肿瘤研究与临床
腫瘤研究與臨床
종류연구여림상
CANCER RESEARCH AND CLINIC
2015年
4期
222-227
,共6页
樊丽君%李美宁%王爽%孙建文%牛万通%程牛亮
樊麗君%李美寧%王爽%孫建文%牛萬通%程牛亮
번려군%리미저%왕상%손건문%우만통%정우량
转录因子激活蛋白2α%microRNA-200c%结肠肿瘤
轉錄因子激活蛋白2α%microRNA-200c%結腸腫瘤
전록인자격활단백2α%microRNA-200c%결장종류
Transcription factor AP-2α%microRNA-200c%Colonic neoplasms
目的 探讨hsa-microRNA-200c对结肠癌细胞增殖能力影响的作用及相关机制.方法 利用生物信息学数据库筛选可能与转录因子激活蛋白2α (AP-2α)特异性结合的microRNA并合成其真核表达质粒与特异干扰序列.利用脂质体介导将质粒PEZX-miR-200c、PEZX-NC、pmir-GLO-AP-2 α3'UTR、pmir-GLO与干扰序列miR-67-inhibtor、miR-200c-inhibitor转染至结肠癌HCT-116、SW480、HEK-293T细胞中.用实时荧光定量PCR法、Westem blot法和免疫细胞化学染色法检测细胞中AP-2α表达情况,用CCK-8法检测细胞增殖情况,用PE标记流式细胞术检测细胞凋亡情况;用双荧光素酶活性检测法判断miR-200c与AP-2 α的相互作用.结果 转染miR-200c-inhibitor的SW480(anti-miR-200c/SW480组)细胞增殖能力低于转染miR-67-inhibitor组(anti-miR-67/SW480组),同时凋亡水平高于anti-miR-67/SW480组[(78±0.7)％比(66±1.1)％,P＜ 0.05],anti-miR-200c/SW480组AP-2α蛋白表达量高于anti-miR-67/SW480组(0.49±0.01比0.09±0.08,P＜0.05).转染PEZX-miR-200c表达质粒组HCT-116细胞(PEZX-miR-200c/HCT-116组)增殖能力高于转染PEZX-NC组(PEZX-NC/HCT-116组),同时AP-2 α的mRNA和蛋白量均降低(0.67±0.07比0.51±0.09,P＜0.05).共转染PEZX-miR-200c与pmirGLO-AP-2 α-3' UTR质粒组(共转实验组)HEK-293T细胞相对荧光素酶活性值明显低于共转染PEZX-miR-200c与pmir-GLO-AP-2α-3' UTR-deleted组(共转对照组)HEK-293T细胞(0.51±0.09比0.98±0.04,P＜ 0.01).结论 结肠癌细胞中miR-200c通过抑制AP-2 α转录后水平的表达活性增强细胞体外增殖能力.
目的 探討hsa-microRNA-200c對結腸癌細胞增殖能力影響的作用及相關機製.方法 利用生物信息學數據庫篩選可能與轉錄因子激活蛋白2α (AP-2α)特異性結閤的microRNA併閤成其真覈錶達質粒與特異榦擾序列.利用脂質體介導將質粒PEZX-miR-200c、PEZX-NC、pmir-GLO-AP-2 α3'UTR、pmir-GLO與榦擾序列miR-67-inhibtor、miR-200c-inhibitor轉染至結腸癌HCT-116、SW480、HEK-293T細胞中.用實時熒光定量PCR法、Westem blot法和免疫細胞化學染色法檢測細胞中AP-2α錶達情況,用CCK-8法檢測細胞增殖情況,用PE標記流式細胞術檢測細胞凋亡情況;用雙熒光素酶活性檢測法判斷miR-200c與AP-2 α的相互作用.結果 轉染miR-200c-inhibitor的SW480(anti-miR-200c/SW480組)細胞增殖能力低于轉染miR-67-inhibitor組(anti-miR-67/SW480組),同時凋亡水平高于anti-miR-67/SW480組[(78±0.7)％比(66±1.1)％,P＜ 0.05],anti-miR-200c/SW480組AP-2α蛋白錶達量高于anti-miR-67/SW480組(0.49±0.01比0.09±0.08,P＜0.05).轉染PEZX-miR-200c錶達質粒組HCT-116細胞(PEZX-miR-200c/HCT-116組)增殖能力高于轉染PEZX-NC組(PEZX-NC/HCT-116組),同時AP-2 α的mRNA和蛋白量均降低(0.67±0.07比0.51±0.09,P＜0.05).共轉染PEZX-miR-200c與pmirGLO-AP-2 α-3' UTR質粒組(共轉實驗組)HEK-293T細胞相對熒光素酶活性值明顯低于共轉染PEZX-miR-200c與pmir-GLO-AP-2α-3' UTR-deleted組(共轉對照組)HEK-293T細胞(0.51±0.09比0.98±0.04,P＜ 0.01).結論 結腸癌細胞中miR-200c通過抑製AP-2 α轉錄後水平的錶達活性增彊細胞體外增殖能力.
목적 탐토hsa-microRNA-200c대결장암세포증식능력영향적작용급상관궤제.방법 이용생물신식학수거고사선가능여전록인자격활단백2α (AP-2α)특이성결합적microRNA병합성기진핵표체질립여특이간우서렬.이용지질체개도장질립PEZX-miR-200c、PEZX-NC、pmir-GLO-AP-2 α3'UTR、pmir-GLO여간우서렬miR-67-inhibtor、miR-200c-inhibitor전염지결장암HCT-116、SW480、HEK-293T세포중.용실시형광정량PCR법、Westem blot법화면역세포화학염색법검측세포중AP-2α표체정황,용CCK-8법검측세포증식정황,용PE표기류식세포술검측세포조망정황;용쌍형광소매활성검측법판단miR-200c여AP-2 α적상호작용.결과 전염miR-200c-inhibitor적SW480(anti-miR-200c/SW480조)세포증식능력저우전염miR-67-inhibitor조(anti-miR-67/SW480조),동시조망수평고우anti-miR-67/SW480조[(78±0.7)％비(66±1.1)％,P＜ 0.05],anti-miR-200c/SW480조AP-2α단백표체량고우anti-miR-67/SW480조(0.49±0.01비0.09±0.08,P＜0.05).전염PEZX-miR-200c표체질립조HCT-116세포(PEZX-miR-200c/HCT-116조)증식능력고우전염PEZX-NC조(PEZX-NC/HCT-116조),동시AP-2 α적mRNA화단백량균강저(0.67±0.07비0.51±0.09,P＜0.05).공전염PEZX-miR-200c여pmirGLO-AP-2 α-3' UTR질립조(공전실험조)HEK-293T세포상대형광소매활성치명현저우공전염PEZX-miR-200c여pmir-GLO-AP-2α-3' UTR-deleted조(공전대조조)HEK-293T세포(0.51±0.09비0.98±0.04,P＜ 0.01).결론 결장암세포중miR-200c통과억제AP-2 α전록후수평적표체활성증강세포체외증식능력.
Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism.Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database,while its eukaryotic expression plasmids and specific inhibitor were synthesized.Plasmids PEZX-miR-200c,PEZX-NC,pmirGLO-AP-2α3'UTR,pmir-GLO and the specific inhibitors miR-67-inhibtor,miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000.The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Westem blot and immunocytochemical staining.CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis.Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c.Results The proliferation activity was significantly decreased in anti-miR-200c/SW480 group,while in PEZX-miR-200c/HCT-116 group,it was higher than that in PEZX-NC/HCT-116 group.The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7) ％ vs (66±1.1) ％,P ＜ 0.05].The expression of AP-2o both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group,while the protein level was increased in Anti-miR-200c/SW480 group.The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3' UTR (0.51±0.09 vs 0.98±0.04,P ＜ 0.01).Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells.
