LincRNA H19与乳腺癌风险的关联及相关功能研究

Breast cancer is a huge burden for women's health. The role of non-coding RNA in the development and progression of breast cancer is becoming more and more evident. Long intergenic non-coding RNA (lincRNA) is a new hot spot in this field after microRNA. Genome-wide association study (GWAS) reported association between lincRNA H19 and breast cancer risk in Caucasians. LincRNA H19 regulates a network of imprinted genes, including IGF2 and IGF2R. miR-675 is located within H19 sequence and regulates several cancer related genes, including IGF1R, RB, and SET8. H19 may be involved in the development and progression of breast cancer through these two mechanisms. In this study, we conduct a large case-control study with 3000 paired breast cancer cases and controls to investigate the association between 15 SNPs in H19 sequence and related functional units and the risk and prognosis of breast cancer, based on previous findings. We also conduct a functional study to investigate the effect of H19 and miR-675 on related genes and cell function, in breast cancer cell lines and tissue samples. The aim of this study is to find new biomarkers for breast cancer risk and prognosis; to demonstrate the function and mechanism for H19 in the development and progression of breast cancer; and to illustrate the relationship between lincRNA and microRNA, and the interaction of the two in cancer.

乳腺癌严重危害女性健康。非编码RNA在恶性肿瘤发生发展中的作用日益凸显，基因间长链非编码RNA（lincRNA）是这一领域内继microRNA之后新的研究热点。全基因组关联研究提示lincRNA H19与白种人乳腺癌发病风险的关联。H19调控一个重要的印记基因网络，包括IGF2和IGF2R。H19序列中包含miR-675，它调控多个肿瘤相关基因，包括IGF1R，RB和SET8。H19可能通过这两种途径影响乳腺癌的发生发展。本项目在前期发现的基础上，通过3000对乳腺癌病例对照样本分析 H19及相关功能元件区域15个独立SNP与乳腺癌发病和预后的关联；通过功能实验研究H19和miR-675对下游基因的作用和对细胞活性的影响。研究结果将有助于发现新的乳腺癌发病和预后分子标志物；有助于明确H19在乳腺癌发生发展中的作用及相关机制；有助于深入了解lincRNA与microRNA在肿瘤中的共同作用。

最近许多研究发现基因组非编码区存在小范围或者大范围的突变，包括染色体易位、拷贝数变异（copy-number alteration，CNV）以及SNP等，发生在调控区域的突变可以引起基因表达的改变，这种改变存在细胞、组织特异性，使突变携带者存在肿瘤易感性。本研究系统性的探讨lincRNA序列SNP与乳腺癌的易感性的关联，探讨lincRNA及其SNP在乳腺癌发生发展中的作用机制，探索乳腺癌发病和预后相关分子标志物。本研究得出以下结论：1.ER+状态下，rs2071095 CA+AA基因型在病例和对照中的分布有统计学意义（P<0.05），能够降低乳腺癌发病风险（OR=0.80; 95%CI: 0.70-0.91）；2.多因素cox回归分析H19 (rs2071095)基因型与乳腺癌预后的关联无统计学意义（OS：CA vs CC, P = 0.842; AA vs CC, P = 0.360；肿瘤特异生存：CA vs CC, P = 0.476; AA vs CC, P = 0.913；DFS：AA vs CC, P =0.594; AA vs CC, P = 0.498）；3.携带H19 (rs2071095) AA基因型（18例）的表达水平要低于携带CA（87例， P < 0.05）基因型、CC（108例, P < 0.001）基因型，同时CA（87例）基因型的表达量低于CC（108例, P < 0.001）基因型；4.在乳腺癌组织样本中发现H19表达与miR-675-5p/3p表达呈正相关，并在TCGA数据中得到验证。在细胞系中过表达H19，miR-675-5p/3p表达随之上调。H19在癌组织中的表达低于癌旁组织，H19过表达可以抑制乳腺癌细胞迁移能力，反之亦然；H19对细胞增殖能力、侵袭能力以及细胞周期的影响不明显。miR-675-5p/3p可以抑制细胞迁移能力，通过将细胞阻滞在G1期、促进细胞凋亡进而抑制细胞增殖。miR-675-3p可以靶向结合IGF1R，下调其蛋白水平的表达，进而发挥抑癌的作用；5.H19高表达的乳腺癌组织ER+比例高H19以及miR-675-5p/3p表达水平与乳腺癌预后关联无统计学意义。综上所述，H19可以通过转录产生miR-675-5p/3p，miR-675-3p可以靶向结合IGF1R，下调其蛋白表达水平，进而抑制乳腺癌的发生和发展。