Identification And Characterization Of Estrogen-induced Circular RNAs (circRNAs) In Human Breast Cancer

Breast cancer has become the second leading cause of death for women worldwide and the first in China due to cancer.Around seventy percent of breast cancers are estrogen receptor(ER)-positive.Aberrant levels of estrogen are thought to be closely relatedtightly linked to the development of breast cancer.Upon estrogen stimulation,estrogen receptor,along with other coactivators binds to estrogen response elements(EREs)on chromatin DNA,and modulates gene transcription,leading to tissue proliferation,and differentiation.Estrogen/estrogen receptor are found to activate a large program of transcriptional units,including coding genes and noncoding RNAs(ncRNAs),such as microRNAs(miRNAs)and long non-coding RNAs(IncRNAs).Circular RNA(circRNA)are emerging as a new family of functional ncRNAs that is highly represented,conserved,and often exhibit tissue-and developmental stage-specific expression in the eukaryotic transcriptome.However,whether circRNAs are subjected to the regulation of estrogen/estrogen receptor,and if so,what are the functions and mechanisms of these circRNAs in estrogen/estrogen receptor-mediated signaling pathways and the development of breast cancer remain unknown;In the current study,through high throughput sequencing in an ER-positive breast cancer cell line,we have identified a large number of circRNAs which are regulated by estrogen,which were further validated by quantitative PCR(qPCR),standard PCR,RNase R-resistant assay and sanger sequencing.We then focused on studying one of the highly-induced circRNA originated from PGR gene,which we named as circPGR.CircPGR was found to localize mainly in the cytosol in the cell.Functional study revealed that circPGR promotes the growth of MCF7 cells and facilities cells transiting from G1 to S phase.To investigate the molecular mechanisms underlying circPGR regulation of cell growth,RNA-seq(RNA sequencing)was performed in MCF7 cells,finding that one of the most enriched gene ontology(GO)terms for genes affected by circPGR was cell cycle regulation.Finally,circPGR was found to be specifically expressed in ER-positive breast cancer cells among in different mammary gland cells of fourteen different types of mammary cells tested.In conclusion,the current study has revealed an estrogen-induced circRNA,circPGR,which is specifically expressed in and promotes the growth of ER-positive cancer cell.Owing to its unique feature of high stability,circRNAs,such as circPGR,may serve as a new diagnosis marker and therapeutic target for breast cancer...