摘要目的 探讨Bit1蛋白对胰腺癌细胞运动、迁移、侵袭等恶性表型的影响及临床相关指标的相关性分析.方法 通过real-time PCR和免疫印迹法验证6株胰腺癌细胞株中Bit1蛋白的表达情况,分别挑选Bit1表达量最高和最低的胰腺癌细胞株作为细胞功能调节的工具细胞.采用polyHEMA铺皿实验构建胰腺癌细胞悬浮培养的模型,检测Bit1对胰腺癌细胞失巢凋亡的影响;利用Transwell实验观察Bit1对胰腺癌细胞运动迁移和侵袭能力的影响;通过免疫印迹法和共焦显微镜下观察Bit1对胰腺癌细胞上皮间质转化(EMT)表型过程相关分子标志物的影响;制作术后胰腺癌患者癌和癌旁组织微阵列并进行免疫组化染色,分析Bit1与胰腺癌患者临床病理特征的相关性.结果Bit1在胰腺癌细胞株PANC1中的表达量(基因:3.13±0.40,蛋白:1.77±1.00)最高,在Mia paca2中的表达量(基因:1.00±0.35,蛋白:0.23±0.45)最低.失巢凋亡检测结果显示,Bit1过表达可促进悬浮培养胰腺癌细胞Mia paca2的凋亡,而在PANC1细胞中敲减Bit1之后失巢凋亡受到抑制;Transwell实验检测结果提示,Bit1敲减之后胰腺癌细胞的运动、迁移及侵袭能力明显大于对照组(P<0.05),而Bit1过表达之后可受到明显抑制(P<0.05);免疫印迹法和共焦显微镜下观察证实Bit1敲减之后EMT过程的间质表型N-cadherin及Vimentin的表达上调,而上皮表型E-cadherin下调,相关转录因子β-catenin的表达也是上调的,Bit1过表达之后结果相反;Bit1的差异性表达与胰腺癌患者肿瘤的神经浸润相关(P<0.05).结论 Bit1可负向调控胰腺癌细胞的失巢凋亡发生及其运动迁移及侵袭能力,其对胰腺癌细胞恶性生物学行为的调控作用与EMT过程密切相关. 胰腺癌患者肿瘤中Bit1蛋白表达与肿瘤的神经浸润存在相关性.

abstractsObjective To investigate the potential role of Bit1 in the pathogenesis of pancreatic ductal cancer cells ( PDAC) and its potential clinical application value. Methods Real-time PCR and Western blot were employed to detect the expression of Bit1 in six pancreatic cancer cells,then the tool cells were selected to further study the function of Bit1. PolyHEMA was used to monitor the suspended cell culture condition in vitro. The invasion and migration abilities of pancreatic cancer cells were detected through Transwell assay. Western blot and confocal assay were used to explore the potential mechanism of Bit1 in the process of metastasis. The expression of Bit1 was detected through tissue microarray,the potential relationship between Bit1 and other clinical factors were analyzed. Results The results of real-time PCR and Western blot indicated that the expression of Bit1 was highest in the PANC1 cells and lowest in the Mia paca2 cells (gene:3. 13±0. 40 vs. 1. 00±0. 35,protein:1. 77±1. 00 vs. 0. 23±0. 45). The shBit1 PANC1 and Bit1-OE ( over expression ) Mia paca2 cells were successfully constructed. Bit1 over expression could promote the anoikis rate of Mia paca2 cells,and Bit knockdown could inhibit the anoikis incidence. Bit1 over expression suppressed the motility and invasion of Mia paca2 cells,but Bit1 knockdown could accelerate the migration and invasion ability of PANC1 cells. Bit1 could potentially affect pancreatic cancer cells′malignant behaviors through epithelial-mesenchymal transition process. Bit1 expression was significantly associated with pancreatic cancer′s neural invasion ( P<0. 05) . Conclusions Bit1 could affect the anoikis incidence of pancreatic cancer,Bit1 negatively affect the migration and invasion abilities of PDAC,the EMT process was potentially involved in the whole modulation process. Bit1 expression is associated with neural invasion in pancreatic cancer patients.