中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
4期
765-768,封3
,共5页
朱智能%朱朝辉%庞自力%兰东阳%王海鹏
硃智能%硃朝輝%龐自力%蘭東暘%王海鵬
주지능%주조휘%방자력%란동양%왕해붕
上皮间质转化%转录因子叉头框蛋白Q1%膀胱癌%RNA干扰
上皮間質轉化%轉錄因子扠頭框蛋白Q1%膀胱癌%RNA榦擾
상피간질전화%전록인자차두광단백Q1%방광암%RNA간우
Epithelial mesenchymal transition%Transcription factor forkhead box protein Q1%Bladder cancer%RNA interference
目的 观察转录因子叉头框蛋白Q1(FOXQl)基因沉默对膀胱癌T24细胞上皮间质转化(EMT)的逆转,探讨其促进肿瘤侵袭转移的作用机制.方法 构建针对FOXQ1基因的短发卡RNA(shRNA)真核表达载体,脂质体Lipofactamine 2000介导FOXQ1干扰质粒转染人膀胱癌细胞株T24细胞；转染48 h后采用逆转录-聚合酶链反应(RT-PCR)、Western blot法检测FOXQ1、上皮间质标志物E-钙黏蛋白(E-Cadherin)、N-钙黏蛋白(N-Cadherin)和波形蛋白(Vimentin)的表达改变；细胞划痕实验、Transwell小室侵袭实验检测体外细胞迁移和侵袭能力；噻唑蓝(MTT)比色法和吖啶橙/溴乙锭(AO/EB)荧光染色法检测shRNA对细胞增殖和凋亡的作用.结果 与转染NC-shRNA阴性对照组比较,转染FOXQ1-shRNA 48 h后的T24细胞,FOXQ1和间质标记基因N-Cadherin、Vimentin的mRNA或蛋白表达下降,而上皮标记基因E-cadherin的mRNA或蛋白表达显著增加；转染后的T24细胞形态向正常上皮细胞转化,体外迁移能力与侵袭力下降[(15.4±1.4)％比(76.5±1.1)％,P＜0.05]；细胞增殖明显抑制(57.8％比82.7％,P＜0.05),同时细胞凋亡率增加(37.6％比14.2％,P＜0.05).结论 靶向沉默FOXQ1基因可以逆转肿瘤细胞的间质表型向正常上皮表型转化,抑制癌细胞迁移和侵袭,降低肿瘤转移潜能.
目的 觀察轉錄因子扠頭框蛋白Q1(FOXQl)基因沉默對膀胱癌T24細胞上皮間質轉化(EMT)的逆轉,探討其促進腫瘤侵襲轉移的作用機製.方法 構建針對FOXQ1基因的短髮卡RNA(shRNA)真覈錶達載體,脂質體Lipofactamine 2000介導FOXQ1榦擾質粒轉染人膀胱癌細胞株T24細胞；轉染48 h後採用逆轉錄-聚閤酶鏈反應(RT-PCR)、Western blot法檢測FOXQ1、上皮間質標誌物E-鈣黏蛋白(E-Cadherin)、N-鈣黏蛋白(N-Cadherin)和波形蛋白(Vimentin)的錶達改變；細胞劃痕實驗、Transwell小室侵襲實驗檢測體外細胞遷移和侵襲能力；噻唑藍(MTT)比色法和吖啶橙/溴乙錠(AO/EB)熒光染色法檢測shRNA對細胞增殖和凋亡的作用.結果 與轉染NC-shRNA陰性對照組比較,轉染FOXQ1-shRNA 48 h後的T24細胞,FOXQ1和間質標記基因N-Cadherin、Vimentin的mRNA或蛋白錶達下降,而上皮標記基因E-cadherin的mRNA或蛋白錶達顯著增加；轉染後的T24細胞形態嚮正常上皮細胞轉化,體外遷移能力與侵襲力下降[(15.4±1.4)％比(76.5±1.1)％,P＜0.05]；細胞增殖明顯抑製(57.8％比82.7％,P＜0.05),同時細胞凋亡率增加(37.6％比14.2％,P＜0.05).結論 靶嚮沉默FOXQ1基因可以逆轉腫瘤細胞的間質錶型嚮正常上皮錶型轉化,抑製癌細胞遷移和侵襲,降低腫瘤轉移潛能.
목적 관찰전록인자차두광단백Q1(FOXQl)기인침묵대방광암T24세포상피간질전화(EMT)적역전,탐토기촉진종류침습전이적작용궤제.방법 구건침대FOXQ1기인적단발잡RNA(shRNA)진핵표체재체,지질체Lipofactamine 2000개도FOXQ1간우질립전염인방광암세포주T24세포；전염48 h후채용역전록-취합매련반응(RT-PCR)、Western blot법검측FOXQ1、상피간질표지물E-개점단백(E-Cadherin)、N-개점단백(N-Cadherin)화파형단백(Vimentin)적표체개변；세포화흔실험、Transwell소실침습실험검측체외세포천이화침습능력；새서람(MTT)비색법화아정등/추을정(AO/EB)형광염색법검측shRNA대세포증식화조망적작용.결과 여전염NC-shRNA음성대조조비교,전염FOXQ1-shRNA 48 h후적T24세포,FOXQ1화간질표기기인N-Cadherin、Vimentin적mRNA혹단백표체하강,이상피표기기인E-cadherin적mRNA혹단백표체현저증가；전염후적T24세포형태향정상상피세포전화,체외천이능력여침습력하강[(15.4±1.4)％비(76.5±1.1)％,P＜0.05]；세포증식명현억제(57.8％비82.7％,P＜0.05),동시세포조망솔증가(37.6％비14.2％,P＜0.05).결론 파향침묵FOXQ1기인가이역전종류세포적간질표형향정상상피표형전화,억제암세포천이화침습,강저종류전이잠능.
Objective To investigate the reversal effect of epithelial-mesenchymal transition (EMT) by silencing transcription factor forkhead box protein Q1 (FOXQ1) in human bladder cancer cell line T24,and study its role in mvasion and metastasis of T24 cells.Methods Short hairpin RNA (shRNA) eukaryotic expression vector (FOXQ1-shRNA) targeting human FOXQ1 gene was transfected into T24 cells with high metastatic potential by lipofectamine 2000.The expression of FOXQ1 and epithelial mesenchymal markers (E-Cadherin,N-Cadherin,vimentin) was detected by using reverse transcriptionpolymerase chain reaction (RT-PCR) and Westem blotting after transfection for 48 h.The migration and metastatic potentials of T24 cells were examined by cell wound model and Transwell chamber assay in vitro respectively,and the proliferation of T24 cells was determined by using MTT assay.Acridine orange/ethidium bromide (AO/EB) fluorescent staining was performed to detect apoptosis.Results In the FOXQ1shRNA transfection group,the expression of FOXQ1,N-Cadherin and Vimentin was decreased in the T24 cells remarkably,and the expression of E-Cadherin was significantly up-regulated (P ＜ 0.05).The morphology of T24 cells was transformed into a normal epithelial phenotype,and the motility and invasion of T24 cells [(15.4± 1.4)％ vs (76.5 ± 1.1)％,P ＜ 0.05] were inhibited,the abilities of proliferation were inhibited significantly (57.8％ vs 82.7％,P＜ 0.05) in vitro,and apoptosis rate (37.6％ vs 14.2％,P ＜ 0.05) was increased notablely as compared with the negative control group (NC-shRNA).Conclusion The silencing of FOXQ1 may reverse mesenchymal morphology into a normal epithelial phenotype,inhibit cancer cell migration and invasion,and suppress tumor metastatic potential.
