微小RNA 216影响胰腺癌细胞增殖凋亡及#br# 对吉西他滨耐药的实验研究#br#

目的 探讨微小RNA 216（miR-216）是否影响胰腺癌细胞增殖凋亡及对吉西他滨耐药。方法 采用实时荧光定量PCR（QPCR）法检测胰腺癌吉西他滨耐药细胞株BxPC-3和非耐药株CFPAC-1中的miR-216表达水平；采用Lipofectamine 2000脂质体法将miR 216模拟物（mimics组）及抑制剂（inhibitor组）分别转染BxPC 3细胞，以进行脂质体转染的BxPC 3细胞为对照组，采用QPCR检测转染48 h后各组miR 216表达水平，CCK-8法检测转染24、48、72 h后各组吸光值以评价增殖率，采用AnnexinⅤ FITC／PI双染流式细胞术检测各组转染48 h后的细胞凋亡率，CCK 8法检测各组对吉西他滨的半数抑制浓度（IC50）。利用生物信息学数据库miRBase预测miR 216的靶基因，利用cytoscape 351及其插件CluGO将其进行Gene Oncology（GO）功能注释。 结果BxPC 3细胞中miR 216表达量为3.010±0.901，高于CFPAC 1细胞的1.049±0.074（P＜0.05）；对照组、mimics组和inhibitor组的miR-216表达量依次为1.130±0.145、4.843±0.782和0256±0.145，差异有统计学意义（P＜0.05）。与对照组比较，mimics组的细胞增殖水平升高而凋亡率降低，inhibitor组的增殖水平降低而凋亡率升高，差异有统计学意义（P＜0.05）。对照组、mimics组和inhibitor组的IC50值为（2.134±0.591）μg／ml、（4.518±0.862）μg／ml和（0.481±0.073）μg／ml，BxPC-3细胞转染inhibitor 后对吉西他滨的耐药性降低，转染mimics后对吉西他滨的耐药性增强（P＜0.05）。miR 216的预测靶基因共有138个，GO功能主要富集于与肿瘤发生发展相关的细胞增殖、凋亡与侵袭迁移过程。结论 miR-216在胰腺癌耐药细胞株中表达上调且可以诱导胰腺癌细胞增殖，暗示其可以发挥类似促癌基因的作用且参与吉西他滨耐药过程，有可能成为胰腺癌早期诊断和新型生物治疗的靶点。

Effect of microRNA 216 on the proliferation and apoptosis of pancreatic cancer cells and the drug resistance to gemcitabin#br#

ObjectiveTo explore the effect of microRNA 216 （miR-216） on the proliferation and apoptosis of pancreatic cancer cells and the drug resistance to gemcitabine. MethodsThe real time quantitative PCR （QPCR） method was used to detect the miR 216 level in the gemcitabine resistant cell line BxPC 3 and the non drug resistant cell line CFPAC 1 of pancreatic cancer. The miR 216 mimics （mimics group） and inhibitor （inhibitor group） were transfected to BxPC 3 cells by Lipofectamine 2000 liposome method. BxPC 3 cells transfected with liposomes were used as the control group. QPCR was used to detect the level of miR 216 at 48 h after transfection. The CCK 8 method was used to detect the absorbance of each group at 24， 48， and 72 h after transfection to evaluate the proliferation rate. Annexin Ⅴ FITC／PI double staining was used to detect the apoptotic rates of each group at 48 h after transfection. The CCK 8 method was used to detect the half inhibitory concentration （IC50） of gemcitabine. The target gene of miR 216 was predicted by bioinformatics database miRBase and Gene Oncology （GO） function was annotated by cytoscape 351 and its plug in CluGO. ResultsThe results of QPCR detection showed that the level of miR 216 in BxPC 3 cells was 3010±0901， higher than 1049±0074 of CFPAC 1 cells （P＜0.05）. The expression levels of miR 216 in the control group， mimics group and inhibitor group were 1130±0.145， 4.843±0.782 and 0.256±0145（P＜0.05）. Compared with the control group， the proliferative rates increased and apoptotic rates decreased in mimics group while the proliferative rates decreased and apoptotic rates increased in inhibitor group （P＜0.05）. The IC50 of gemcitabine were （2.134±0.591）μg／ml， （4.518±0.862）μg／ml and （0481±0073）μg／ml in the control group， mimics group and inhibitor group. The resistance of BxPC 3 cells to gemcitabine decreased after transfection of miR 216 inhibitor， and the resistance to gemcitabine increased after transfection of miR 216 mimics （P＜0.05）. There were 138 predicted target genes of miR-216. GO functions are mainly enriched in proliferation， apoptosis， invasion and migration. ConclusionThe expression of miR 216 is up regulated in pancreatic cancer drug resistant cells and can induce the proliferation of cancer cells， suggesting that it can play a similar role in promoting tumor genes and participate in the process of gemcitabine resistance， and it may become a target for early diagnosis and new biological treatment of pancreatic cancer.