微小RNA-216影响胰腺癌细胞增殖凋亡及对吉西他滨耐药的实验研究

Effect of microRNA-216 on the proliferation and apoptosis of pancreatic cancer cells and the drug resistance to gemcitabine

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Bx PC-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Bx PC-3细胞,以进行脂质体转染的Bx PC-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Clu GO将其进行Gene Oncology(GO)功能注释。结果 Bx PC-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IC_(50)值为(2.134±0.591)μg/ml、(4.518±0.862)μg/ml和(0.481±0.073)μg/ml,Bx PC-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。

Objective To explore the effect of microRNA-216( miR-216) on the proliferation and apoptosis of pancreatic cancer cells and the drug resistance to gemcitabine. Methods The real-time quantitative PCR( QPCR) method was used to detect the miR-216 level in the gemcitabine resistant cell line Bx PC-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 Bx PC-3 cells by Lipofectamine 2000 liposome method. Bx PC-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( IC_(50)) 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 Clu GO. Results The results of QPCR detection showed that the level of miR-216 in Bx PC-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 were1. 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 Bx PC-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. Conclusion The 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.