MicroRNA-200b在吉西他滨诱导的胰腺癌细胞株MiaPaCa-2上皮间质转化中的作用

Effect of miR-200b on gemcitabine induced epithelial mesenchymal transition in pancreatic cancer cell line MiaPaCa-2

目的探讨MicroRNA-200b（miR-200b）在吉西他滨诱导的胰腺癌MiaPaCa-2细胞上皮间质转化（EMT）过程中的作用。方法应用不同浓度的吉西他滨诱导MiaPaCa-2细胞，选择50％细胞生长抑制时的药物浓度（IC50），获取耐药MiaPaCa-2细胞。采用脂质体法将miR-200b和无意义小分子片段（阴性对照）分别转染MiaPaCa-2细胞，再用IC50的吉西他滨诱导细胞，获取转染miR-200b的耐药MiaPaCa-2细胞及阴性对照的耐药MiaPaCa．2细胞。倒置显微镜下观察细胞形态变化；Transwell小室测定细胞侵袭能力；实时定量PCR检测细胞miR-200b表达；蛋白质印迹法检测细胞E—cadherin、Vimentin、Zebl、Zeb2蛋白表达。结果吉西他滨处理后细胞体积逐渐缩小，呈纺锤样，细胞间连接减少，伪足增多，呈现间质细胞特征。耐药MiaPaCa-2细胞的穿膜数从亲本细胞的（26±3）个上升至（85±6）个，Vimentin、Zebl、Zeb2表达分别上升至亲本细胞的（1．87±0．17）、（2．57±0．21）、（5．24±0．83）倍，miR-200b表达下降至亲本细胞的（0．36±0．01）倍，E-cadherin表达下降至亲本细胞的（0．47±0．05）倍。而转染miR-200b的耐药MiaPaCa-2细胞的穿膜数下降至（42±4）个，Zebl、Zeb2表达下降至阴性对照的耐药MiaPaCa-2细胞的（0．36±0．07）、（0．08±0．01）倍。结论吉西他滨诱导胰腺癌MiaPaCa-2细胞过程中细胞出现EMT，其机制可能与miR-200b表达下调有关。

Objective To investigate the role of miR-200b on gemcitabine induced epithelial- mesenchymal transition （EMT） in pancreatic cancer cell line MiaPaCa-2. Methods Different concentrations of gemcitabine were used to induce MiaPaCa-2, and the concentration of 50% cell proliferation inhibited （ ICs0 ） was applied to obtain drug-resistant MiaPaCa-2 cells. MiR-200b or nonsense small molecular fragments （ negative control, NC） was transfected into MiaPaCa-2 cells by liposomes, then gemcitabine of ICs0 was used to induce ceils to obtain drug-resistant MiaPaCa-2 cells transfected with miR-200b or NC. The morphological characteristics of MiaPaCa-2 cells were observed by inverted microscope. Invasion of ceils were detected by transwell chamber. The expression of miR-200b was measured by using real-time PCR. The expressions of E- cadherin, Vimentin, Zebl, Zeb2 proteins were determined by Western blot. Results After gemcitabine treatment, the ceils＇ size gradually diminished, intercellular junctions decreased, pseudopodium increased, which presented the characteristics of mesenchymal morphology. The invaded cell number increased from （26 ± 3 ） to （85 ±6）, and the expression of Vimentin Zebl, Zeb2 was increased to （ 1.87 ± 0.17）, （2.57± 0.21 ）, （5.24 ±0.83） folds of the parent cells. The expression of miR-200b was decreased to （0.36 ±0.01 ） folds of the parent cells, and the expression of E-cadherin was decreased to 0.47 ＋ 0.05 folds of the parent cells, while the invaded cell number of drug-resistant MiaPaCa-2 transfected with miR-200b was decreased to （42± 4） , and the expression of Zebl, Zeb2 was decreased to （0.36 ± 0.07）, （0.08 ± 0.01 ） folds of drug- resistant MiaPaCa-2 transfected with NC. Conclusions The occurrence of EMT is observed in pancreatic cancer cell line MiaPaCa-2 during gemcitabine induction, and miR-200b down-regulation may be a possible mechanism.