中国肿瘤临床
中國腫瘤臨床
중국종류림상
CHINESE JOURNAL OF CLINICAL ONCOLOGY
2013年
9期
500-504
,共5页
李文静%石萌%王亚东%郑浩轩
李文靜%石萌%王亞東%鄭浩軒
리문정%석맹%왕아동%정호헌
结肠癌%上皮间质转化%Fas通路
結腸癌%上皮間質轉化%Fas通路
결장암%상피간질전화%Fas통로
colon cancer%epithelial-mesenchymal transition (EMT)%fas signaling
目的:探索Fas通路在结肠癌细胞中诱导上皮间质转化(epithelial-mesenchymal transition,EMT)的分子机制.方法:分别对结肠癌细胞SW480及DLD1予以低剂量FasL(12.5 ng/mL)处理.作用3d后分别提取实验组和对照组细胞的总蛋白、总RNA,并进行Western blot、RT-PCR检测,分析FasL作用下结肠癌细胞的上皮标记物、间质标记物以及EMT相关的转录因子的表达状况.在低剂量FasL作用3d后行免疫荧光检测,观察EMT相关转录因子在细胞内的分布情况.建立稳定敲除Snail及Twist的结肠癌细胞系,再予以低剂量FasL刺激,采用Western blot、RT-PCR检测是否发生EMT过程.对结肠癌细胞SW480予以低剂量FasL(12.5 ng/mL)处理后,通过Western blot检测实验组和对照组细胞ERK1/2通路及p38通路的激活状况.对SW480细胞进行信号通路抑制剂的预处理,再予以低剂量FasL刺激,采用Western blot、RT-PCR检测是否发生EMT过程,从而探索Fas通路诱导EMT的可能机制.结果:低剂量FasL可使结肠癌SW480和DLD1细胞的上皮标记物表达下调,间质标记物表达上调,EMT相关转录因子在细胞核周聚集,细胞发生梭形改变,提示发生EMT.而将结肠癌细胞的Snail或Twist基因敲除后,FasL的上述诱导作用明显减弱.低剂量FasL可激活结肠癌细胞的ERK1/2通路激活,而ERK抑制剂可减弱FasL诱导的EMT过程.结论:Fas通路可能通过激活ERK1/2通路诱导结肠癌细胞发生EMT.
目的:探索Fas通路在結腸癌細胞中誘導上皮間質轉化(epithelial-mesenchymal transition,EMT)的分子機製.方法:分彆對結腸癌細胞SW480及DLD1予以低劑量FasL(12.5 ng/mL)處理.作用3d後分彆提取實驗組和對照組細胞的總蛋白、總RNA,併進行Western blot、RT-PCR檢測,分析FasL作用下結腸癌細胞的上皮標記物、間質標記物以及EMT相關的轉錄因子的錶達狀況.在低劑量FasL作用3d後行免疫熒光檢測,觀察EMT相關轉錄因子在細胞內的分佈情況.建立穩定敲除Snail及Twist的結腸癌細胞繫,再予以低劑量FasL刺激,採用Western blot、RT-PCR檢測是否髮生EMT過程.對結腸癌細胞SW480予以低劑量FasL(12.5 ng/mL)處理後,通過Western blot檢測實驗組和對照組細胞ERK1/2通路及p38通路的激活狀況.對SW480細胞進行信號通路抑製劑的預處理,再予以低劑量FasL刺激,採用Western blot、RT-PCR檢測是否髮生EMT過程,從而探索Fas通路誘導EMT的可能機製.結果:低劑量FasL可使結腸癌SW480和DLD1細胞的上皮標記物錶達下調,間質標記物錶達上調,EMT相關轉錄因子在細胞覈週聚集,細胞髮生梭形改變,提示髮生EMT.而將結腸癌細胞的Snail或Twist基因敲除後,FasL的上述誘導作用明顯減弱.低劑量FasL可激活結腸癌細胞的ERK1/2通路激活,而ERK抑製劑可減弱FasL誘導的EMT過程.結論:Fas通路可能通過激活ERK1/2通路誘導結腸癌細胞髮生EMT.
목적:탐색Fas통로재결장암세포중유도상피간질전화(epithelial-mesenchymal transition,EMT)적분자궤제.방법:분별대결장암세포SW480급DLD1여이저제량FasL(12.5 ng/mL)처리.작용3d후분별제취실험조화대조조세포적총단백、총RNA,병진행Western blot、RT-PCR검측,분석FasL작용하결장암세포적상피표기물、간질표기물이급EMT상관적전록인자적표체상황.재저제량FasL작용3d후행면역형광검측,관찰EMT상관전록인자재세포내적분포정황.건립은정고제Snail급Twist적결장암세포계,재여이저제량FasL자격,채용Western blot、RT-PCR검측시부발생EMT과정.대결장암세포SW480여이저제량FasL(12.5 ng/mL)처리후,통과Western blot검측실험조화대조조세포ERK1/2통로급p38통로적격활상황.대SW480세포진행신호통로억제제적예처리,재여이저제량FasL자격,채용Western blot、RT-PCR검측시부발생EMT과정,종이탐색Fas통로유도EMT적가능궤제.결과:저제량FasL가사결장암SW480화DLD1세포적상피표기물표체하조,간질표기물표체상조,EMT상관전록인자재세포핵주취집,세포발생사형개변,제시발생EMT.이장결장암세포적Snail혹Twist기인고제후,FasL적상술유도작용명현감약.저제량FasL가격활결장암세포적ERK1/2통로격활,이ERK억제제가감약FasL유도적EMT과정.결론:Fas통로가능통과격활ERK1/2통로유도결장암세포발생EMT.
Objective:The present study aimed to examine the presence and mechanism of Fas-induced epithelial-mesenchymal transition (EMT) in human colon cancer cells. Methods:Colon cancer cells, namely, SW480 and DLD1, were treated with low-dose FasL (12.5 ng/mL) for 3 d. Total protein and total RNA levels of the experimental and control groups were extracted, and Western blot and RT-PCR were performed to measure both the transcriptional and translational levels of epithelial markers such as CDH1 and Villin, mesenchymal markers such as CHD2 and Vimentin, and EMT transcriptional factors such as Snail and Twist. Immunofluorescence was performed to observe the cellular distribution of EMT transcriptional factors on the third day of treatment. Snail and Twist stably knocked-down colon cancer cell lines were established and confirmed by Western blot. Colon cancer cells with low-expressing Snail or Twist were treated with low-dose FasL for 3 d. Western blot and RT-PCR were performed as described above to study the dependence of Fas-induced EMT on either Snail or Twist. Colon cancer cells, specifically SW480, were treated with low-dose FasL for 1 h, and Western blot was performed to test the activation status of the extra-cellular regulated protein kinases 1/2 (ERK1/2) pathway and the p38 pathway at 15 min, 30 min, and 1 h. SW480 was pretreated for 2 h with the signaling pathway inhibitor, which was demonstrated efficiently by Western blot, and then treated with low-dose FasL for 3 d. Western blot and RT-PCR were performed as described above to explore the possible mechanisms of Fas-induced EMT. Results:The transcriptional and translational levels of the epithelial markers decreased, whereas those of the mesenchymal markers and of the EMT transcriptional factors increased in colon cancer cells, namely, SW480 and DLD1, after treatment of low-dose FasL for 3 d. The resulting levels were accompanied by the nucleus translocation of EMT transcription factors and cellular morphological changes from paving-stone-like shape into spindle-like shape. This result confirms the occurrence of EMT. The stable knock-down of Snail or Twist eliminated the Fas-induced EMT process mentioned above. Low-dose FasL activated the ERK1/2 pathway, whereas the ERK inhibitor weakened the FasL-induced EMT process. Conclusion:Low-dose FasL can induce the EMT process in colon cancer cells, namely, SW480 and DLD1, through the activation of the ERK1/2 signaling pathway.
