【作者】 杨晨；

【导师】 邵志敏；

【作者基本信息】 复旦大学 ， 肿瘤学， 2011， 博士

【摘要】 第一部分趋化因子诱饵受体基因多态与乳腺癌患者区域淋巴结转移的关联分析趋化因子诱饵受体DARC和D6通过清除血管生成性和炎性趋化因子而抑制乳腺癌的侵袭和转移。在人体内,DARC广泛表达于红细胞、血管和淋巴管的内皮细胞,D6表达于淋巴管内皮细胞。乳腺癌主要通过淋巴道和血道转移。机体微环境中DARC和D6形成了一道机体遏抑乳腺癌转移的屏障。DARC和D6编码基因的多态可能会引起相应蛋白表达或功能差异,从而影响到乳腺癌患者的转移风险。我们首先采用关联分析,选取了DARC基因和CCBP2基因(编码D6蛋白)上共十个单核苷酸多态(single nucleotide polymorphism, SNP)位点,研究其和乳腺癌患者区域淋巴结转移的关系。在785例区域淋巴结阴性和678例区域淋巴结阳性的乳腺癌患者中,我们发现DARC基因的多态rs3027012和rs12075, CCBP2基因的多态rs2228468和rs1366046与乳腺癌区域淋巴结转移相关。经过false discovery rate校正,rs12075依旧显示了统计学意义,其A型等位基因能显著减少乳腺癌区域淋巴结转移的风险,OR为0.5(95% CI为0.35-0.69,P=4.0×10-4)；rs2228468显示了具有统计学意义的趋势(P=0.052)。这提示DARC和D6编码基因的多态与乳腺癌患者区域淋巴结转移风险相关。第二部分趋化因子诱饵受体基因多态对乳腺癌转移潜能的功能学研究关联分析发现DARC基因和CCBP2基因的四个等位基因型与乳腺癌患者区域淋巴结转移相关。其中两个位于基因外显子区的多态DARC-rs12075(G42A)和CCBP2-rs2228468 (S373Y)均为错义多态。我们分别构建了pcDNA3.1-DARC-42G、pcDNA3.1-DARC-42A、pcDNA3.1-D6-373S和pcDNA3.1-D6-373Y的表达质粒,由MDA-MB-231细胞分别建立了高表达DARC的231-DARC-42G和231-DARC-42A乳腺癌细胞株、高表达D6的231-D6-373S和231-D6-373Y乳腺癌细胞株、同时高表达DARC和D6的231-DARC-42G-D6-373S和231-DARC-42A-D6-373Y乳腺癌细胞株。含不同等位基因的DARC质粒和D6质粒在乳腺癌细胞中的表达无明显差异。质粒双重稳定转染对DARC和D6在乳腺癌细胞中的表达无明显交互作用。然后通过体外、体内实验观察SNP位点对乳腺癌细胞清除趋化因子和诱导老化等生物学行为的影响。针对rs12075(G42A),231-DARC-42A上清液中人CXCL8、CCL2和CCL5的浓度比231-DARC-42G上清液中的浓度分别降低30%-35%不等(P=0.031,P=0.028和P=0.027)。针对rs2228468(S373Y),231-D6-373Y上清液中CCL2的浓度比231-D6-373S上清液中的浓度低了16.4%,差异具有统计学意义的趋势(P=0.069)。DARC-rs12075对诱导乳腺癌细胞老化无明显差异。体内实验发现,第5周时,231-DARC-42A所形成的移植瘤平均体积显著小于231-DARC-42G所形成的移植瘤平均体积,两者分别为1.80cm3和2.97 cm3。但是231-D6-373S和231-D6-373Y所形成的移植瘤体积无显著差异。231-DARC-42A所形成的移植瘤内的小鼠CCL2和CCL5的浓度显著低于231-DARC-42G所形成的移植瘤内的浓度(P=0.034和P=0.038)。231-D6-373Y所形成的移植瘤内的小鼠CCL2的浓度比231-D6-373S所形成的移植瘤内的浓度低11.4%,差异具有统计学意义的趋势(P=0.075)。我们在同时高表达DARC和D6的231-DARC-42A-D6-373Y细胞及移植瘤中观察到两个SNP位点对其蛋白清除趋化因子具有联合效应。此外,42GA型红细胞体外清除细胞上清液中趋化因子的能力显著高于42GG型红细胞。这部分结果提示,DARC和CCBP2基因多态通过影响其编码蛋白清除趋化因子的能力,调节乳腺癌的转移潜能。更多还原

【Abstract】 PartⅠAssociation between genetic polymorphisms within chemokine decoy receptor genes and regional lymph node metastasis in patients with breast cancerThe inhibitory effect of two chemokine decoy receptors (CDRs), DARC and D6, on breast cancer metastasis is mainly due to their capability in sequestrating angiogenic and inflammatory chemokines. DARC is widely expressed on erythrocytes and vascular endothelial cells, and D6 is mainly expressed on lymphatic endothelial cells. Lymphatic and hematogenous dissemination are two common ways for metastasis of breast cancer. Thus, DARC and D6 serve as systemic barrier to metastasis. We hypothesized that the genetic polymorphisms within CDR genes may influence the gene expression and protein activity of CDRs, thus leading to different metastasis potential of breast cancer. To test our hypothesis, we investigated the relationship between regional lymph node metastasis (RLNM) and ten genetic variations in DARC and CCBP2 (encoding D6) in 785 breast cancer patients with negative RLNM and 678 patients with positive RLNM. There were significant association between RLNM with four studied single nucleotide polymorphisms (SNPs), i.e., rs3027012 and rs 12075 in DARC, and rs2228468 and rs 1366046 in CCBP2. After correction of multiple comparisons by false discovery rate, SNP rs 12075 continued to be significantly associated with RLNM with its A-allele significantly decreasing the risk of RLNM (OR=0.5,95% CI,0.35-0.69, P=4.0×10-4), and SNP rs2228468 tended to be significantly associated with RLNM (P=0.052). The data suggest that genetic polymorphisms within CDR genes may affect the risk of RLNM in patients with breast cancer.Part IIThe functional role of genetic polymorphisms within chemokine decoy receptor genes in metastasis potential of breast cancer The data in Part I have shown the association between RLNM in patients with breast cancer and the four genetic variants in DARC and CCBP2. rs 12075 (G42A) and rs2228468 (S373Y) located in the exons are non-synonymous SNPs, respectively. We generated’pcDNA3.1-DARC-42G’,’pcDNA3.1-DARC-42A’,’pcDNA3.1-D6-373S’ and’pcDNA3.1-D6-373Y’plasmid constructs. Stable transfectants of 231-DARC-42G,231-DARC-42A,231-D6-373S,231-D6-373Y,231-DARC-42A-D6-373Y, 231-DARC-42G-D6-373S and 231-vector were generated from MDA-MB-231 cells. Significant SNPs in DARC and CCBP2 had no differential effects on gene expression in breast cancer cells in vitro, respectively. No interaction effect of these two SNPs on gene expression was observed, either. The effects of the two SNPs on their capability in sequestrating chemokines and in inducing senescence were further investigated both in vitro and in vivo. Regarding the effect of rs 12075 (G42A), the levels of CXCL8, CCL2, and CCL5 in the supernatant of 231-DARC-42A transfectants were about 30-35% lower than those of 231-DARC-42G transfectants (P=0.031, P=0.028, and P=0.027, respectively). Regarding the effect of rs2228468 (S373Y), the level of CCL2 in the supernatant of 231-D6-373Y transfectants was 16.4% lower than that of 231-D6-373S transfectants (a borderline P-value of 0.069). DARC-rs 12075 has no differential effect on senescence in vitro. The mean tumor volumes were 2.97cm3 for 231-DARC-42G xenografts and 1.80cm3 for 231-DARC-42A xenografts in five-weeks, respectively. But the difference in tumor volume for 231-D6-373Y xenografts and 231-D6-373S xenografts was not obvious. The levels of mouse CCL2 and CCL5 in 231-DARC-42A xenografts were significantly lower than those in 231-DARC-42G xenografts (P=0.034 and P=0.038, respectively). A trend was noted that the level of mouse CCL2 in 231-D6-373Y xenografts was about 11.4% lower than that in 231-D6-373S xenografts (P=0.075). The synthetic effects on chemokine sequestration were also observed in 231-DARC-42A-D6-373Y cells and xenografts expressing high levels of both DARC and D6. Furthermore, erythrocytes of 42GA heterozygote sequestrated significantly more chemokines than those of 42GG homozygote ex vivo. Taken together, our data suggest that genetic polymorphisms in DARC and CCBP2 might affect metastasis potential of breast cancer by altering the chemokine sequestrating capability of their proteins.更多还原