CAJ | 학술논문

[目的]研究内皮素-3(ET-3)对人恶性黑素瘤(MM )A375细胞上皮基质转化(EMT)的影响.[方法]体外培养A375细胞,分别设立3组:空白对照组、100 nmol/L ET-3组、100 nmol/L ET-3和100 μmol/L BQ788(内皮素受体B阻断剂)组.采用Transwell小室检测细胞转移,细胞爬片技术检测细胞形态变化,实时PCR和Western印迹检测上皮基质转化相关分子上皮细胞钙黏蛋白、波形蛋白及转录因子( Twist、Slug)表达情况,使用方差分析及Scheffe法对结果进行分析.[结果]各干预条件中,与空白对照组比较,ET-3可以促进A375细胞的转移,BQ788可阻断该效应(3组穿膜细胞数分别为4.200±0.837、9.400±0.548、3.400±0.894,F=88.44,P＜ 0.01)；ET-3可以促进A375细胞由上皮型向成纤维细胞样形态转变,促进A375上皮细胞钙黏蛋白表达下调(3组分别为0.330±0.002、0.280±0.007、0.420±0.008,F=329.98,P＜ 0.01),波形蛋白表达上调(0.830±0.014、1.160±0.003、0.750±0.030,F=262.94,P＜ 0.01),而BQ788可阻断这种效应.ET-3可以促进上皮基质转化相关转录因子Slug mRNA(F=376.94,P＜ 0.01)及Twist mRNA(F=215.62,P＜0.01)及其蛋白水平(Fslug=288.87,P＜ 0.01；Ftwist=156.96,P＜ 0.05)上的表达上调.[结论]ET-3/ETRB通过上调波形蛋白,下调上皮细胞钙黏蛋白的表达,并上调转录因子(Twist、Slug)的表达,促进黑素瘤A375细胞上皮基质转化.
[목적]연구내피소-3(ET-3)대인악성흑소류(MM )A375세포상피기질전화(EMT)적영향.[방법]체외배양A375세포,분별설립3조:공백대조조、100 nmol/L ET-3조、100 nmol/L ET-3화100 μmol/L BQ788(내피소수체B조단제)조.채용Transwell소실검측세포전이,세포파편기술검측세포형태변화,실시PCR화Western인적검측상피기질전화상관분자상피세포개점단백、파형단백급전록인자( Twist、Slug)표체정황,사용방차분석급Scheffe법대결과진행분석.[결과]각간예조건중,여공백대조조비교,ET-3가이촉진A375세포적전이,BQ788가조단해효응(3조천막세포수분별위4.200±0.837、9.400±0.548、3.400±0.894,F=88.44,P＜ 0.01)；ET-3가이촉진A375세포유상피형향성섬유세포양형태전변,촉진A375상피세포개점단백표체하조(3조분별위0.330±0.002、0.280±0.007、0.420±0.008,F=329.98,P＜ 0.01),파형단백표체상조(0.830±0.014、1.160±0.003、0.750±0.030,F=262.94,P＜ 0.01),이BQ788가조단저충효응.ET-3가이촉진상피기질전화상관전록인자Slug mRNA(F=376.94,P＜ 0.01)급Twist mRNA(F=215.62,P＜0.01)급기단백수평(Fslug=288.87,P＜ 0.01；Ftwist=156.96,P＜ 0.05)상적표체상조.[결론]ET-3/ETRB통과상조파형단백,하조상피세포개점단백적표체,병상조전록인자(Twist、Slug)적표체,촉진흑소류A375세포상피기질전화.
[Objective] To explore the role of endothelin-3 (ET-3) on epithelial-to-mesenchymal transition in a malignant melanoma cell line A375.[Methods] A375 cells were cultured in vitro and classified into 3 groups to be treated with ET-3 at 100 nmol/L (ET-3 group),co-cultured with ET-3 at 100 nmol/L and endothelin receptor B (ETRB) antagonist BQ788 at 100 μmo1/L (ETRB antagonist group),or to remain untreated (blank control group).After additional 24-hour culture,Transwell chamber assay was used to detect the invasive capability of A375 cells,real time-PCR to measure the mRNA expressions of Twist and Slug,and Western blot to determine the protein expression of E-cadherin,vimentin,Twist and Slug.The changes in the morphology of A375 cells were observed.Data were assessed by analysis of variance and Scheffe's method.[Results] In the Transwell assay,the number of A431 cells permeating through the basement membrane was 4.200 ± 0.837,9.400 ± 0.548 and 3.400 ± 0.894 respectively in the blank control group,ET-3 group and ETRB antagonist group (F =88.44,P ＜ 0.01 ),suggesting that ET-3 could promote the metastasis of A375 ceils,while BQ788 could block the promotive effect of ET-3.The epithelial-to-mesenehymal transition was obvious in cells treated with ET-3 alone,but was inapparent in cells treated with ET-3 and BQ788.The ET-3 at 100 nmol/Lsignificantly decreased the protein expression of E-cadhefin from 0.33 ± 0.002 (blank control group) to 0.28 ±0.007,but increased that of vimentin from 0.83 ± 0.014 (blank control group) to 1.16 ± 0.003,while BQ788upregulated the E-cadherin expression to 0.42 ± 0.008 and downregulated the vimentin expression to 0.75 ±0.030,and significant differences were observed in the E-cadherin expression and vimentin expression among the ET-3 group,ETRB antagonist group and blank control group (F =329.98,262.94,respectively,both P ＜ 0.01 ).A significant increase was observed in the mRNA and protein expression of Slug (F=376.94,288.87,both P＜ 0.01 )and Twist (F=215.62,156.96,P＜ 0.01 and 0.05) in A375 cells after treatment with ET-3.[Conclusion] ET3/ETRB axis may promote the epithelial-to-mesenchymal transition in A375 cells likely by regulating the expression of E-cadherin,vimentin and two important transcription factors Twist and Slug.