microRNA-301在胰腺癌中的表达及其临床意义

目的 分析miR-301在胰腺癌组织中的表达,探讨其在胰腺癌侵袭转移中的意义.方法 用FQ-PCR方法从细胞水平检测5种胰腺癌细胞系(PANC-1、PaCa-2、AsPC-1、Hs-766T、BxPC-3)中miR-301的表达;进一步用免疫组织化学方法从组织水平检测胰腺癌组织芯片(含60份胰腺癌组织、10份癌旁组织和10份正常胰腺组织)中miR-301的表达;在证实miR-301高表达后,进一步研究其在胰腺癌侵袭转移中的临床意义,用100 nmol/L miR-301抑制剂(anti-miR-301)或阴性对照(Anti-miR~(TM) Negative Control#1)处理对数生长的胰腺癌细胞系PANC-1、PaCa-2,用WB检测胰腺癌细胞系中侵袭转移相关分子COX-2、MMP-2的表达,并用Transwell技术检测胰腺癌细胞的侵袭转移能力.结果 FQ-PCR检测结果显示,在5种胰腺癌细胞系PANC-1、PaCa-2、AsPC-1、Hs-766T、BxPC-3中miR-301的相对表达量分别为33.09±4.21、30.76±3.18、47.57±3.56、20.20±1.21、76.75±13.51;而正常胰腺细胞中为1.00±0.08;5种胰腺癌细胞系分别与正常胰腺细胞相比,差异有统计学意义(t值分别为8.86、9.53、6.39、6.77、11.18,P均<0.01).免疫组织化学结果亦显示,胰腺癌组织、癌旁组织及正常胰腺组织中miR-301的相对表达量分别为0.88±0.09、0.22±0.04、0.14±0.05;胰腺癌组织分别与癌旁组织及正常胰腺组织相比,差异有统计学意义(t值分别为15.1、10.6,P均<0.01);正常胰腺组织与癌旁组织相比,差异无统计学意义(t=1.32,P=0.22).用miR-301抑制剂抑制胰腺癌细胞系PANC-1、PaCa-2中miR-301的表达后,WB检测结果显示,侵袭转移相关分子COX-2、MMP-2的表达下调;细胞侵袭转移能力试验结果显示,miR-301抑制剂处理后跨膜转移的细胞数分别为PANC-1(587±27)个、PaCa-2(363±13)个,而阴性对照处理后跨膜转移的细胞数为:PANC-1(1 091±15)个、PaCa-2(737±44)个;miR-301抑制剂处理与阴性对照处理相比,细胞侵袭转移能力亦显著下降(t值分别为7.89、7.56,P均<0.01).结论 胰腺癌细胞系和组织中miR-301高表达;且与胰腺癌的侵袭转移密切相关,抑制miR-301的表达能够有效抑制胰腺癌细胞的侵袭转移,miR-301有望成为抗胰腺癌侵袭转移治疗的新分子靶标和胰腺癌早期诊断的新分子标志.

The expression and its clinical significance of microRNA-301 in pancreatic carcinoma

Objective To study the expression of microRNA-301 in pancreatic carcinoma andvalidate the significance of miR-301 in invasion and metastasis of pancreatic carcinoma.Methods miR-301 expression were detected by FQ-PCR in 5 pancreatic cancer eell lines(PANC-1,PaCa-2,AsPC-1,Hs766T.BxPC-3).Further immunohistochemistry in pancreatic cancer tissue microarrays was detected miR-301 expression,which contained 60 pancreatic cancer specimens along with 10 normal adjacent tissues and 10 normal pancreas tissues.After high expression of miR-301 in pancreatic carcinoma being confirmed.the clinical significance of high expression of miR-301 in invasion and metastasis of pancreatic carcinoma were studed.Pancreatic cancer cell lines(PANC-1.PaCa-2)were transfected by 100 nmoml/L miR-301 inhibitor(anti-miR-301)or negative eontrol(Anti-miR~(TM) Negative Control#1).COX-2 and MMP-2 protein expression in pancreatic cancer cell lines were detected by WB.and cell migration assays were performed using transwell technology.Results FQ-PCR resuhs indicated that miR-301 expression was higher in pancreatic cancer cell lines than normal pancreatic cells.The relative level of miR-301 in 5 pancreatic cancer cell lines(PANC-1,PaCa-2,AsPC-1,Hs-766T,BxPC-3)and normal pancreatic cell were 33.09± 4.21,30.76±3.18,47.57±3.56,20.20 ±1.21,76.75±13.51 and 1.00±0.08 respectively.The miR-301 level in all 5 pancreatic cancer cells were significantly higher than those of normal pancreatic cell(t=8.86,9.53,6.39,6.77,11.18,P<0.01).Immunohistochemistry results also showed miR-301 expression was higher in pancreatic carcinoma tissues than those in the cancer adjacent tissues and normal pancreatic tissues.The relative levels of miR-301 in pancreatic carcinoma tissues.normal adjacent tissues and normal pancreas tissues were 0.88±0.09,0.22±0.04 and 0.14±0.05 respectively.The miR-301 levels in pancreatic carcinoma tissues were significantly higher than those of normal adjacent tissues and normal pancreatic tissues(t=15.1,10.6,P<0.01).There was no significant difference between normal adjacent tissues and normal pancreas tissues(t=1.32,P=0.22).After miR-301 inhibitor was introduced into pancreatic cancer cells PANC-1 and PaCa-2.miR-301 levels were reduced while the protein levels of COX-2 and MMP-2.which were invasion and metastasis related factors,were down-regulated.The cell migration assay indicated the numbers of PANC-1 and PaCa-2 cells,which migrated to lower chamber.were 587±27 and 363±13 respectively after miR-301 inhibitor was applied.The numbers of migrated cells were 1091 4-15.737±44 when the netative control was applied.The cell invasion ability was decreased significantly in the inhibitor group compared with the negative group(t=7.89,7.56,P<0.01).Conclusions miR-301 is highly expressed in pancreatic cancer cell lines and pancreatic cancer tissues.Inhibition of miR-301 expression can effectively supress the invasion of pancreatic cancer cells.miR-301 may serve as a new biomarker for early detection of pancreatic cancer and molecular target for early treatment of pancreatic cancer.