中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2010年
1期
62-67
,共6页
陈卫群%陈和明%孔德勇%曹阳%詹燏%卢忠心
陳衛群%陳和明%孔德勇%曹暘%詹燏%盧忠心
진위군%진화명%공덕용%조양%첨율%로충심
胰腺肿瘤%微小RNAs%肿瘤转移%肿瘤标记%生物学
胰腺腫瘤%微小RNAs%腫瘤轉移%腫瘤標記%生物學
이선종류%미소RNAs%종류전이%종류표기%생물학
Pancreatic neoplasms%MicroRNAs%Neoplasm metastasis%Tumor markers,biological
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.
目的 分析miR-301在胰腺癌組織中的錶達,探討其在胰腺癌侵襲轉移中的意義.方法 用FQ-PCR方法從細胞水平檢測5種胰腺癌細胞繫(PANC-1、PaCa-2、AsPC-1、Hs-766T、BxPC-3)中miR-301的錶達;進一步用免疫組織化學方法從組織水平檢測胰腺癌組織芯片(含60份胰腺癌組織、10份癌徬組織和10份正常胰腺組織)中miR-301的錶達;在證實miR-301高錶達後,進一步研究其在胰腺癌侵襲轉移中的臨床意義,用100 nmol/L miR-301抑製劑(anti-miR-301)或陰性對照(Anti-miR~(TM) Negative Control#1)處理對數生長的胰腺癌細胞繫PANC-1、PaCa-2,用WB檢測胰腺癌細胞繫中侵襲轉移相關分子COX-2、MMP-2的錶達,併用Transwell技術檢測胰腺癌細胞的侵襲轉移能力.結果 FQ-PCR檢測結果顯示,在5種胰腺癌細胞繫PANC-1、PaCa-2、AsPC-1、Hs-766T、BxPC-3中miR-301的相對錶達量分彆為33.09±4.21、30.76±3.18、47.57±3.56、20.20±1.21、76.75±13.51;而正常胰腺細胞中為1.00±0.08;5種胰腺癌細胞繫分彆與正常胰腺細胞相比,差異有統計學意義(t值分彆為8.86、9.53、6.39、6.77、11.18,P均<0.01).免疫組織化學結果亦顯示,胰腺癌組織、癌徬組織及正常胰腺組織中miR-301的相對錶達量分彆為0.88±0.09、0.22±0.04、0.14±0.05;胰腺癌組織分彆與癌徬組織及正常胰腺組織相比,差異有統計學意義(t值分彆為15.1、10.6,P均<0.01);正常胰腺組織與癌徬組織相比,差異無統計學意義(t=1.32,P=0.22).用miR-301抑製劑抑製胰腺癌細胞繫PANC-1、PaCa-2中miR-301的錶達後,WB檢測結果顯示,侵襲轉移相關分子COX-2、MMP-2的錶達下調;細胞侵襲轉移能力試驗結果顯示,miR-301抑製劑處理後跨膜轉移的細胞數分彆為PANC-1(587±27)箇、PaCa-2(363±13)箇,而陰性對照處理後跨膜轉移的細胞數為:PANC-1(1 091±15)箇、PaCa-2(737±44)箇;miR-301抑製劑處理與陰性對照處理相比,細胞侵襲轉移能力亦顯著下降(t值分彆為7.89、7.56,P均<0.01).結論 胰腺癌細胞繫和組織中miR-301高錶達;且與胰腺癌的侵襲轉移密切相關,抑製miR-301的錶達能夠有效抑製胰腺癌細胞的侵襲轉移,miR-301有望成為抗胰腺癌侵襲轉移治療的新分子靶標和胰腺癌早期診斷的新分子標誌.
목적 분석miR-301재이선암조직중적표체,탐토기재이선암침습전이중적의의.방법 용FQ-PCR방법종세포수평검측5충이선암세포계(PANC-1、PaCa-2、AsPC-1、Hs-766T、BxPC-3)중miR-301적표체;진일보용면역조직화학방법종조직수평검측이선암조직심편(함60빈이선암조직、10빈암방조직화10빈정상이선조직)중miR-301적표체;재증실miR-301고표체후,진일보연구기재이선암침습전이중적림상의의,용100 nmol/L miR-301억제제(anti-miR-301)혹음성대조(Anti-miR~(TM) Negative Control#1)처리대수생장적이선암세포계PANC-1、PaCa-2,용WB검측이선암세포계중침습전이상관분자COX-2、MMP-2적표체,병용Transwell기술검측이선암세포적침습전이능력.결과 FQ-PCR검측결과현시,재5충이선암세포계PANC-1、PaCa-2、AsPC-1、Hs-766T、BxPC-3중miR-301적상대표체량분별위33.09±4.21、30.76±3.18、47.57±3.56、20.20±1.21、76.75±13.51;이정상이선세포중위1.00±0.08;5충이선암세포계분별여정상이선세포상비,차이유통계학의의(t치분별위8.86、9.53、6.39、6.77、11.18,P균<0.01).면역조직화학결과역현시,이선암조직、암방조직급정상이선조직중miR-301적상대표체량분별위0.88±0.09、0.22±0.04、0.14±0.05;이선암조직분별여암방조직급정상이선조직상비,차이유통계학의의(t치분별위15.1、10.6,P균<0.01);정상이선조직여암방조직상비,차이무통계학의의(t=1.32,P=0.22).용miR-301억제제억제이선암세포계PANC-1、PaCa-2중miR-301적표체후,WB검측결과현시,침습전이상관분자COX-2、MMP-2적표체하조;세포침습전이능력시험결과현시,miR-301억제제처리후과막전이적세포수분별위PANC-1(587±27)개、PaCa-2(363±13)개,이음성대조처리후과막전이적세포수위:PANC-1(1 091±15)개、PaCa-2(737±44)개;miR-301억제제처리여음성대조처리상비,세포침습전이능력역현저하강(t치분별위7.89、7.56,P균<0.01).결론 이선암세포계화조직중miR-301고표체;차여이선암적침습전이밀절상관,억제miR-301적표체능구유효억제이선암세포적침습전이,miR-301유망성위항이선암침습전이치료적신분자파표화이선암조기진단적신분자표지.
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.
