目的：探讨microRNA-10b（miR-10b）与胰腺癌细胞吉西他滨（gemcitabine,GEM）化疗耐药的相关性及可能机制。方法：RT-qPCR检测吉西他滨相对耐药的胰腺癌细胞株PANC-1及吉西他滨相对敏感的CFPAC-1中miR-10b的表达情况;用不同浓度的吉西他滨作用于上述细胞,RT-qPCR检测二者miR-10b的表达变化;CFPAC-1细胞转染miR-10b mimics上调miR-10b表达量,CCK-8法及流式细胞仪检测细胞对吉西他滨药物敏感性的变化,Western blot检测抗凋亡相关蛋白（如PI3K、p-Akt、Bcl-2、Survivin）的表达,RT-qPCR检测PTEN基因的表达变化。结果：PANC-1细胞中miR-10b的表达显著高于CFPAC-1细胞,且上述两种细胞中miR-10b的表达量与吉西他滨呈浓度梯度依赖;高表达miR-10b后CFPAC-1细胞对吉西他滨的敏感性下降,PI3K、p-Akt、Bcl-2、Survivin蛋白的表达升高,PTEN mRNA的表达水平降低。结论：miR-10b可能通过负性调控PTEN的表达水平,从而增加PI3K、p-Akt、Bcl-2、Survivin蛋白的表达,减少凋亡来降低CFPAC-1对吉西他滨的敏感性,最终导致胰腺癌细胞对吉西他滨化疗耐受。
Objectives：To reveal the relationship between mieroRNA-10b and gemcitabine （GEM） resistance of pancreatic cancer and its possible mechanism. Methods： The expression of mieroRNA-10b in PANC-1 cells （which are relatively resistant to GEM） and CFPAC-1 cells （which are relatively sensitive to GEM） were detected by RT-qPCR. PANC-1 and CFPAC-1 cells were treated with dif- ferent doses of GEM,and then the cells were harvested. The expression of mieroRNA-10b was detected by RT-qPCR. CCK-8 assay and flow eytometry were performed to evaluate the drug sensitivity of CFPAC-1 cells,which were transfected with mieroRNA-10b mimics to GEM. The protein expressions of PI3K, p-Akt, Bcl-2 and Survivin in CFPAC-1 cells transfected with microRNA-10b mimics or negative control were demonstrated using Western blotting assay. The gene expressions of PTEN in CFPAC-1 cells transfected with microRNA-10b mimics or negative control were detected by RT-qPCR. Results： PANC-1 cells showed significantly increased expres- sion of microRNA-10b as compared to CFPAC-I cells. The expression of microRNA-10b both in PANC-1 and CFPAC-1 cells in- creased with the rising of GEM concentration. The sensitivity of CFPAC-l cells to GEM decreased when microRNA-10b was overex- pressed and the expressions of PI3K, p-Akt, Bcl-2, and Survivin were much higher than their control groups. The expression of PTEN mRNA decreased as microRNA-10b increased. Conclusions： MicroRNA-10b might negatively regulate the gene expression of PTEN, which increases the protein expressions of PI3K,p-Akt,Bcl-2 and Survivin,thus reduces the sensitivity of CFPAC-1 cells to GEM,and as a result,pancreatic cancer cells become resistant to GEM.
Acta Universitatis Medicinalis Nanjing