微小RNA-9-5p干扰RhoA表达调控胰腺癌细胞增殖、侵袭及迁移

MicroRNA-9-5p Affects Proliferation, Invasion and Migration of Pancreatic Ductal Adenocarcinoma Cells Through RhoA

[目的]探讨微小RNA(miR)-9-5p在胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)中表达及影响其生物学行为的机制。[方法]实时定量反转录聚合酶链反应(qRT-PCR)测定40例PDAC与正常人胰腺细胞系HPDE6-C7中miR-9-5p的表达。miR-9-5p模拟物转染PDAC细胞株PANC-1;甲基噻唑蓝(MTT)比色法检测细胞增殖活性;划痕实验、Transwell侵袭实验测定细胞侵袭、迁移能力。qPCR及蛋白印迹实验检测RhoA、ROCK1、PCNA、CyclinD1、MMP-2、MMP-9及p21表达。双荧光素酶报告基因分析实验检测miR-9-5p对RhoA的调控功能。[结果](1)临床样本中miR-9-5p在PDAC组织中的表达显著性低于癌旁组织(0.34±0.08 vs 1.01±0.24,t=13.222,P<0.001)。miR-9-5p表达降低与胰腺癌淋巴结转移、肿瘤分期存在相关性。(2)miR-9-5p在胰腺癌细胞系PANC-1中表达明显低于正常人胰腺细胞系HPDE6-C7(0.28±0.04 vs 0.53±0.05,t=7.204,P=0.019)。(3)双荧光素酶报告基因分析结果表明,野生型RhoA的荧光素酶活性在转染miR-9-5p模拟物后较对照组降低(0.99±0.08 vs0.22±0.02,P<0.001)。(4)转染后,转染组细胞增殖活性降低(0.53±0.07 vs 1.09±0.11、1.13±0.08,P均<0.001),侵袭距离减少(0.28±0.07μm vs 0.66±0.05μm、0.64±0.07μm,P均<0.001),迁移细胞数减少(32.83±2.99 vs 70.00±4.29、65.67±5.79,P均<0.001)。(5)与阴性对照组及Scramble组比较,miR-9-5p模拟物转染组RhoA、ROCK1、PCNA、Cyclin D1、MMP-2、MMP-9表达显著性降低,p21表达升高(P均<0.05)。[结论]胰腺导管腺癌细胞中miR-9-5p表达降低;过表达miR-9-5p后能明显抑制PANC-1细胞的体外增殖、侵袭及迁移,其调控机制可能与RhoA信号通路相关。

[Objective]To explored the effect and mechanisms of microRNA-9-5p(miR-9-5p)on proliferation,invasion and migration of pancreatic ductal adenocarcinoma cells(PDAC).[Methods]The expression of miR-9-5p was detected by qRT-PCR in tumor tissues and pericancerous tissues of 40 patients with pancreatic ductal adenocarcinoma,the correlation between miR-9-5p expression and clinicopathological features of PDAC was analyzed.The levels of miR-9-5p were detected in pancreatic ductal adenocarcinoma cell lines and normal human pancreatic cell line HPDE6-C7.PDAC PANC-1 cells were transfected with miR-9-5p mimics.The cell proliferation activity was detected with MTT assay,cell invasion and migration ability was determined with wound healing assay and Transwell assay,the expression of RhoA,ROCK1,PCNA,Cyclin D1,MMP-2,MMP-9 and p21 was detected with RT-PCR and Western blot,and the regulatory function of miR-9-5p on RhoA was verified with dual luciferase reporter gene analysis.[Results]①The expression of miR-9-5p in PDAC tissues was significantly lower than that in paracancerous tissues(0.34±0.08 vs 1.01±0.24,t=13.222,P<0.001).Low miR-9-5p expression was correlated with lymph node metastasis and higher tumor stage in PDAC.②The expression of miR-9-5p in PANC-1 cells was significantly lower than that in HPDE6-C7 cells(0.28±0.04 vs 0.53±0.05,t=7.204,P=0.019).③Dual luciferase reporter gene analysis showed that the luciferase activity of wild-type RhoA was reduced after transfection with miR-9-5p mimics compared to negative controls group(0.99±0.08 vs 0.22±0.02,t=20.976,P<0.001).④The cell proliferation activity(0.53±0.07 vs 1.09±0.11,1.13±0.08,P all<0.001),invading distance(0.28±0.07μm vs 0.66±0.05μm,0.64±0.07μm,P all<0.001),and the number of migration cells(32.83±2.99 vs 70.00±4.29,65.67±5.79,P all<0.001)were all reduced in PANC-1 cells after transfected with miR-9-5p mimics.⑤Compared with the negative control and Scramble group,miR-9-5p mimics transfected group showed significantly lower expression of RhoA,ROCK1,PCNA,Cyclin D1,MMP-2,MMP-9 and higher expression of p21(P all<0.05).[Conclusion]The miR-9-5p is down-regulated in pancreatic ductal adenocarcinoma cells,overexpression of miR-9-5p significantly inhibits the proliferation,invasion and migration of PANC-1 cells in vitro,and its regulatory mechanism may be related to the RhoA signaling pathway.