shRNA靶向沉默ST8SIA4基因表达对乳腺癌细胞侵袭和迁移的影响

目的 探讨短发夹RNA（shRNA）靶向沉默α2， 8-唾液酸转移酶4（ST8SIA4）表达对乳腺癌BT549细胞侵袭、迁移的影响。方法 采用实时荧光定量PCR（QPCR）和Western blotting检测乳腺癌BT549细胞和乳腺正常上皮MCF-10A细胞中ST8SIA4 mRNA和蛋白水平。采用LipofectamineTM2000向BT549细胞随机转染成功构建的靶向沉默ST8SIA4表达的shRNA载体片段（沉默组）或无义shRNA片段（对照组），转染48 h后采用QPCR检测ST8SIA4 mRNA水平，Western blotting检测ST8SIA4、磷酸化丝／苏氨酸蛋白激酶（p-Akt）、神经纤毛蛋白2（NRP2）和肿瘤坏死因子-α（TNF-α）的表达情况，Transwell实验和划痕实验分别检测细胞的侵袭和迁移能力。结果 与MCF-10A细胞相比，BT549细胞的ST8SIA4 mRNA和蛋白水平均升高（P＜0.05）。转染48 h后，沉默组的ST8SIA4 mRNA和蛋白水平分别为0.19±0.02和0.13±0.02，均低于对照组的0.98±0.11和0.52±0.05（P＜0.05）；沉默组的侵袭细胞数、迁移率及p-Akt、NRP2和TNF-α蛋白相对表达量分别为（39.5±4.2）个、（16.9±1.3）％、0.22±0.03、0.31±0.04和0.27±0.06，均低于对照组的（91.3±8.5）个、（38.4±3.9）％、0.58±0.07、0.55±0.05和0.46±0.05，差异有统计学意义（P＜0.05）。结论 ST8SIA4基因在乳腺癌BT549细胞中高表达，沉默其表达可抑制侵袭和迁移过程，可能与上调p-Akt、NRP2和TNF-α蛋白表达有关。

Relationship between ERCC5 promoter polymorphism and chemosensitivity of oxaliplatin-based chemotherapy in patients with advanced colorectal cancer

Objective：To study the relationship between single nucleotide polymorphisms（SNPs） in the promoter of ERCC5 （ excision repair cross complementation group 5） gene and ehemosensitivity of oxaliplatin-based chenlotherapy in Chinese patients with advanced colorectal cancer. Methods：DNA was extracted from peripheral blood cells before oxaliplatin-based chemotherapy in 83 advanced eolorectal cancer patients. Three polymorphisms in the ERCC5 gene promoter , -1415 C 〉 T （ rs2094258 ） ,-763A 〉 G （rs2016073） and -413C 〉 T（ rs943245 ） were detected by PCR-LDR（ polymerase chain reaction-ligation detection reaction）. Statistical analysis was conducted to investigate the association between polymorphism and ehemosensitivity. Results：The genotype distribution for each polymorphism was found to be in Hardy-Weinberg equilibrium in the study population. Only CC genotype was ohserved at site -413C 〉 T and no genetic variation was observed. The response rate among patients with -763GG genotypes（72.7% ） was significantly higher than that with other genotypes（22. 2% for -763AA genotype, X2 = 8. 568, P = 0. 008 and 37.2%） for -763AG genotype, .g2 = 4. 475,P = 0. 046）. Individuals with -763A allele had higher risk of non-response comparing to those carrying -763G allele （OR 2. 390, 95% CI ： 1. 241-4. 601, P = 0. 009）. No significant association or trend was found between -1415C 〉 T polymorphism and chemosensitivity. Conclusion：The results suggested that ERCC5-763A 〉 G polymorphism may be associated with chemosensitivity of oxali- platin-based chemotherapy in Chinese patients with advanced coloreetal cancer.