微小RNA-618通过靶向羧基末端结合蛋白2抑制胰腺癌细胞增殖、侵袭和迁移

MicroRNA-618 inhibits proliferation,invasion and migration of pancreatic cancer cells by targeting C-terminal binding protein 2

目的探讨微小RNA(miR)-618及羧基末端结合蛋白2(CtBP2)对胰腺癌增殖、侵袭和迁移的影响及其作用机制。方法采用实时定量反转录聚合酶链反应(RT-qPCR)检测胰腺癌细胞中miR-618的表达;分别转染miR-618 inhibitor和mimics后,采用细胞计数试剂盒(CCK-8)、克隆集落形成及Transwell实验检测miR-618对胰腺癌MIA PaCa-2和SW1990细胞增殖、侵袭和迁移能力的影响;运用在线数据库预测能够与miR-618结合的靶基因;RT-qPCR检测转染miR-618 inhibitor和mimics后相关靶基因的表达;蛋白质印迹法(Western blot)检测miR-618 inhibitor组和mimics组中CtBP2的表达量;双荧光素酶报告基因实验检测miR-618和CtBP2的靶向结合情况;CCK-8、克隆形成和Transwell实验检测同时转染si-CtBP2和miR-618 inhibitor较单独转染si-CtBP2后对胰腺癌增殖及侵袭迁移的影响。组间比较采用t检验,组内两两比较采用LSD-t检验。结果BxPC-3(0.65±0.01)、MIA PaCa-2(0.45±0.07)、PANC-1(0.51±0.04)和SW1990(0.53±0.01)中miR-618的表达量低于正常胰腺导管上皮细胞(HPDE)(1.01±0.13,t=4.371、8.859、6.794、6.171,P<0.01),差异有统计学意义;干扰miR-618的表达后可显著促进胰腺癌MIA PaCa-2和SW1990的增殖、侵袭及迁移能力;同时在线数据库预测miR-618的潜在靶基因,结合RT-qPCR验证转染mimics和inhibitor后各候选基因的表达,结果提示仅有CtBP2与miR-618的表达呈负相关;进一步Western blot结果显示,转染miR-618 mimics后MIA PaCa-2(0.32±0.06)和SW1990(0.45±0.03)中CtBP2的表达量低于对照组(1.02±0.05和0.99±0.05,t=9.850、10.087,P<0.01),差异有统计学意义;双荧光素酶报告结果显示miR-618与CtBP2靶向结合(t=6.573,P<0.05),差异有统计学意义;功能回复实验结果显示共转染miR-618 inhibitor及si-CtBP2组可逆转单独干扰CtBP2后对胰腺癌细胞增殖、侵袭和迁移能力的抑制作用。结论miR-618靶向CtBP2抑制胰腺癌细胞增殖、侵袭及迁移。

Objective To investigate the effects of microRNA(miR)-618 and C-terminal binding protein 2 on proliferation,invasion and migration of pancreatic cancer and the underlying mechanisms.Methods Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-618 in pancreatic cancer cells.After transfection of miR-618 inhibitor or/and mimics,the effect of miR-618 inhibitor and mimics on proliferation,invasion and migration of pancreatic cancer cells MIA PaCa-2 and SW1990 was measured by cell counting kit-8 assay(CCK-8),clone colony formation and Transwell assay.Online database was used to predict the target genes that could bind to miR-618.The expression of relevant target genes after transfection with miR-618 inhibitor and mimics was detected by RT-qPCR.The expression of CtBP2 in the miR-618 inhibitor-treated group and the mimics group was detected by Western blotting.The interaction between miR-618 and CtBP2 was detected by dual luciferase reporter gene assay.CCK-8,clone colony formation and Transwell were utilized to test the effect of si-CtBP2 and miR-618 inhibitor co-transfection on the proliferation,invasion and migration of pancreatic cancer compared with the single transfection of si-CtBP2.T test was used for comparison between groups,and LSD-t test was used for pairwise comparison within groups.Results The expression level of miR-618 in BxPC-3(0.65±0.01),MIA PaCa-2(0.45±0.07),PANC-1(0.51±0.04)and SW1990(0.53±0.01)was lower than that in normal pancreatic ductal epithelial cells(HPDE,1.01±0.13,t=4.371,8.859,6.794,6.171,P<0.05).Interference of miR-618 expression can significantly promote the proliferation,invasion and migration of pancreatic cancers MIA PaCa-2 and SW1990.At the same time,the potential target genes of miR-618 were predicted through online database,and the expression of the candidate genes after the transfection of mimics and inhibitors was verified by RT-qPCR.The results showed that only CtBP2 was negatively correlated with the expression of miR-618.Western blotting results showed that the expression of CtBP2 in MIA PaCa-2(0.32±0.06)and SW1990(0.45±0.03)after transfection with miR-618 mimics was lower than that in control group(1.02±0.05,0.99±0.05,t=9.850,10.087,P<0.01).Dual luciferase reporter gene assay showed that CtBP2 was the target gene that bound to miR-618(t=6.573,P<0.05).Functional recovery assay results showed that the inhibition of proliferation,invasion and migration of pancreatic cancer cells after CtBP2 interference alone was reversed in the miR-618 inhibitor and si-CtBP2 co-transfected group.Conclusion miR-618 inhibits proliferation,invasion and migration of pancreatic cancer cells through targeting CtBP2.