shRNA靶向沉默ST8SIA4基因表达对乳腺癌细胞侵袭和迁移的影响

摘要

Objective To investigate the effect of short hairpin RNA (shRNA)targeted silencing of α2,8-sialyltransferase 4 (ST8SIA4)gene on invasion and migration of breast cancer BT549 cells. Methods Real-time quantitative PCR (QPCR)and Western blotting were used to detect the mRNA and protein levels of ST8SIA4 in BT549 cells and MCF-10A cells. BT549 cells were randomly transfected with the successfully constructed shRNA vector fragments targeting ST8SIA4 gene (silencing group)or nonsense shRNA fragments (control group)via LipofectamineTM2000.ST8SIA4 mRNA expression was detected after 48-h transfection by QPCR. The expressions of ST8SIA4,phosphorylated Akt (p-Akt),neuropilin2 (NRP2)and tumor necrosis factor-α (TNF-α)were detected by Western blotting. The invasion and migration abilities of cells were detected by Transwell assay and scratch assay,respectively. Re-sults Compared with MCF-10A cells,the relative expression level of ST8SIA4 mRNA and protein in BT549 cells increased signifi-cantly (P＜0.05).After 48-h transfection,the mRNA and protein levels of ST8SIA4 in the silencing group were 0.19±0.02 and 0.13± 0.02,lower than 0.98±0.11 and 0.52±0.05 in the control group (P＜0.05). The number of invasive cells,migration rate and rela-tive expressions of p-Akt,NRP2 and TNF-α were 39.5±4.2,(16.9±1.3)%,0.22±0.03,0.31±0.04 and 0.27±0.06 in the silen-cing group,all lower than 91.3±8.5,(38.4±3.9)%,0.58±0.07,0.55±0.05 and 0.46±0.05 in the control group(P＜0.05).Con-clusion ST8SIA4 gene is highly expressed in breast cancer BT549 cells. Silencing its expression may inhibit invasion and migration, which may be related to up-regulation of p-Akt,NRP2 and TNF-a protein expression.%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.