ml, 24 hours). (B) XTT cell proliferation assay revealed that depletion of miR-199b-5p increased cell viability from 11% to 25% in A2780s cells and 12% to 88% in OV2008 upon cisplatin treatment (1 μg/ml) at 24 h and 72 h compared with the negative control. (C & D) XTT cell proliferation assay demonstrated that the stable expression of miR-199b-5p inhibited cell proliferation by 15% in A2780cp cells and 13% in SKOV3 cells but further inhibited the cell viability 19-26% in A2780cp and 13-18% in SKOV3 upon cisplatin treatment (3 μg/ml). (E) The focus formation assay revealed that stable expression of miR-199b-5p caused a 40% reduction in foci formation in C13* and SKOV3 cells and an 80% reduction in A2780cp cells upon cisplatin treatment (3 μg/ml, 14 days). The results are reported as the mean ± SD of three independent experiments performed in triplicate, *, P<0.05, **, P<0.01. (F) miR-199b-5p stable expression (C2) and vector control C13* cells were inoculated subcutaneously into nude mice. The mice were i.p. injected with cisplatin (5 mg/kg) every three days beginning on day 7 when the palpable tumor was formed. The tumor size was calculated and reported as the mean tumor volume ± SE of each group of five mice. The miR-199b-5p stable expression transfectant (C2) resulted in a significantly slower tumor growth rate compared with the vector control. Vertical arrows indicate the time of cisplatin injection. Points, mean of 5 mice. Bars, SD. N=5, ** P<0.01.

Fig 4

Fig 4. JAG1 is a direct target of mir199b-5p and a key factor mediating Notch1 signaling activity in chemoresistant ovarian cancer

(A) A schematic diagram indicating the wild-type and mutated type binding sites of miR-199b-5p in the JAG1 3'UTR. (B) The luciferase reporter assay demonstrated that co-transfection of the JAG1 3'UTR wild-type pmir-GLO-JAG1-WT but not the mutated pmirGLO-JAG1-MUT plasmids (100 ng) with pmR-ZsGreen1-miR-199b-5p (400 ng) resulted in a 38% reduction in the luciferase activity compared with the vector control. *, P=0.025. (C) Both western blot and Q-PCR results indicated a reciprocal expression pattern of JAG1 and miR-199b-5p in a panel of ovarian cancer cell lines. (D) Transient transfection of pmR-ZsGreen1-miR-199b-5p reduced the expression of JAG1 in HEK293 cells in a dose–dependent manner and in chemoresistant C13* and SKOV3 cells. (E) Co-transfection of miR-199b-5p reduced HES1 luciferase activity by 35% compared with the vector control in SKOV3, expressing a relatively higher level of JAG1. (F) Representative pictures showing an inverse relationship between the expression levels of miR-199b-5p and JAG1 examined by ISH and IHC analyses in a commercial ovarian cancer tissue array (OVC1021, N=97, P=0.013). Two-fold and 5-fold changes in expression were used as cutoff level for miR-199b-5p and JAG1, respectively, in the statistical analysis.

Fig 5

Fig 5. JAG1 enhances ovarian cancer cell growth and cisplatin-resistance

(A) Western blot analysis demonstrating the transient transfection of JAG1 (pCD-HA/JAG1) in A2780s cells. (B) XTT cell proliferation assay demonstrated that enforced expression of JAG1 increased the cell proliferation rate by 29% and increased cell viability by up to 40% in A2780s cells upon cisplatin treatment (1 μg/ml). (C) Western blot analysis revealed that two out of three siRNAs (siRNA2 and siRNA3) remarkably reduced JAG1 expression by 80-85% in C13* and 70-75% in SKOV3 cells compared with scrambled controls (NC) (left panel). XTT cell proliferation assay demonstrated that depletion of JAG1 by siRNA reduced cell viability by 15-20% in C13* cells and 10-15% in SKOV3 cells upon cisplatin treatment (3 μg/ml) (right panel). The results are expressed as the mean ± SD of three independent experiments performed in triplicate. *, P<0.05, **, P<0.01.

Fig 6

Fig 6. JAG1 enhances the chemoresistance of ovarian cancer cells by activating Notch1 signaling activity

(A) HES1 luciferase reporter assay demonstrated that JAG1 was able to increase HES1 luciferase activity by 30% compared with the vector control in OV2008. (B) Western blot analysis revealed that GSI inhibited the expression of the Notch1 active domain by 60-95% in A2780cp, C13*, SKOV3 and ES-2 cells. (C) XTT cell proliferation assay demonstrated that GSI or cisplatin reduced cell viability by 5-10% in A2780cp, C13*, SKOV3 and ES-2 cells after 24 to 72 hrs. Co-treatment with GSI and cisplatin dramatically reduced cell viability by 17-38% in the above cell lines after 24 hrs to 72 hrs when compared with the DMSO control. Dose of DMSO, 10 μM; GSI, 10 μM; cisplatin, 3 μg/ml; GSI, 10 μM, + cisplatin, 3 μg/ml. The results are reported as the mean ± SD of three independent experiments performed in triplicate. *, P<0.05, **, P<0.01. (D) Blocking Notch1 signaling by GSI enhances cisplatin-mediated cell cytotoxicity in a tumor xenograft mouse model. ES-2 cells were injected subcutaneously into nude mice. The tumor-bearing nude mice were separated into 4 test groups on day 7 and were i.p. injected with DMSO, GSI (200 ng/kg), cisplatin (5 mg/kg) or a combination of GSI and cisplatin every two days. The tumor size was monitored and calculated as the mean tumor volume ± SE of each group. The group receiving both GSI and cisplatin exhibited the slowest tumor growth rate. The representative picture shows the tumor size of each group. Vertical arrows indicate the time of drug injection. Points show the mean of 3 mice of each group. Bars, SD. N=3, * P<0.05; ** P<0.01.

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