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Cited by 118MicroRNA in disease regulationOvarian cancer diagnosis and treatmentCancer-related molecular mechanisms research

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Upregulation of miR-21 in Cisplatin Resistant Ovarian Cancer via JNK-1/c-Jun Pathway

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Echevarría-Vargas, Ileabett M., Valiyeva, Fatma, Vivas-Mejía, Pablo E.·2014·PLoS ONE
DOI 10.1371/journal.pone.0097094PMC PMC4035252
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Abstract

Cisplatin has been the most accepted drug for the treatment of ovarian cancer for almost 40 years. Although the majority of patients with ovarian cancer respond to front-line platinum combination chemotherapy, many patients will develop cisplatin-resistance disease, which is extremely rapid and fatal. Although various mechanisms of cisplatin resistance have been postulated, the key molecules involved in such resistance have not been identified. MiRNAs are endogenously expressed small non-coding RNAs, which are evolutionarily conserved and function as post-transcriptional regulators of gene expression. Dysregulation of miRNAs have been associated with cancer initiation, progression and drug resistance. The oncogenic miRNA-21, one of the best-studied miRNAs, is upregulated in almost all human cancers. However, the regulation of miR-21 in cisplatin resistant ovarian cancer cells has not been assessed. In this study, we measured the miR-21 expression by real-time PCR and found upregulation of miR-21 in cisplatin resistant compared with cisplatin sensitive ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated the association of the c-Jun transcription factor to the pri-mir-21 DNA promoter regions. Blocking the JNK-1, the major activator of c-Jun phosphorylation, reduced the expression of pre-mir-21 and increased the expression of its well-known target gene, PDCD4. Overexpression of miR-21 in cisplatin sensitive cells decreased PDCD4 levels and increased cell proliferation. Finally, targeting miR-21 reduced cell growth, proliferation and invasion of cisplatin resistant ovarian cancer cells. These results suggest that the JNK-1/c-Jun/miR-21 pathway contributes to the cisplatin resistance of ovarian cancer cells and demonstrated that miR-21 is a plausible target to overcome cisplatin resistance.

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Figure 1 RT-PCR and western blot analysis of miRNA-21-related molecules. (A) Validation of microarrays by RT-PCR. (B) MiR-21 levels in a panel of ovarian cancer cells. MiR-21 levels were expressed relative to the A2780 cells miR-21 levels. IC50s were calculated after 72-hr treatment of cells with different concentrations of cisplatin as described in the “Material and Methods” section. (C) Evaluation of c-Jun and p-c-Jun protein expression in A2780 and A2780CP20 cells. (D) Protein expression analysis of MAPKs in total and nuclear fractions of A2780 and A2780CP20 cells. Expression level in Figures A, C and D are without cisplatin treatment. *p<0.05, **p<0.01, ***p<0.001 compared to control. Columns represent the means of triplicates ± S.E.M.
Figure 1 RT-PCR and western blot analysis of miRNA-21-related molecules. (A) Validation of microarrays by RT-PCR. (B) MiR-21 levels in a panel of ovarian cancer cells. MiR-21 levels were expressed relative to the A2780 cells miR-21 levels. IC50s were calculated after 72-hr treatment of cells with different concentrations of cisplatin as described in the “Material and Methods” section. (C) Evaluation of c-Jun and p-c-Jun protein expression in A2780 and A2780CP20 cells. (D) Protein expression analysis of MAPKs in total and nuclear fractions of A2780 and A2780CP20 cells. Expression level in Figures A, C and D are without cisplatin treatment. *p<0.05, **p<0.01, ***p<0.001 compared to control. Columns represent the means of triplicates ± S.E.M.CC BY · PubMed Central
Figure 2 Effect of JNK-1 inhibition in miR-21 and PDCD4 expression. A2780CP20 cells were treated with 10 µM SP600125. (A) Western blot shown the inhibition of p-c-Jun after treatment with SP600125 in A2780CP20 cells compared to control (DMSO). (B) SYBR-I-based real-time PCR was performed to calculate the relative pre-mir-21 expression in A2780CP20 cells after treatment with SP600125 inhibitor. (C) Western blot and densitometric analysis of PDCD4 protein expression levels after treatment of A2780CP20 cells with SP600125. (D) PDCD4 protein expression levels after transfection of A2780CP20 with miR-21 oligonucleotide inhibitor. (E) A2780 CP20 cells were transiently transfected with two c-Jun-targeted siRNAs as described in the “Materials and Methods” section. Western blot analysis shows that both c-Jun-siRNAs decreased the c-Jun levels. SYBR-I-based real-time PCR was performed (see “Materials and Methods” section) to calculate the relative pre-mir-21 expression levels in A2780CP20 cells after siRNA-mediated c-Jun silencing. *p<0.05, **p<0.01, ***p<0.001 compared to control. Columns represent the means of triplicates ± S.E.M.
Figure 2 Effect of JNK-1 inhibition in miR-21 and PDCD4 expression. A2780CP20 cells were treated with 10 µM SP600125. (A) Western blot shown the inhibition of p-c-Jun after treatment with SP600125 in A2780CP20 cells compared to control (DMSO). (B) SYBR-I-based real-time PCR was performed to calculate the relative pre-mir-21 expression in A2780CP20 cells after treatment with SP600125 inhibitor. (C) Western blot and densitometric analysis of PDCD4 protein expression levels after treatment of A2780CP20 cells with SP600125. (D) PDCD4 protein expression levels after transfection of A2780CP20 with miR-21 oligonucleotide inhibitor. (E) A2780 CP20 cells were transiently transfected with two c-Jun-targeted siRNAs as described in the “Materials and Methods” section. Western blot analysis shows that both c-Jun-siRNAs decreased the c-Jun levels. SYBR-I-based real-time PCR was performed (see “Materials and Methods” section) to calculate the relative pre-mir-21 expression levels in A2780CP20 cells after siRNA-mediated c-Jun silencing. *p<0.05, **p<0.01, ***p<0.001 compared to control. Columns represent the means of triplicates ± S.E.M.CC BY · PubMed Central
Figure 3 Chromatin immunoprecipitation assay (ChIP). ChIP assay was performed as described in the “Materials and Methods” section. (A) SYBR-I-based real-time PCR amplification of the region containing the c-Jun recognition sequence in the pri-miR-21 DNA. The phospho-c-Jun levels bound to the pri-miR-21 promoter was higher in A2780CP20 cells compared with A2780 cells. (B) SYBR-I-based real-time PCR amplification of a DNA region far of the pre-mir-21 promoter was performed as a control. *p<0.05 compared to control. Columns represent the means of triplicates ± S.E.M.
Figure 3 Chromatin immunoprecipitation assay (ChIP). ChIP assay was performed as described in the “Materials and Methods” section. (A) SYBR-I-based real-time PCR amplification of the region containing the c-Jun recognition sequence in the pri-miR-21 DNA. The phospho-c-Jun levels bound to the pri-miR-21 promoter was higher in A2780CP20 cells compared with A2780 cells. (B) SYBR-I-based real-time PCR amplification of a DNA region far of the pre-mir-21 promoter was performed as a control. *p<0.05 compared to control. Columns represent the means of triplicates ± S.E.M.CC BY · PubMed Central
Figure 4 Effect of pre-mir-21 overexpression in A2780 cells. (A) A2780 cells were stably transfected with pCMV-miR21 or empty pCMV-EV vectors. The miR-21 expression was quantified by qRT-PCR. (B) Western blot analysis shows a decreased expression of PDCD4 levels in A2780-miR-21 compared with A2780-EV cells. (C) Overexpression of miR21 increased cell proliferation (13%, ***p<0.001) in A2780-miR-21 compared with A2780-EV cells. (D) A2780-miR-21 overexpressed clones were more resistant to cisplatin-induced cell death compared with untransfected A2780 cells or with the A2780-EV cells. **p<0.01. Columns represent the means of triplicates ± S.E.M.
Figure 4 Effect of pre-mir-21 overexpression in A2780 cells. (A) A2780 cells were stably transfected with pCMV-miR21 or empty pCMV-EV vectors. The miR-21 expression was quantified by qRT-PCR. (B) Western blot analysis shows a decreased expression of PDCD4 levels in A2780-miR-21 compared with A2780-EV cells. (C) Overexpression of miR21 increased cell proliferation (13%, ***p<0.001) in A2780-miR-21 compared with A2780-EV cells. (D) A2780-miR-21 overexpressed clones were more resistant to cisplatin-induced cell death compared with untransfected A2780 cells or with the A2780-EV cells. **p<0.01. Columns represent the means of triplicates ± S.E.M.CC BY · PubMed Central
Figure 5 Effect of miR-21 inhibition in cell growth, proliferation and invasion. (A) A2780CP20 cells were transiently transfected with a miR-21 antagomir or with a negative antagomir (-) as described in the “Materials and Methods” section. Eight and 24 hours after transfection cells were collected and RNA (including miRNAs) was isolated as described in the “Materials and Methods” section. (B) MiR-21 inhibition reduced cell growth compared to untreated cells (NT) or with the negative control inhibitor (NC-Inh). (C) A2780CP20 cells were transfected as in 5A. A thousand cells were seeded in Petri dishes. Ten days later, the colonies were stained and counted. Inhibition of miR-21 decreased the ability of cells to undergo unlimited division compared with the NC-Inh. (D) Cell invasion was carried out as described in the “Materials and Methods” section. The number of invaded cells was expressed in percentages, taken the NC-Inh as 100%. *p<0.05, **p<0.01 compared to NC-Inh. Columns represent the means of triplicates ± S.E.M.
Figure 5 Effect of miR-21 inhibition in cell growth, proliferation and invasion. (A) A2780CP20 cells were transiently transfected with a miR-21 antagomir or with a negative antagomir (-) as described in the “Materials and Methods” section. Eight and 24 hours after transfection cells were collected and RNA (including miRNAs) was isolated as described in the “Materials and Methods” section. (B) MiR-21 inhibition reduced cell growth compared to untreated cells (NT) or with the negative control inhibitor (NC-Inh). (C) A2780CP20 cells were transfected as in 5A. A thousand cells were seeded in Petri dishes. Ten days later, the colonies were stained and counted. Inhibition of miR-21 decreased the ability of cells to undergo unlimited division compared with the NC-Inh. (D) Cell invasion was carried out as described in the “Materials and Methods” section. The number of invaded cells was expressed in percentages, taken the NC-Inh as 100%. *p<0.05, **p<0.01 compared to NC-Inh. Columns represent the means of triplicates ± S.E.M.CC BY · PubMed Central

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Metadata

Authors
Echevarría-Vargas, I. M., Valiyeva, F., & Vivas-Mejía, P. E.
Affiliations
  • University of Puerto Rico, Medical Sciences Campus (PR)
  • University of Puerto Rico System (PR)
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-05-27
Volume
9
Issue
5
Pages
e97094
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0097094
PMC
PMC4035252
OpenAlex
W2077670883
Cited by
118
References
53
Citations by year
2026: 4 · 2025: 4 · 2024: 6 · 2023: 13 · 2022: 14 · 2021: 13 · 2020: 12 · 2019: 10 · 2018: 8 · 2017: 8 · 2016: 8 · 2015: 16 · 2014: 2
FWCI
3.63
Type
journal article
Language
en
Update notices
correction (2014-12-26)
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health
  • National Cancer Institute (K22CA166226)
  • National Institute of General Medical Sciences (R25GM061838)
  • National Institute on Minority Health and Health Disparities (U54MD008149)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Health Sciences › Medicine › Oncology › Ovarian cancer diagnosis and treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
Keywords
miR-21, cisplatin resistance, ovarian cancer, JNK1, c-Jun, JNK/c-Jun signaling pathway, PDCD4, microRNA regulation, cell proliferation, cell invasion, drug resistance
MeSH terms
Antineoplastic Agents, Cell Movement, Cisplatin, Female, Humans, Ovarian Neoplasms, Promoter Regions, Genetic, RNA, Messenger, Tumor Cells, Cultured, Blotting, Western, RNA-Binding Proteins, Apoptosis, Drug Resistance, Neoplasm, Reverse Transcriptase Polymerase Chain Reaction, Chromatin Immunoprecipitation, JNK Mitogen-Activated Protein Kinases, Mitogen-Activated Protein Kinase 8, Cell Proliferation, Apoptosis Regulatory Proteins, Real-Time Polymerase Chain Reaction

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