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. 2014 Apr 1;20(7):1990-2000.
doi: 10.1158/1078-0432.CCR-13-2805. Epub 2014 Feb 10.

V体育平台登录 - Reduced expression of miRNA-27a modulates cisplatin resistance in bladder cancer by targeting the cystine/glutamate exchanger SLC7A11

Ross M Drayton  1 , Ewa Dudziec, Stefan Peter, Simone Bertz, Arndt Hartmann, Helen E Bryant, James Wf Catto
Affiliations

Reduced expression of miRNA-27a modulates cisplatin resistance in bladder cancer by targeting the cystine/glutamate exchanger SLC7A11

"VSports在线直播" Ross M Drayton et al. Clin Cancer Res. .

Abstract

Purpose: Resistance to cisplatin-based chemotherapy is a major obstacle to bladder cancer treatment VSports手机版. We aimed to identify microRNAs (miRNA) that are dysregulated in cisplatin-resistant disease, ascertain how these contribute to a drug-resistant phenotype, and how this resistance might be overcome. .

Experimental design: miRNA expression in paired cisplatin-resistant and -sensitive cell lines was measured. Dysregulated miRNAs were further studied for their ability to mediate resistance. The nature of the cisplatin-resistant phenotype was established by measurement of cisplatin/DNA adducts and intracellular glutathione (GSH) V体育安卓版. Candidate miRNAs were examined for their ability to (i) mediate resistance and (ii) alter the expression of a candidate target protein (SLC7A11); direct regulation of SLC7A11 was confirmed using a luciferase assay. SLC7A11 protein and mRNA, and miRNA-27a were quantified in patient tumor material. .

Results: A panel of miRNAs were found to be dysregulated in cisplatin-resistant cells. miRNA-27a was found to target the cystine/glutamate exchanger SLC7A11 and to contribute to cisplatin resistance through modulation of GSH biosynthesis. In patients, SLC7A11 expression was inversely related to miRNA-27a expression, and those tumors with high mRNA expression or high membrane staining for SLC7A11 experienced poorer clinical outcomes. Resistant cell lines were resensitized by restoring miRNA-27a expression or reducing SLC7A11 activity with siRNA or with sulfasalazine. V体育ios版.

Conclusion: Our findings indicate that miRNA-27a negatively regulates SLC7A11 in cisplatin-resistant bladder cancer, and shows promise as a marker for patients likely to benefit from cisplatin-based chemotherapy. SLC7A11 inhibition with sulfasalazine may be a promising therapeutic approach to the treatment of cisplatin-resistant disease. VSports最新版本.

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Figures (V体育2025版)

Figure 1
Figure 1. Characterization of cisplatin resistant cell lines and microRNA expression
(a) Clonogenic survival assays of cisplatin-treated bladder cancer cell lines and their cisplatin-resistant derivatives (designated as respective cell line followed by R). Error bars indicate SD, n=4 (b) Fold change in expression of c. 160 microRNAs in three paired sensitive and resistant lines. (c) Hierarchical clustering using 32 selected miRs (red = low expression, green = high expression) stratifies cells according to cisplatin resistance (orange – resistant, white – sensitive) regardless of cell line or method of derivation.
Figure 2
Figure 2. Genotoxicty of cisplatin and measurement of cisplatin/DNA adducts in sensitive and resistant cells
(a) Measurement of 1,2 (G,G) Intrastrand Cisplatin-DNA adducts in genomic DNA of EJ and EJ-R cells following a two hour treatment with a range of cisplatin concentrations. Error bars indicate SD, n=3. p<0.001 for all concentrations of cisplatin above 1 μM (b) Densitromic plot of extent of 1,2 (G,G) Intrastrand Cisplatin-DNA adduct repair in EJ / EJ-R for 48 hours following a two hour treatment with 12 μM cisplatin. Error bars indicate SD, n=3 (c) Example dot blot used to generate data visualized in Fig 3B.
Fig 3
Fig 3. Analysis of candidate pathways mediating cisplatin resistance in sensitive and resistant cells
(a) Change in expression of three mRNAs concerned with cisplatin import (CTR1) and export (ATP7A, ATP7B) in three paired cisplatin sensitive (EJ, D4, G7) and resistant (EJ-R, D4-R, G7-R) cell lines. (b) Measurement of intracellular glutathione (Reduced glutathione/GSH – black bars, Total glutathione GSH and GSSG, white bars) in EJ bladder cancer cells and their cisplatin-resistant derivative line EJ. Error bars indicate SD, n=3, * p<0.01 **p<0.0005. (c) Change in expression of six mRNAs concerned with glutathione biosynthesis in three paired cisplatin sensitive (EJ, D4, G7) and resistant (EJ-R, D4-R, G7-R) cell lines. (d) Western blot of proteins concerned with cisplatin import and export (ATP7A (163 kDa), CTR1 (35 kDa), SLC7A11 (55 kDa) against α-tubulin (55kDa)) in three paired cisplatin sensitive (EJ, D4, G7) and resistant (EJ-R, D4-R, G7-R) cell lines.
Figure 4
Figure 4. Effect of microRNA-27a manipulation on SLC7A11 expression, cisplatin sensitivity and cisplatin genotoxicity in resistant bladder and ovarian cancer cells
(a) Western blot of SLC7A11 expression following transfection of cisplatin-resistant cells with microRNA-27a, microRNA-25 or microRNA-32, (mIR-27a, -25, -32). (b) Effect of increased microRNA-27a expression on cisplatin sensitivity of EJ-R cells compared to a scrambled control oligo as measured by clonogenic assay. Error bars indicate SD, n=3 (c) Effect of increased microRNA-27a expression of level of cisplatin induced DNA damage in EJ-R cells compared to untreated and scrambled RNA controls. Error bars indicate SD, n=3 (d) Effect of increased microRNA-27a expression on levels of reduced (black bars) and total (white bars) glutathione *= p<0.01. Error bars indicate SD, n=3 (e) Effect on luciferase expression of a microRNA-27a inhibitor in HEK293 cells compared to scrambled control . The luciferase gene was augmented with a portion of the 3′UTR of SLC7A11 containing the microRNA-27a binding site, or a similar construct with the microRNA-27a abolished by site-directed mutagenesis (mutant). (f) Effect of increased microRNA-27a expression (miR-27a) or transfection with a scrambled control (Scrm) on cisplatin sensitivity the cisplatin resistant ovarian cancer cell line A2780-cDDP as measured by clonogenic assay. Error bars indicate SD, n=3
Figure 5
Figure 5. Effect of SLC7A11 knockdown and inhibition on cisplatin sensitivity
(a) Use of siRNA directed against SLC7A11 to reduce SLC7A11 protein levels in EJ-R cells 48 hours post transfection. (b) Effect of SLC7A11 knockdown compared with scrambled RNA control on cisplatin sensitivity of EJR cells as measured by clonogenic assay. Error bars indicate SD, n=3. *** = p<0.005 (e) Effect of sulfasalazine pretreatment on cisplatin sensitivity of EJ-R cells as measured by clonogenic assay. Error bars indicate SD, n=3 * p<0.1
Fig 6
Fig 6. Clinical significance of decreased miR-27a/b expression and increased SLC7A11 expression in bladder tumours
(a) Expression of miR-27a/27b and SLC7A11 mRNA in bladder cancer samples as determined by real-time PCR. (b). Correlation between low microRNA-27a expression (red line) and an aggressive tumor phenotype as shown by progression to invasion and metastases following treatment in 139 freshly frozen tumors. (c). High membranous SLC7A11 protein expression was identified in 10.5% of tumors (examples shown of high (100x and 630x) and absent expression (630x)) in a tissue array of tumors from patients treated with adjuvant chemotherapy for invasive bladder cancer. (d) SLC7A11 protein expression stratifies progression free and cancer specific survival (CSS) in patients treated with this agent within an RCT of cisplatin-based regimens. (*P<0.05, ** P<0.001)
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