br Abbreviations lncRNA long noncoding RNA LNA locked nucleic

Abbreviations: lncRNA, long noncoding RNA; LNA, locked nucleic acid; BCAG, breast cancer associated genes
Corresponding author at: Department of Biochemistry, College of Veterinary Medicine, Seoul National University, Gwanak-ro1, Gwanak-gu, Seoul 151-742, South Korea.
1 Both authors contributed equally to this study.
immune responses, and publicly shared the resulting Compound C profiles in diverse cancers including breast cancer (Quinn and Chang, 2016). There indeed are several publicly available databases that allow users to search for and download lncRNA sequences and functional char-acteristics, as well as expression profiles in various diseases, including cancers (Amaral et al., 2011; Li et al., 2015; Wapinski and Chang, 2011). Yet, despite growing in silico resources regarding expression profiles of lncRNAs in various cancers, the functions of most lncRNAs in breast cancer remain unclear. Thus, determining the target protein coding genes regulated by oncogenic lncRNA might provide a starting point for an extensive study.
The locally associated expression interference relationship between anti-sense RNAs and protein coding genes has been reported (Fu et al., 2015; Vance et al., 2014). The naturally occurring antisense RNAs can modulate the expression level of the sense mRNA through processes such as transcriptional interference, RNA splicing, editing and short RNAs. The antisense RNAs are often subdivided into cis- and trans-, depending on the regulation mechanism of the target gene's expression. Although potential regulatory mechanisms of both cis- and trans-anti-sense RNAs are unknown, cis- antisense RNA's mechanism used to be defined as the suppressive influence of one transcriptional process on an adjacent transcriptional process through the interfering RNA poly-merase (RNAP) DNA trafficking (Wierzbicki, 2012). Moreover, anti-sense RNAs transcribed from opposite strands of the DNA tend to have a potential to bind with corresponding sense transcripts resulting in transcription attenuation, RNA degradation or translational inhibition (Zhang et al., 2018). Directly- or reversely-correlated lncRNA expres-sions with the expression of neighboring protein coding genes have been reported in several different processes of development and dis-eases (Lin et al., 2016; Xue et al., 2016). Thus, unravelling the profiles of these lncRNAs in cancer is essential and urgent.
The aim of this study was to identify putative oncogenic lncRNAs, of which the expression might be linked to adjacent genes that were previously documented as oncogenic driver genes or are associated with the tumorigenesis and prognosis of breast cancer. In this study, we surveyed lncRNAs closely located near the genes associated with breast cancer, and validated their expression in five different human breast cancer cell lines. Thus, this study and approach may give a new and efficient insight to initiate the functional study of lncRNA in breast cancer.
2. Materials and methods
2.1. In silico lncRNA survey
Breast cancer associated genes were identified from the literature review, using “breast cancer,” “gene,” or “gene expression” search terms in PubMed, UCSC and Ensembl. Public cancer gene expression databases, such as LNCpedia and Malacards, were also surveyed (https://lncipedia.org/), (https://www.malacards.org/). LncRNAs within a 15 kb up- and downstream region of the breast cancer asso-ciated genes were analyzed. The overall procedure and conceptual scheme were illustrated in Fig. 1, and breast cancer associated genes (BCAG) and adjacent lncRNAs found in this study were summarized in Table S1.
MCF-7, MDA-MB-231 and MDA-MB-436 cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) (HyClone, GE Healthcare Life Sciences, PA, USA), containing 10% fetal bovine serum (FBS) (JCBIO Co.LTD, Korea) and 1% antimycotic antibiotic (Gibco, MA, USA), while MCF-10A was maintained in DME/ F12(HyClone) containing 10% FBS and 1% antimycotic antibiotic. SK-BR3 cell lines were maintained in RPMI with 10% FBS and 1% antimycotic antibiotic.