Role of MIR-29B-1 and MIR-29A in endocrine-resistant breast cancer.

miRNA-29b-1;miRNA-29a;breast cancer;endocrine-resistance;estrogen receptor;tamoxifen;RNA-seq

Penn Muluhngwi

University:University of Louisville

Department:Biochemistry and Molecular Biology

关键词: miRNA-29b-1;    miRNA-29a;    breast cancer;    endocrine-resistance;    estrogen receptor;    tamoxifen;    RNA-seq;   

Others  :  https://ir.library.louisville.edu/cgi/viewcontent.cgi?article=3732&context=etd

美国|英语

来源: The Universite of Louisville's Institutional Repository

【 摘 要 】

Therapies targeting estrogen receptor α (ERα) including selective estrogen receptor modulators (SERMs), e.g., tamoxifen (TAM); selective estrogen receptor downregulators (SERDs), e.g., fulvestrant (ICI 182,780); and aromatase inhibitors (AI), e.g., letrozole, are successfully used in treating breast cancer patients whose initial tumor expresses ERα. Unfortunately, the effectiveness of endocrine therapies is limited as ~ 40% of breast cancer patients will eventually acquire resistance to them. The role of miRNAs in the progression of endocrine-resistant breast cancer is of keen interest in developing biomarkers and therapies to counter metastatic disease. This dissertation begins with a review on miRNAs implicated in breast cancer, their bona fide gene targets, and associated pathways promoting endocrine resistance. Although microRNAs are dysregulated in breast cancer, their contribution to endocrine-resistance is not yet fully understood. Previous microarray analysis identified miR-29a and miR-29b-1 as repressed by TAM in MCF-7 endocrine-sensitive breast cancer cells but stimulated by TAM in LY2 endocrine-resistant breast cancer cells. Here we examined the mechanism for the differential regulation of these miRs by TAM in MCF-7 versus TAM-resistant LY2 and LCC9 breast cancer cells and the functional role of these microRNAs in these cells. Knockdown studies revealed that ERα is responsible for TAM regulation of miR-29b-1/a transcription. Transient overexpression of miR-29b-1/a decreased MCF-7, LCC9, and LY2 proliferation and inhibited LY2 cell migration and colony formation but did not sensitize LCC9 or LY2 cells to TAM. Furthermore, TAM reduced DICER1 mRNA and protein in LY2 cells, a known target of miR-29. Supporting this observation, anti-miR-29b-1 or anti-miR-29a inhibited the suppression of DICER by 4-OHT. These results suggest that miR-29b-1/a have tumor suppressor activity in TAM-resistant cells and do not appear to play a role in mediating TAM resistance. The target genes mediating miR-29b-1/a tumor suppressor activity were unknown. Here, using RNA sequencing, we identify miR-29b-1 and miR-29a target transcripts in both MCF-7 and LCC9 cells. We find that miR-29b-1 and miR-29a regulate common and unique transcripts in each cell line. The cell-specific and common downregulated genes were characterized using the MetaCore Gene Ontology (GO) enrichment analysis algorithm. LCC9-sepecific miR-29b-1/a-regulated GO processes include oxidative phosphorylation, ATP metabolism, and apoptosis. Extracellular flux analysis of cells transfected with anti- or pre- miR-29a confirmed that miR-29a inhibits mitochondrial bioenergetics in LCC9 cells. qPCR and luciferase reporter assays also verified the ATP synthase subunit genes ATP5G1 and ATPIF1 as bona fide miR29b-1/a targets. Our results suggest that miR-29 repression of TAM-resistant breast