长链非编码RNA-TRA调控乳腺癌内分泌耐药的机制研究

Endocrine therapy with tamoxifen targeting the estrogen receptor (ER) pathway is highly effective in ER positive breast cancer, but its efficacy is limited by intrinsic and acquired resistance. Multiple explanatory mechanisms through the study of different models emphasize the importance of multiple proteins, microRNAs and metabolites. However, the contributions of long non-coding RNAs (lncRNAs) to endocrine resistance in breast cancer remain elusive. To address this issue, we performed a genome-wide lncRNA microarray analysis in tamoxifen resistant breast cancer cell lines (MCF-7/LCC2 and MCF-7/LCC9) and parental MCF-7, and delineated the distinct transcription patterns. A specific differentially regulated lncRNA (Tamoxifen Resistance Associated, termed TRA) was also validated in both resistant cell lines. TRA is derived from intergenic region between DKK1 and MBL2, predicted to contain several stable hairpins in secondary structure and has less conservative sequences. Interestingly, TRA expression is significantly down-regulated in parental sensitive MCF-7 cells after receiving tamoxifen treatment. To further elucidate the underlying mechanisms by which TRA functions, we will investigate its subcellular location by RNA-FISH analysis, target signaling pathways and molecules by co-expression and microarray analysis, potential TRA-protein interactions by RIP and ChIRP, modulation patterns by luciferase reporter assay, in vitro effect on cell dynamics by proliferation, cell cycle and cell apoptosis analysis, in vivo effect of TRA by xenograft mouse model, and the correlation between TRA, clinical characteristics and survival in breast cancer patients receiving tamoxifen by real-time PCR and statistical analysis. Overall, our study indicates novel insights of endocrine resistance in lncRNA level and potential development of therapeutic strategies targeting TRA for endocrine resistant breast cancer patients.

内分泌治疗是激素受体阳性乳腺癌的重要治疗方式，他莫昔芬是其一线治疗药物，然而其有效性常受到药物耐受的制约。现阶段针对他莫昔芬耐药的分子机制研究主要侧重于蛋白、代谢物及微小RNA，迄今对长链非编码RNA（lncRNA）与乳腺癌内分泌耐药的关系仍知之甚少。我们的预实验结果揭示了内分泌耐药细胞中lncRNA表达谱特征，并鉴定出一个新的内分泌耐药相关的lncRNA：TRA。但其作用机制还有待进一步探讨。为此，我们提出假说：TRA可能通过多水平多通路调节，影响内分泌治疗的敏感性。为了验证这一假说，我们将通过经典的他莫昔芬耐药细胞模型：LCC2和LCC9，采用共表达分析、基因芯片、ChIRP、腺病毒载体转染等手段，从分子、细胞、动物及临床样本多方面探讨和明确TRA在他莫昔芬耐药发生中的调控机制。本研究将从lncRNA这个新视点为揭示他莫昔芬耐药的发生机制奠定基础，为内分泌耐药乳腺癌的防治提供新的思路。

内分泌治疗是雌激素受体阳性乳腺癌的重要治疗方式，他莫昔芬是其一线治疗药物，然而其有效性通常受到原发性及获得性耐药的制约。现阶段针对他莫昔芬耐药的分子机制的研究主要侧重于蛋白、代谢物及microRNA，迄今对长链非编码RNA（lncRNA）与乳腺癌内分泌耐药的关系仍知之甚少。我们的实验结果揭示了内分泌耐药细胞中lncRNA表达谱特征，并鉴定出一个新的内分泌耐药相关的lncRNA：TRA (Tamoxifen Resistance Associated), 其编码基因位于10号染色体上，位于蛋白编码基因DKK1和MBL2之间，为基因间lncRNA（lincRNA）；保守性较弱，二级结构含有较多稳定的发卡结构。在他莫昔芬的刺激下，他莫昔芬敏感株的lncRNA-TRA具有时间依赖性下调的趋势。采用Coexpression分析、基因芯片、腺病毒载体转染等手段，从分子、细胞、组织、动物及临床样本多方面探讨和明确TRA在他莫昔芬耐药发生中的调控机制。表达谱芯片分析lncRNA-TRA调节基因的研究中，本研究也揭示了lncRNA-TRA对细胞的信号通路和分子功能的影响是全方面的，且主要影响P53信号通路及周期凋亡通路，结合BIK的相应变化趋势，本研究认为lncRNA-TRA通过激活P53通路，上调BIK的表达，促进他莫昔芬的凋亡诱导作用，从而达到抑癌作用。在裸鼠模型中lncRNA-TRA参与调控移植瘤对他莫昔芬的药物敏感性。接受他莫昔芬治疗的ER受体阳性乳腺癌新鲜冰冻标本分析lncRNA-TRA的表达水平，生存分析提示lncRNA-TRA低表达患者无进展生存较高表达者明显缩短。本研究从lncRNA这个新视点为进一步解释乳腺癌他莫昔芬耐药的发生机制奠定基础，为内分泌耐药乳腺癌的防治提供新的思路。