Notch-1和微小RNA-200家族在胰腺癌上皮-间充质转化中的作用

The mechanisms of Notch - 1 and microRNA - 200 family in epithelial - mesenchymal transition of pancreatic cancer

目的观察Noteh-1和微小RNA（miRNA，miR）-200家族在吉西他滨（Gem）诱导的人胰腺癌PANC-1细胞上皮-间充质转化（EMT）中的作用。方法体外培养PANC-1细胞并诱导细胞EMT，Western blot实验和反转录．聚合酶链反应（RT-PCR）检测Notch—1、E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）、相关转录因子及miR-200家族成员的表达；Transwell小室检测具有EMT改变的PANC-1细胞迁移和侵袭能力。结果Gem诱导PANC-1细胞EMT最适药物浓度为10nmol／L，最适时间为9d。Western blot灰度分析显示未经Gem处理的亲代细胞（parental）组Noteh-1、E—cadherin、Vimentin、slug、snail、twist、E盒结合锌指蛋白1（ZEBl）的表达分别为0．840±0．002、0．995±0．008、1．158±0．001、0．253±0．004、0．528±0．003、0．346±0．002，0．29l±0．006，Gem（＋）组分另0为1．599±0．00l、0．483±0．001、1．821±0．003、0．719±0．004、0．919±0．005、0．762±0．002、0．70l±0．004，两组比较差异有统计学意义（P〈0．05）。RT—PCR检测parental组Notch-1、E—cadherin、Vimentin、slug、snail、twist、ZEB1 mRNA的表达分别为（2．200±0．010）×10-3、（1．540±0．002）×10-4、1．003±0．004、（8．580±0．003）×10-4、（5．040±0．007）×10-3、（6．930±0．001）×10-5、（2．010±0．001）×10-4，Gem（＋）组分别为（5．870±0．004）×10-5、（1．430±0．001）×10-5、1．660±0．007、（4．030±0．002）×10-3、（8．980±0．005）×10-3、（3．300±0．020）×10-6、（1．120±0．003）×10-3，两组比较差异有统计学意义（P〈0．05）。RT—PCR法检测parental组miR-200a、miR-200b、miR-200e、miR-141和miR-429的表达水平分别为（2．340±0．002）×10-5、（4．280±0．001）×10-6、（3．250±0．003）×10-5、（2．470±0．004）×10-5、（6．230±0．005）×10-4，Cem（＋）组分别为（0．801±0．000）×10-5、（2．030±0．004）×10-6、（5．000±0．050）×10-6、（3．670±0．003）×10-6、（1．310±0．002）×10-4，两组问比较差异有统计学意义（P〈0．05）。PANC-1细胞EMT改变后侵袭能力明显增强，parental组迁移和侵袭实验细胞计数分别为（25．70±3．67）个和（16．30±2．21）个，Gem（＋）组分别为（77．40±3．01）个和（68．00±8．76）个，两组间比较差异有统计学意义（P〈0．05）。结论Gem可诱导PANC-1细胞EMT，Noteh-1可能是该过程的重要信号通路，并参与miR-200家族的调控。

Objective To investigate the mechanisms of Notch - 1 and microRNA （miRNA, miR） -200 family in epithelial- mesenehymal transition （EMT） induced by gemcitabine （Gem）. Meth- ods EMT phenotype of PANC - 1 cells was induced by Gem, as well as the expression of Notch - 1, E - cadherin, Vimentin, relevant transcription factors, and miR - 200 family were observed by Western blotting and reverse transcriptasc - polymerase chain reaction （ RT - PCR）. Migration and invasion abilities were detected by transwell assay. Results The most appropriate drug concentration for EMT was 10 nmol/L, and the suitable time was 9 days. Western blotting analysis showed that the expression of Notch - 1, E - cadherin, Vimentin, slug, snail, twist, zinc finger E -box binding protein 1 （ZEB1） in parental group were 0. 840 ± 0.002,0.995 ±0.008, 1.158 ±0.001, 0.253±0.004, 0.528 ±0.003, 0.346 ±0.002, 0.291 ± 0. 006, and the Gem（ ＋ ） group were 1. 599 ±0. 001, 0. 483 ±0. 001, 1. 821 ±0. 003, 0. 719 ±0. 004, 0. 919 ±0. 005, 0. 762 ± 0. 002, 0. 701± 0. 004, there was significant difference between the two group （P 〈0. 05）. RT - PCR analysis of the mRNA of Notch - 1 , E - cadherin, Vimentin, slug, snail, twist,ZEB1 in parental group were （2.200 ±0.010） × 10-5, （1.540 ±0.002） × 10-4, 1.003 ±0.004, （8.580±0.003） × 10-4, （5.040 ＋0.007） x 10-3, （6.930 ±0.001） × 10-5, （2.010 ±0.001） × 10-4, and the Gem（＋） were （5. 870 ＋0.004） × 10-5, （1.430±0.001） × 10-5, 1.660 ±0.007, （4.030 ±0.002） ×10-3, （8.980 ±0.005） x10-3, （3.300＋0.020） ×10-4, （1.120±0.003） ×10-3, there was significant difference between the two group （ P 〈 0. 05 ）. RT - PCR analysis of miR - 200a, miR -200b, miR -200c, miR - 141 and miR -429 were （2. 340＋0. 002） × 10-5, （4. 280±0. 001 ） × 10-6, （3.250±0.003） ×10 5, （2.470±0.004） ×10-5, （6.230±0.005） ×10-4, and Gem （＋） group were （0.801±0.000） ×10-5, （2.030±0.004） ×10-6, （5.000±0.050） ×10-6, （3.670± 0. 003） x 10-6, （ 1. 310 ± 0. 002） × 10-4, there was significant difference between the two group （ P 〈 0. 05 ）. The migration and invasion abilities of gemcitabine resistance cells were enhanced. The cell content found that the parental group of Transwell migration and invasion assay were 25.70± 3.67, 16. 30 ± 2. 21, and the Gem （ ＋ ） group were 77.40 ± 3.01 , 68.00 ± 8.76, there was significant difference between the two group（ P 〈 0. 05 ）. Coneluslon Gem was a critical drug for EMT phenotype of PANC - 1 cells. Notch - 1 signaling pathway may be mechanistically linked with EMT phenotype and the regulation of miR - 200 family.