BRRIAR lncRNA alters breast cancer risk by modulating interferon signaling in cis and in trans through BHLHE40 and RIG-I

Interferons (IFNs) are key regulators of cell proliferation and anti-tumor immunity. We identified a breast cancer-associated long noncoding RNA (lncRNA), BRRIAR, that modulates IFN signaling in estrogen receptor-positive (ER+) breast cancer. BRRIAR is transcribed from an 11 kb enhancer cluster at 3p26, and its reduced expression is linked to breast cancer GWAS risk variants. Primarily expressed in ER+ breast tumors, BRRIAR exhibits dual functionality, acting both in cis and in trans. Nuclear BRRIAR regulates BHLHE40 expression through its enhancer, while cytoplasmic BRRIAR binds to the pattern recognition receptor RIG-I, modulating its activation. BRRIAR RNA overexpression activates RIG-I signaling, inducing IFN responses that selectively trigger apoptosis in ER+ breast tumor cells in vitro and in vivo, while promoting immune activation in human peripheral blood mononuclear cells. These findings emphasize the complex regulatory mechanisms at GWAS risk regions, reveal the critical role of lncRNAs as modulators of tumor immunity and identify BRRIAR as a promising RNA-based therapeutic for ER+ breast cancer. H3K27ac ChIPseq data in T47D cells after CRISPRi-BRRIAR or using gRNAs targeted to the 3’ end of the enhancer cluster. T47D cells were cross-linked with 1% formaldehyde (Merck) at 37oC for 10 min, rinsed with phosphate buffered saline (PBS; TMO) containing 5% bovine serum albumin (BSA; Scientifix) and harvested in PBS containing protease inhibitor cocktail (PIC; Roche). Harvested cells were centrifuged for 2 min at 3,000 rpm. Cell pellets were resuspended in lysis buffer (1% sodium dodecyl sulfate (SDS; Merck), 10 mM EDTA (TMO), 50 mM Tris-HCl, pH 8.1, PIC) and sonicated three times for 15 s at 70% duty cycle (Branson SLPt) then centifuged at 13,000 rpm for 15 min. Supernatants were resuspended in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1). Antibodies detecting H3K27ac (2 μg; Abcam) or an IgG control (2 μg; Abcam) were prebound for 6 h to protein G Dynabeads (TMO), then incubated with the chromatin for 16 h. The complexes were then washed six times in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate (Merck), 0.1% SDS, 1 mM DTT, PIC). To reverse cross-links, the complexes were incubated at 65oC for 16 h in elution buffer (1% SDS, 0.1 M NaHCO3), then DNA fragments purified using the QIAquick spin kit (Qiagen). Libraries were prepared using the NEBNext Ultra II DNA library prep kit (NEB), according to manufacturer’s instructions.

干扰素（Interferons, IFNs）是细胞增殖与抗肿瘤免疫的关键调控因子。本研究鉴定出一种与乳腺癌相关的长链非编码RNA（long noncoding RNA, lncRNA）BRRIAR，其可在雌激素受体阳性（estrogen receptor-positive, ER+）乳腺癌中调控IFN信号通路。BRRIAR由3p26染色体区域的11 kb增强子簇转录而来，其表达下调与乳腺癌全基因组关联研究（genome-wide association study, GWAS）的风险变异相关。BRRIAR主要在ER+乳腺肿瘤中表达，兼具顺式与反式调控双重功能。定位于细胞核的BRRIAR可通过其增强子调控BHLHE40的表达，而胞质BRRIAR则可结合模式识别受体RIG-I并调控其激活。过表达BRRIAR RNA可激活RIG-I信号通路，诱导IFN应答，进而在体内外选择性诱导ER+乳腺肿瘤细胞发生凋亡，同时可促进人外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）的免疫活化。本研究结果阐明了GWAS风险区域存在的复杂调控机制，揭示了长链非编码RNA作为肿瘤免疫调控因子的关键作用，并确定BRRIAR是一种颇具前景的ER+乳腺癌RNA靶向治疗手段。本数据集涵盖经CRISPRi干扰BRRIAR，或使用靶向增强子簇3'端的向导RNA（guide RNA, gRNA）处理的T47D细胞的H3K27乙酰化染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）数据，具体实验流程如下：T47D细胞经1%甲醛（Merck）于37℃交联10分钟后，用含5%牛血清白蛋白（bovine serum albumin, BSA; Scientifix）的磷酸盐缓冲液（phosphate buffered saline, PBS; TMO）冲洗，随后收集于含蛋白酶抑制剂混合物（protease inhibitor cocktail, PIC; Roche）的PBS中。收集的细胞以3000 rpm离心2分钟。细胞沉淀重悬于裂解缓冲液（1%十二烷基硫酸钠sodium dodecyl sulfate, SDS; Merck、10 mM EDTA (TMO)、50 mM Tris-HCl，pH 8.1及PIC）中，随后以70%占空比超声破碎3次，每次15秒（Branson SLPt超声仪），再以13000 rpm离心15分钟。上清液重悬于稀释缓冲液（1% Triton X-100、2 mM EDTA、150 mM NaCl、20 mM Tris-HCl，pH 8.1）中。将识别H3K27ac的抗体（2 μg; Abcam）或IgG对照抗体（2 μg; Abcam）与蛋白G磁珠（Dynabeads; TMO）预结合6小时，随后与染色质样本共孵育16小时。复合物随后用RIPA缓冲液（50 mM Tris-HCl，pH 8.0、150 mM NaCl、1% IGEPAL CA-630、0.5%脱氧胆酸钠sodium deoxycholate; Merck、0.1% SDS、1 mM DTT及PIC）洗涤6次。为逆转交联，将复合物置于洗脱缓冲液（1% SDS、0.1 M NaHCO3）中65℃孵育16小时，随后使用QIAquick离心柱试剂盒（Qiagen）纯化DNA片段。按照试剂盒说明书，使用NEBNext Ultra II DNA文库制备试剂盒（NEB）构建测序文库。