中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2014年
6期
413-416
,共4页
丁克云%徐娟%满昌峰%范钰
丁剋雲%徐娟%滿昌峰%範鈺
정극운%서연%만창봉%범옥
黑色素瘤%RNA干扰%基因,PLK1%肿瘤浸润
黑色素瘤%RNA榦擾%基因,PLK1%腫瘤浸潤
흑색소류%RNA간우%기인,PLK1%종류침윤
Melanoma%RNA interference%Genes,PLK1%Neoplasm invasiveness
目的 探讨RNA干扰技术下调PLK1基因对恶性黑素瘤细胞侵袭的影响和可能机制.方法 用PLK1小干扰核糖核酸分子(siRNA)转染人恶性黑素瘤A375细胞后,分别用实时定量PCR和Western印迹法检测PLK1 mRNA和蛋白表达,Transwell方法检测细胞侵袭能力,以平板集落形成方法了解癌细胞集落形成情况,分别用琼脂糖凝胶电泳和TUNEL方法检测癌细胞失巢凋亡情况.结果 恶性黑素瘤A375细胞经PLK1 siRNA转染后,PLK1 mRNA和蛋白表达明显下调;siRNA转染组癌细胞生长明显受到抑制.与空白对照组和脂质体对照组比较,siRNA细胞转染组侵袭性明显下降.失巢凋亡检测发现,琼脂糖凝胶电泳出现明显的梯度图谱.TUNEL结果显示,PLK1 siRNA可诱导恶性黑素瘤A375细胞凋亡,siRNA细胞转染组较空白对照组和脂质体对照组凋亡指数明显升高[3.86％±0.35％(3.125 nmol/L siRNA组),7.35％±0.36％(6.250 nmol/LsiRNA组),17.56％±0.38％(12.500 nmol/L siRNA组)比1.15％±0.25％和1.18％±0.22％),均P＜ 0.01].结论 PLK1 siRNA可抑制恶性黑素瘤细胞的侵袭,其机制可能与诱导失巢凋亡有关.
目的 探討RNA榦擾技術下調PLK1基因對噁性黑素瘤細胞侵襲的影響和可能機製.方法 用PLK1小榦擾覈糖覈痠分子(siRNA)轉染人噁性黑素瘤A375細胞後,分彆用實時定量PCR和Western印跡法檢測PLK1 mRNA和蛋白錶達,Transwell方法檢測細胞侵襲能力,以平闆集落形成方法瞭解癌細胞集落形成情況,分彆用瓊脂糖凝膠電泳和TUNEL方法檢測癌細胞失巢凋亡情況.結果 噁性黑素瘤A375細胞經PLK1 siRNA轉染後,PLK1 mRNA和蛋白錶達明顯下調;siRNA轉染組癌細胞生長明顯受到抑製.與空白對照組和脂質體對照組比較,siRNA細胞轉染組侵襲性明顯下降.失巢凋亡檢測髮現,瓊脂糖凝膠電泳齣現明顯的梯度圖譜.TUNEL結果顯示,PLK1 siRNA可誘導噁性黑素瘤A375細胞凋亡,siRNA細胞轉染組較空白對照組和脂質體對照組凋亡指數明顯升高[3.86％±0.35％(3.125 nmol/L siRNA組),7.35％±0.36％(6.250 nmol/LsiRNA組),17.56％±0.38％(12.500 nmol/L siRNA組)比1.15％±0.25％和1.18％±0.22％),均P＜ 0.01].結論 PLK1 siRNA可抑製噁性黑素瘤細胞的侵襲,其機製可能與誘導失巢凋亡有關.
목적 탐토RNA간우기술하조PLK1기인대악성흑소류세포침습적영향화가능궤제.방법 용PLK1소간우핵당핵산분자(siRNA)전염인악성흑소류A375세포후,분별용실시정량PCR화Western인적법검측PLK1 mRNA화단백표체,Transwell방법검측세포침습능력,이평판집락형성방법료해암세포집락형성정황,분별용경지당응효전영화TUNEL방법검측암세포실소조망정황.결과 악성흑소류A375세포경PLK1 siRNA전염후,PLK1 mRNA화단백표체명현하조;siRNA전염조암세포생장명현수도억제.여공백대조조화지질체대조조비교,siRNA세포전염조침습성명현하강.실소조망검측발현,경지당응효전영출현명현적제도도보.TUNEL결과현시,PLK1 siRNA가유도악성흑소류A375세포조망,siRNA세포전염조교공백대조조화지질체대조조조망지수명현승고[3.86％±0.35％(3.125 nmol/L siRNA조),7.35％±0.36％(6.250 nmol/LsiRNA조),17.56％±0.38％(12.500 nmol/L siRNA조)비1.15％±0.25％화1.18％±0.22％),균P＜ 0.01].결론 PLK1 siRNA가억제악성흑소류세포적침습,기궤제가능여유도실소조망유관.
Objective To investigate the effects of down-regulation of polo-like kinase-1 (PLK1) gene by RNA interference (RNAi) on the invasion of a human malignant melanoma cell line A375 and their possible mechanisms.Methods Cultured A375 cells were classified into several groups:blank control group receiving no treatment,liposome group transfected with lipofectamine only,and three siRNA groups transfected with three concentrations of a small interference RNA (siRNA) targeting PLK1 respectively.After additional culture,real time quantitative PCR and Western blot analysis were performed to quantify the expressions of PLK1 mRNA and protein in A375 cells respectively,Transwell invasion assay to evaluate the invasive capacity of A375 cells,agarose gel electrophoresis and terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling (TUNEL) to detect anoikis in A375 cells.The colony-forming capacity was also evaluated for A375 cells.Statistical analysis was carried out by one-factor analysis of variance.Results There was a significant decrease in PLK1 mRNA and protein expressions as well as in colony-forming units in the siRNA groups compared with the blank control group (all P ＜ 0.05).The invasive capacity of A375 cells was significantly inhibited in the siRNA groups with the number of migrating cells in Transwell assay being 39 ± 5,19 ± 5 and 9 ± 3 in A375 cells transfected with 3.125,6.250 and 12.500 nmol/L siRNAs respectively,compared to 56 ± 5 in the blank control group (all P ＜ 0.05).A characteristic DNA ladder was observed on agarose gel electrophoresis in the siRNA (6.250 nmol/L) group.Compared with the blank control group and liposome group,the three siRNA groups showed increased apoptotic index (3.86％ ± 0.35％ (3.125 nmol/L siRNA),7.35％ ± 0.36％ (6.250 nmol/L siRNA) and 17.56％ ± 0.38％ (12.500 nmol/L siRNA) vs.1.15％ ± 0.25％ (blank control group) and 1.18％ ± 0.22％ (liposome group),all P ＜ 0.05).Conclusions PLK1 siRNA can inhibit the invasion of malignant melanoma cells,likely by inducing anoikis in these cells.
