Racial differences in RAD51 expression are regulated by miRNA-214-5P and its inhibition synergizes with olaparib in triple-negative breast cancer.
Racial differences in RAD51 expression are regulated by miRNA-214-5P and its inhibition synergizes with olaparib in triple-negative breast cancer.
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DOI：
10.1186/s13058-023-01615-6
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发表时间：
2023-04-20
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--
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三阴性乳腺癌（TNBC）影响年轻女性，是乳腺癌（BC）中最具侵袭性的亚型。与其他种族相比，TNBC对非裔美国人（AA）血统的妇女的影响更大。我们已经确定了DNA修复基因RAD 51作为TNBC的不良预后标志物，并通过microRNAs（miRNAs）进行转录后调控。本研究旨在描述导致RAD 51上调的机制，并开发新的治疗组合以有效治疗TNBC并减少临床结果的差异。使用UALCAN门户和PrognoScan分析BC群组的TCGA数据，鉴定了TNBC中的RAD 51过表达。miRNA测序鉴定了RAD 51靶向miRNA miR-214- 5 P和miR-142- 3 P的显著下调。RT-PCR测定用于验证miRNA和RAD 51的水平，并且免疫组织化学和免疫印迹技术类似地用于TNBC组织和细胞系中的RAD 51蛋白水平。在RAD 51 3 '-UTR的控制下进行荧光素酶测定以证实miR-214- 5 P调节RAD 51表达。为了检查miR-214- 5 P介导的RAD 51下调对TNBC细胞中同源重组（HR）的影响，进行Dr-GFP报告基因测定。为了评估miR-214- 5 P中奥拉帕尼诱导的DNA损伤应答的水平，使用转染的细胞、免疫印迹和免疫荧光测定。此外，使用COMET测定来测量DNA损伤，并进行集落测定来评估BRCA活性TNBC细胞对奥拉帕尼的敏感性。计算机模拟分析将RAD 51的上调鉴定为TNBC中的不良预后标志物。miRNA-seq数据显示，与高加索-美国（CA）女性相比，源自AA女性的TNBC细胞系中miR-214- 5 P和miR-142- 3 P显著下调。在这些细胞系中，miR-214- 5 P模拟下调的RAD 51表达并诱导HR缺陷，如通过Dr-GFP测定所测量的。基于这些结果，我们使用克隆形成存活测定设计了miR-214- 5 P和奥拉帕尼在HR-熟练AA TNBC细胞系中的组合治疗。在这些细胞系中，与单独处理相比，miR-214- 5 P和奥拉帕尼的组合显示出协同致死性。我们的研究鉴定了miR-214- 5 P对TNBC中的RAD 51的新的表观遗传调节，这表明涉及miR-214- 5 P和奥拉帕尼的新的组合疗法用于治疗HR熟练的TNBC并减少治疗结果的种族差异。在线版本包含补充材料，可通过10.1186/s13058-023-01615-6获得。
Triple-negative breast cancer (TNBC) affects young women and is the most aggressive subtype of breast cancer (BC). TNBCs disproportionally affect women of African-American (AA) descent compared to other ethnicities. We have identified DNA repair gene RAD51 as a poor prognosis marker in TNBC and its posttranscriptional regulation through microRNAs (miRNAs). This study aims to delineate the mechanisms leading to RAD51 upregulation and develop novel therapeutic combinations to effectively treat TNBCs and reduce disparity in clinical outcomes. Analysis of TCGA data for BC cohorts using the UALCAN portal and PrognoScan identified the overexpression of RAD51 in TNBCs. miRNA sequencing identified significant downregulation of RAD51-targeting miRNAs miR-214-5P and miR-142-3P. RT-PCR assays were used to validate the levels of miRNAs and RAD51, and immunohistochemical and immunoblotting techniques were used similarly for RAD51 protein levels in TNBC tissues and cell lines. Luciferase assays were performed under the control of RAD51 3’-UTR to confirm that miR-214-5P regulates RAD51 expression. To examine the effect of miR-214-5P-mediated downregulation of RAD51 on homologous recombination (HR) in TNBC cells, Dr-GFP reporter assays were performed. To assess the levels of olaparib-induced DNA damage responses in miR-214-5P, transfected cells, immunoblots, and immunofluorescence assays were used. Furthermore, COMET assays were used to measure DNA lesions and colony assays were performed to assess the sensitivity of BRCA-proficient TNBC cells to olaparib. In-silico analysis identified upregulation of RAD51 as a poor prognostic marker in TNBCs. miRNA-seq data showed significant downregulation of miR-214-5P and miR-142-3P in TNBC cell lines derived from AA women compared to Caucasian-American (CA) women. miR-214-5P mimics downregulated RAD51 expression and induces HR deficiency as measured by Dr-GFP assays in these cell lines. Based on these results, we designed a combination treatment of miR-214-5P and olaparib in HR-proficient AA TNBC cell lines using clonogenic survival assays. The combination of miR-214-5P and olaparib showed synergistic lethality compared to individual treatments in these cell lines. Our studies identified a novel epigenetic regulation of RAD51 in TNBCs by miR-214-5P suggesting a novel combination therapies involving miR-214-5P and olaparib to treat HR-proficient TNBCs and to reduce racial disparity in therapeutic outcomes. The online version contains supplementary material available at 10.1186/s13058-023-01615-6.
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影响因子：
64.8
作者：
Ray Chaudhuri A;Callen E;Ding X;Gogola E;Duarte AA;Lee JE;Wong N;Lafarga V;Calvo JA;Panzarino NJ;John S;Day A;Crespo AV;Shen B;Starnes LM;de Ruiter JR;Daniel JA;Konstantinopoulos PA;Cortez D;Cantor SB;Fernandez-Capetillo O;Ge K;Jonkers J;Rottenberg S;Sharan SK;Nussenzweig A
通讯作者：
Nussenzweig A
DOI：
10.1186/s40661-017-0039-8
发表时间：
2017
期刊：
Gynecologic oncology research and practice
影响因子：
--
作者：
Frey MK;Pothuri B
通讯作者：
Pothuri B
影响因子：
50.5
作者：
Cardoso, F.;Harbeck, N.;Gelmon, K. A.
通讯作者：
Gelmon, K. A.
影响因子：
3.1
作者：
Christiansen, Neal;Chen, Lei;Szabo, Stephen
通讯作者：
Szabo, Stephen
影响因子：
11.2
作者：
Carey JPW;Karakas C;Bui T;Chen X;Vijayaraghavan S;Zhao Y;Wang J;Mikule K;Litton JK;Hunt KK;Keyomarsi K
通讯作者：
Keyomarsi K