【作者】 范蕾；

【导师】 邵志敏； 狄根红；

【作者基本信息】 复旦大学 ， 肿瘤学， 2010， 博士

【摘要】 Toll样受体(Toll like receptors, TLRs)家族是近年新发现一类信号通路基因,早先被认为是天然免疫模式识别的主要受体,并控制着由天然免疫向获得性免疫的转变。TLR信号通路并不是单一的直线传导,而是与多个信号通路之间存在着交联和融合,组成了复杂的信号网络。最近的研究显示,不仅外源性分子,许多内源性分子也能经TLRs信号传导途径激活免疫应答。其家族成员之一,Toll样受体3(TLR3)所介导的通路中有两个关键基因核转录因子kappaB (NF-kappaB)和B细胞编码k轻链多肽基因抑制激酶E(IKBKE)被证实与乳腺癌的进展密切相关,但对于该通路关键基因TLR3和IKBKE在乳腺癌发生中的研究尚处于空白。因此在第一部分研究中我们选取TLR3所介导的通路中的三个关键基因TLR3、IKBKE和Toll样受体接头分子1(TICAM1)作为乳腺癌易感候选基因,并选取三个基因中的17个SNP位点进行分型及关联分析。首先,我们通过一期病例对照研究(n=2,303例)发现三个TLR3基因多态与散发性乳腺癌显著相关。这三个位点为rs5743305、rs5743312、rs3775296,分别位于TLR3启动子区、内含子区和5’非翻译区(5’UTR)。其中’rLR3启动子区域SNP rs5743305的-926bpA等位基因能显著增加乳腺癌发生风险,OR为1.220(95%CI为1.059-1.407,P=0.007)；rs5743312的T等位基因型能显著增加乳腺癌的发生风险,OR为1.173(95%CI为1.046-1.315,P=0.006),rs3775296的+95T等位基因型能显著增加乳腺癌的发生风险,OR为1.159(95%CI为1.036-1.298,P=0.011)。这些一期研究中的发现,在第二期以家族性／早发性人群为主的病例对照研究中得到了进一步验证(n=l,248)。两期研究(n=3,551)的联合P值分别是1.97×10-4(rs5743305)、7.01×10-5(rs5743312)和8.69×10-5(rs3775296)。我们发现风险位点仅集中在TLR3基因中,进而我们研究了位于该基因启动子-926bp区域具有潜在功能意义的位点—rs5743305的作用机制。通过瞬时转录和荧光活性检测,我们在不同的细胞系里均观察到相似的趋势,即风险等位基因-926A相对于保护型等位基因-926T能够使转录降低活性30-50%。在MCF-7和MDA-MB-231细胞系中,这种差别具有统计学差异。第一部分的研究提示TLR3是一个乳腺癌易感基因,TLR3的基因多态性与乳腺癌的发生相关,其启动子区域的-926A>T多态的低转录活性等位基因(A-allele)与分子流行病学的风险型等位基因(A-allele)一致,提示TLR3高表达可能降低乳腺癌风险。第一部分研究对TLR3介导的通路中的关键基因TLR3、IKBKE、TICAM1与乳腺癌的发生进行了关联分析及功能论证,初步揭示该通路中与乳腺癌发生的风险基因主要集中在TLR3基因上。通过对其启动子区域风险多态-926bpA>T的功能学研究发现,TLR3表达降低与乳腺癌风险增加相关。这向我们提示TLR3基因本身对乳腺癌的发生发展具有一定的影响。目前TLR3的研究多数集中在TLR3信号参与的肿瘤免疫调节,主要通过MyD88-IRAK-TRAF6-NF-kappaB途径影响免疫相关基因的表达,发挥抗肿瘤作用。近期越来越多的研究发现TLR3在肿瘤发生的不同阶段、不同肿瘤甚至相同肿瘤不同细胞系中发挥不同的作用。关于TLR3在乳腺癌中的地位的研究几近空白,因此有理由对TLR3基因在乳腺癌中的地位及可能的作用机制进行进一步研究。该部分中,我们构建了TLR3表达质粒,建立人乳腺癌TLR3稳定转染细胞株,通过体内、体外实验研究观察TLR3对人乳腺癌细胞增殖、侵袭、转移等生物学行为的影响。并在临床标本中观察TLR3基因的表达对生物学表型的影响。体外实验结果显示：TLR3稳定高表达的细胞出现明显的增殖抑制；细胞S期比例降低,细胞发生G1期阻滞；运动与侵袭能力减弱。体内实验得到了进一步证明：高表达TLR3基因细胞的移植瘤成瘤的时间晚于对照组,生长速度和肿瘤体积明显减小。进一步对机制的研究发现：TLR3的高表达可通过Caspase家族和Bcl-2家族共同调控促进凋亡,并抑制EGFR/PI3K/AKT通路活性。最后,我们在组织标本中证实正常乳腺组织中比癌组织中TLR3表达水平更高。总之,我们的研究提示TLR3至少通过抑制EGFR/PI3K/AKT增殖通路及激活凋亡途径,遏制乳腺癌的进展。更多还原

【Abstract】 Toll-like receptors (TLRs) have been established to play an essential role in the activation of innate immunity by recognizing specific patterns of microbial components. TLRs play a crucial role in defending against pathogenic microbial infection through the induction of inflammatory cytokines and typeⅠinterferons. Furthermore, TLRs also play roles in shaping pathogen-specific humoral and cellular adaptive immune responses. Two key factors in TLR3 pathway, NF-kappaB and IKBKE have been reported to have tight relationship with the progression of breast cancer. However, no research has been done to analysis the function of TLR3 and IKBKE on breast cancer development. Therefore, seventeen SNP points in three genes, TLR3, IKBKE and TICAM1, were selected to address the association between polymorphisms withinTLR3 and breast cancer. We performed case-control studies to investigate the contributions of genetic variants/haplotypes of the TLR3 gene to breast cancer risk. In the first hospital-based study (n=2,303), we observed significant associations between the incidence of breast cancer and rs5743305, rs5743312, and rs3775296 polymorphism. Increased risk was associated with the D-allele of-926A (odds ratio (OR),1.220; P=0.0007) and the rs5743312 T (OR,1.173; P=0.006). The associations were successfully replicated in an independent population set (familial/early-onset breast cancer cases and community-based controls, n=1,248). The combined P-values of the two studies (n=3,551) are 1.97×10-4 (rs5743305), 7.01×10-5 (rs5743312) and 8.69×10-5 (rs3775296). Using promoter reporter-gene assays and electrophoretic-mobility-shift assays, our present work demonstrated that the other risk-allele,-926A>T-allele of rs5743305. Furthermore, an ex vivo study showed that normal breast tissues harboring protective genotypes expressed significantly higher levels of TLR3 mRNA than those in normal breast tissues harboring risk genotypes. Taken together, the data presented here strongly suggest that TLR3 is a susceptibility gene for breast carcinogenesis.We have shown that TLR3 is a susceptibility gene for breast carcinogenesis. Using promoter reporter-gene assays and electrophoretic-mobility-shift assays, our present work demonstrated that the other risk-allele,-926A>T-allele of rs5743305. Furthermore, an ex vivo study showed that normal breast tissues harboring protective genotypes expressed significantly higher levels of TLR3 mRNA than those in normal breast tissues harboring risk genotypes. Human TLR3 cDNA was reintroduced into the breast cancer cells by stable transfection. Effects of TLR3 on the proliferation and invasion of the cells were investigated by MTT, flow cytometry and transwell. Human breast cancer cells were implanted orthotopically into mice. Expression of Bcl-2 and Caspase were detected by Western blot. The results show that human TLR3 cDNA was reintroduced into the breast cancer cells. We demonstrated that TLR3 over-expression induced inhibition of tumorigenesis and/or metastasis through interfering with the tumor angiogenesis in vivo. This inhibition is associated with Bcl-2 protein levels and through inhibition of EGFR/PI3K/AKT pathway. In breast cancer samples, we also demonstrated that higher level of the TLR3 mRNA. TLR3 is a negative regulator of growth in breast cancer, mainly by inhibition of EGFR/PI3K/AKT proliferation pathway and activation of apoptosis.更多还原