Identification And Characterization Of Estrogen-induced Long Non-coding RNAs(lncRNAs) In Human Breast Cancer

Breast cancer is now the leading cause of death due to cancer in women worldwide.Treatment decisions which were based predominantly on the anatomic extent of the disease in the past are now shifting to the underlying molecular mechanisms.While new approaches are continuously being introduced to treat breast cancer,many people are still suffering from this disease.Deeper understanding of the molecular mechanisms underlying the initiation and development of this disease would help to achieve more effective measures for the early prevention,diagnosis,and treatment.ER-positive breast carcinoma is one of the most commonly diagnosed subtypes and often treated with ER antagonists or inhibitors designed for estrogen synthesis,but therapy resistance is often developed in most tumors after treatment.In this respect,we need to better illustrate and understand the signaling pathways and transcriptional programs underlying the proliferative and tumorigenic roles of estrogen and estrogen receptor.Long non-coding RNAs(lncRNAs),which are widely distributed in mammals,are a class of RNA molecules with more than 200 nucleotides that function as RNAs with little or no protein-coding capacity.Large-scale analyses of the mammalian transcriptome have shown that the number and types of lncRNAs far exceed those of protein-coding mRNAs.Increasing evidence indicates that lncRNAs play important roles in a variety of biological processes through complicated mechanisms.Aberrant expression of IncRNAs has shown to be associated with several subtypes of cancer,especially in breast carcinoma research.However,a small proportion of IncRNAs has been characterized to exert cellular biological functions,especially in hormone-induced breast carcinogenesis..Therefore,investigation of the biological roles of these lncRNAs will futher advance our understanding of breast cancer.In this study,we presented a comprehensive list of estrogen-induced lncRNAs in a ER-positive human breast cancer cell line,MCF7,by applying RNA sequencing(RNA-seq)and global run-on sequencing(GRO-seq).It was found that,in addition to more than 4,000 protein-coding genes,estrogen and estrogen receptor regulated approximately 700 IncRNAs,with half of them were positively-regulated.Through a series of functional assays including estrogen response element-driven luciferase reporter assay,cell proliferation assay and cell cycle analysis,we further examined the function of the top-induced lncRNAs.It was found that knockdown of one of the lncRNAs,LINC02568,resulted in attenuated estrogen target gene expression,delayed cell cycle progression and reduced cell proliferation in MCF7 cells.Subcellular fraction shows about 60%of LINC02568 transcripts are located in the nucleus.We further demonstrated that LINC02568 is a direct transcriptional target of estrogen receptor,and its expression was highly specific in breast tumor samples,particularly ER-positive breast cancer subtypes.In summary,a comprehensive list of estrogen-regulated lncRNAs was uncovered by using high throughput sequencing coupled with bioinformatics analysis.Further functional characterization revealed that some of these lncRNAs,such as LINC02568,are involved in estrogen/ER-mediated gene transcriptional regulation and breast cancer cell proliferation.Significantly,LINC02568 was highly expressed in ER-positive breast cancer subtypes,supporting its role in breast carcinogenesis.The identification of the estrogen-induced lncRNA program and their functional importance warrants future investigation,which will provide potential biomarkers and therapeutic targets for ER-positive breast cancers.