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Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1-miR-593-5p-MFF axis.

Fan S, Liu B, Sun L, Lv XB, Lin Z, Chen W, Chen W, Tang Q, Wang Y, Su Y, Jin S, Zhang D, Zhong J, Li Y, Wen B, Zhang Z, Yang P, Zhou B, Liang Q, Yu X, Zhu Y, Hu P, Chu J, Huang W, Feng Y, Peng H, Huang Q, Song E, Li J - Oncotarget (2015)

Bottom Line: The BRCA1-miR-593-5p-MFF axis also affected cisplatin sensitivity in vivo.Importantly, in a retrospective analysis of multiple centers, we further found that the BRCA1-miR-593-5p-MFF axis was significantly associated with cisplatin sensitivity and the survival of patients with TSCC.Together, our data reveal a model for mitochondrial fission regulation at the transcriptional and post-transcriptional levels; we also reveal a new pathway for BRCA1 in determining cisplatin sensitivity through the mitochondrial fission program.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.

ABSTRACT
Cisplatin has been widely employed as a cornerstone chemotherapy treatment for a wide spectrum of solid neoplasms; increasing tumor responsiveness to cisplatin has been a topic of interest for the past 30 years. Strong evidence has indicated that mitochondrial fission participates in the regulation of apoptosis in many diseases; however, whether mitochondrial fission regulates cisplatin sensitivity remains poorly understood. Here, we show that MFF mediated mitochondrial fission and apoptosis in tongue squamous cell carcinoma (TSCC) cells after cisplatin treatment and that miR-593-5p was downregulated in this process. miR-593-5p attenuated mitochondrial fission and cisplatin sensitivity by targeting the 3' untranslated region sequence of MFF and inhibiting its translation. In exploring the underlying mechanism of miR-593-5p downregulation, we observed that BRCA1 transactivated miR-593-5p expression and attenuated cisplatin sensitivity in vitro. The BRCA1-miR-593-5p-MFF axis also affected cisplatin sensitivity in vivo. Importantly, in a retrospective analysis of multiple centers, we further found that the BRCA1-miR-593-5p-MFF axis was significantly associated with cisplatin sensitivity and the survival of patients with TSCC. Together, our data reveal a model for mitochondrial fission regulation at the transcriptional and post-transcriptional levels; we also reveal a new pathway for BRCA1 in determining cisplatin sensitivity through the mitochondrial fission program.

No MeSH data available.


Related in: MedlinePlus

BRCA1 transactivates miR-593-5pA, BRCA1 was analyzed using immunoblotting in Cal-27 cells under cisplatin treatment. B, BRCA1 attenuated the cisplatin-induced decrease of miR-593-5p. Cal-27 cells were transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) and then treated with cisplatin for 24h. miR-593-5p were detected using qRT-PCR (upper panel), whereas BRCA1 was analyzed using immunoblotting (lower panel). *P< 0.01 versus cisplatin alone. C, ChIP-qPCR analysis of BRCA1 binding to the promoter of miR-593-5p in the BS3 region. **P< 0.001. D, ChIP-qPCR analysis of the association levels of BRCA1 with the miR-593-5p promoter in the BS3 region under cisplatin treatment. E, A luciferase assay indicated that cisplatin induced a reduction of miR-593-5p promoter activity in the BS3 region. Cal-27 cells were transfected with the wild-type promoter (wt) in the BS3 or empty vector (pGL3-Basic). F, A luciferase assay indicated that BRCA1 activated miR-593-5p promoter activity in the BS3 region. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) were treated with the wild-type promoter (wt) or a promoter with mutations in the BS3 (mut). **P< 0.001.
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Figure 4: BRCA1 transactivates miR-593-5pA, BRCA1 was analyzed using immunoblotting in Cal-27 cells under cisplatin treatment. B, BRCA1 attenuated the cisplatin-induced decrease of miR-593-5p. Cal-27 cells were transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) and then treated with cisplatin for 24h. miR-593-5p were detected using qRT-PCR (upper panel), whereas BRCA1 was analyzed using immunoblotting (lower panel). *P< 0.01 versus cisplatin alone. C, ChIP-qPCR analysis of BRCA1 binding to the promoter of miR-593-5p in the BS3 region. **P< 0.001. D, ChIP-qPCR analysis of the association levels of BRCA1 with the miR-593-5p promoter in the BS3 region under cisplatin treatment. E, A luciferase assay indicated that cisplatin induced a reduction of miR-593-5p promoter activity in the BS3 region. Cal-27 cells were transfected with the wild-type promoter (wt) in the BS3 or empty vector (pGL3-Basic). F, A luciferase assay indicated that BRCA1 activated miR-593-5p promoter activity in the BS3 region. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) were treated with the wild-type promoter (wt) or a promoter with mutations in the BS3 (mut). **P< 0.001.

Mentions: First, we observed that BRCA1 was downregulated under cisplatin stress in Cal-27 cells (Figure 4A). BRCA1 siRNA (Supplementary Figure S5B) or the enforced expression of BRCA1 (Supplementary Figure S5C) downregulated or upregulated miR-593-5p expression, respectively, and expressing exogenous BRCA1 attenuated the cisplatin-induced downregulation of miR-593-5p (Figure 4B). Furthermore, a chromatin immunoprecipitation (ChIP) quantitative PCR (qPCR) assay revealed that BRCA1 bound to binding site 3 (BS3) but not to the other binding sites under physiological conditions (Figure 4C). Cisplatin treatment led to a reduced association of BRCA1 with the miR-593-5p promoter in the BS3 region (Figure 4D); the luciferase reporter assay also demonstrated reduced miR-593-5p promoter activity after cisplatin exposure (Figure 4E). BRCA1 overexpression increased miR-593-5p promoter activity; this enhancement was reversed through mutations introduced into the BS3 region (Figure 4F). miR-593-5p is located within the intron of the SND1 gene, but SND1 mRNA levels were not substantially altered after cisplatin treatment (Supplementary Figure S5D) or BRCA1 overexpression (Supplementary Figure S5E). Together, these data suggest that BRCA1 can positively regulate miR-593-5p expression in Cal-27 cells.


Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1-miR-593-5p-MFF axis.

Fan S, Liu B, Sun L, Lv XB, Lin Z, Chen W, Chen W, Tang Q, Wang Y, Su Y, Jin S, Zhang D, Zhong J, Li Y, Wen B, Zhang Z, Yang P, Zhou B, Liang Q, Yu X, Zhu Y, Hu P, Chu J, Huang W, Feng Y, Peng H, Huang Q, Song E, Li J - Oncotarget (2015)

BRCA1 transactivates miR-593-5pA, BRCA1 was analyzed using immunoblotting in Cal-27 cells under cisplatin treatment. B, BRCA1 attenuated the cisplatin-induced decrease of miR-593-5p. Cal-27 cells were transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) and then treated with cisplatin for 24h. miR-593-5p were detected using qRT-PCR (upper panel), whereas BRCA1 was analyzed using immunoblotting (lower panel). *P< 0.01 versus cisplatin alone. C, ChIP-qPCR analysis of BRCA1 binding to the promoter of miR-593-5p in the BS3 region. **P< 0.001. D, ChIP-qPCR analysis of the association levels of BRCA1 with the miR-593-5p promoter in the BS3 region under cisplatin treatment. E, A luciferase assay indicated that cisplatin induced a reduction of miR-593-5p promoter activity in the BS3 region. Cal-27 cells were transfected with the wild-type promoter (wt) in the BS3 or empty vector (pGL3-Basic). F, A luciferase assay indicated that BRCA1 activated miR-593-5p promoter activity in the BS3 region. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) were treated with the wild-type promoter (wt) or a promoter with mutations in the BS3 (mut). **P< 0.001.
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Figure 4: BRCA1 transactivates miR-593-5pA, BRCA1 was analyzed using immunoblotting in Cal-27 cells under cisplatin treatment. B, BRCA1 attenuated the cisplatin-induced decrease of miR-593-5p. Cal-27 cells were transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) and then treated with cisplatin for 24h. miR-593-5p were detected using qRT-PCR (upper panel), whereas BRCA1 was analyzed using immunoblotting (lower panel). *P< 0.01 versus cisplatin alone. C, ChIP-qPCR analysis of BRCA1 binding to the promoter of miR-593-5p in the BS3 region. **P< 0.001. D, ChIP-qPCR analysis of the association levels of BRCA1 with the miR-593-5p promoter in the BS3 region under cisplatin treatment. E, A luciferase assay indicated that cisplatin induced a reduction of miR-593-5p promoter activity in the BS3 region. Cal-27 cells were transfected with the wild-type promoter (wt) in the BS3 or empty vector (pGL3-Basic). F, A luciferase assay indicated that BRCA1 activated miR-593-5p promoter activity in the BS3 region. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) were treated with the wild-type promoter (wt) or a promoter with mutations in the BS3 (mut). **P< 0.001.
Mentions: First, we observed that BRCA1 was downregulated under cisplatin stress in Cal-27 cells (Figure 4A). BRCA1 siRNA (Supplementary Figure S5B) or the enforced expression of BRCA1 (Supplementary Figure S5C) downregulated or upregulated miR-593-5p expression, respectively, and expressing exogenous BRCA1 attenuated the cisplatin-induced downregulation of miR-593-5p (Figure 4B). Furthermore, a chromatin immunoprecipitation (ChIP) quantitative PCR (qPCR) assay revealed that BRCA1 bound to binding site 3 (BS3) but not to the other binding sites under physiological conditions (Figure 4C). Cisplatin treatment led to a reduced association of BRCA1 with the miR-593-5p promoter in the BS3 region (Figure 4D); the luciferase reporter assay also demonstrated reduced miR-593-5p promoter activity after cisplatin exposure (Figure 4E). BRCA1 overexpression increased miR-593-5p promoter activity; this enhancement was reversed through mutations introduced into the BS3 region (Figure 4F). miR-593-5p is located within the intron of the SND1 gene, but SND1 mRNA levels were not substantially altered after cisplatin treatment (Supplementary Figure S5D) or BRCA1 overexpression (Supplementary Figure S5E). Together, these data suggest that BRCA1 can positively regulate miR-593-5p expression in Cal-27 cells.

Bottom Line: The BRCA1-miR-593-5p-MFF axis also affected cisplatin sensitivity in vivo.Importantly, in a retrospective analysis of multiple centers, we further found that the BRCA1-miR-593-5p-MFF axis was significantly associated with cisplatin sensitivity and the survival of patients with TSCC.Together, our data reveal a model for mitochondrial fission regulation at the transcriptional and post-transcriptional levels; we also reveal a new pathway for BRCA1 in determining cisplatin sensitivity through the mitochondrial fission program.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.

ABSTRACT
Cisplatin has been widely employed as a cornerstone chemotherapy treatment for a wide spectrum of solid neoplasms; increasing tumor responsiveness to cisplatin has been a topic of interest for the past 30 years. Strong evidence has indicated that mitochondrial fission participates in the regulation of apoptosis in many diseases; however, whether mitochondrial fission regulates cisplatin sensitivity remains poorly understood. Here, we show that MFF mediated mitochondrial fission and apoptosis in tongue squamous cell carcinoma (TSCC) cells after cisplatin treatment and that miR-593-5p was downregulated in this process. miR-593-5p attenuated mitochondrial fission and cisplatin sensitivity by targeting the 3' untranslated region sequence of MFF and inhibiting its translation. In exploring the underlying mechanism of miR-593-5p downregulation, we observed that BRCA1 transactivated miR-593-5p expression and attenuated cisplatin sensitivity in vitro. The BRCA1-miR-593-5p-MFF axis also affected cisplatin sensitivity in vivo. Importantly, in a retrospective analysis of multiple centers, we further found that the BRCA1-miR-593-5p-MFF axis was significantly associated with cisplatin sensitivity and the survival of patients with TSCC. Together, our data reveal a model for mitochondrial fission regulation at the transcriptional and post-transcriptional levels; we also reveal a new pathway for BRCA1 in determining cisplatin sensitivity through the mitochondrial fission program.

No MeSH data available.


Related in: MedlinePlus