中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2012年
2期
139-143
,共5页
β-catenin%siRNA%SK-NEP-1%肾母细胞瘤
β-catenin%siRNA%SK-NEP-1%腎母細胞瘤
β-catenin%siRNA%SK-NEP-1%신모세포류
β-catenin%siRNA%SK-NEP-1%Nephroblastoma
目的 采用RNAi技术抑制Wnt/β-catenin信号通路中β-catenin基因(CTNNB1)的表达,观察对肾母细胞瘤细胞株SK-NEP-1细胞增殖的影响,并检测通路下游相关基因及β-catenin蛋白表达水平的变化及意义.方法 将两种不同序列β-catenin特异性siRNA瞬时转染到SK-NEP-1细胞中,实验分为4组:未转染组(UT)、阴性对照组(NS)、siRNA1组(S1)、siRNA2组(S2),分别在转染后的0、24、48、72 h用CCK-8试剂盒检测比较各组光吸收度值(AV)；在转染24 h后检测各组中CTNNB1、CCND1、MYC基因的表达水平；转染48 h后免疫荧光法检测各组中β-catenin蛋白的表达水平.结果 转染优化实验表明,100 nmol/L的β-catenin特异siRNA1和siRNA2均可获得有效抑制效应.瞬时转染24 h,UT组的AV值(0.62±0.03)分别是S1组(0.49±0.02)和S2组(0.50±0.02)的1.25倍和1.23倍(P＜0.01),48 h后UT组的AV值(1.03±0.07)分别是S1组(0.69±0.04)和S2组(0.72±0.03)的1.48倍和1.43倍(P＜0.01),转染组细胞增殖明显受到抑制.转染24h后S1和S2组CTNNB1的mRNA水平分别被敲减了67％和73％,S1、S2组与UT组比较,下游基因CC-ND1、MYC的mRNA表达水平也明显降低,CCND1组间比较P＜0.01；MYC组间比较P＜0.05,而UT组与NS组比较,CTNNB1、CCND1、MYC基因的P值均＞0.05.转染48 h后,积分光密度/面积(IOD/Area)值分析比较各组β-catenin荧光强度,S1组(0.50±0.36)、S2组(0.58±0.20)较UT组(2.06±0.30)、NS组(2.03±0.53)明显减少,P＜0.01.结论 瞬时转染β-catenin特异性siRNA能抑制SK-N EP-1细胞的增殖,使细胞中β-catenin蛋白的表达降低,Wnt/β-catenin信号通路下游相关基因MYC及CCND1表达下调.β-catenin是肾母细胞瘤分子靶向治疗的一个潜在新靶点.
目的 採用RNAi技術抑製Wnt/β-catenin信號通路中β-catenin基因(CTNNB1)的錶達,觀察對腎母細胞瘤細胞株SK-NEP-1細胞增殖的影響,併檢測通路下遊相關基因及β-catenin蛋白錶達水平的變化及意義.方法 將兩種不同序列β-catenin特異性siRNA瞬時轉染到SK-NEP-1細胞中,實驗分為4組:未轉染組(UT)、陰性對照組(NS)、siRNA1組(S1)、siRNA2組(S2),分彆在轉染後的0、24、48、72 h用CCK-8試劑盒檢測比較各組光吸收度值(AV)；在轉染24 h後檢測各組中CTNNB1、CCND1、MYC基因的錶達水平；轉染48 h後免疫熒光法檢測各組中β-catenin蛋白的錶達水平.結果 轉染優化實驗錶明,100 nmol/L的β-catenin特異siRNA1和siRNA2均可穫得有效抑製效應.瞬時轉染24 h,UT組的AV值(0.62±0.03)分彆是S1組(0.49±0.02)和S2組(0.50±0.02)的1.25倍和1.23倍(P＜0.01),48 h後UT組的AV值(1.03±0.07)分彆是S1組(0.69±0.04)和S2組(0.72±0.03)的1.48倍和1.43倍(P＜0.01),轉染組細胞增殖明顯受到抑製.轉染24h後S1和S2組CTNNB1的mRNA水平分彆被敲減瞭67％和73％,S1、S2組與UT組比較,下遊基因CC-ND1、MYC的mRNA錶達水平也明顯降低,CCND1組間比較P＜0.01；MYC組間比較P＜0.05,而UT組與NS組比較,CTNNB1、CCND1、MYC基因的P值均＞0.05.轉染48 h後,積分光密度/麵積(IOD/Area)值分析比較各組β-catenin熒光彊度,S1組(0.50±0.36)、S2組(0.58±0.20)較UT組(2.06±0.30)、NS組(2.03±0.53)明顯減少,P＜0.01.結論 瞬時轉染β-catenin特異性siRNA能抑製SK-N EP-1細胞的增殖,使細胞中β-catenin蛋白的錶達降低,Wnt/β-catenin信號通路下遊相關基因MYC及CCND1錶達下調.β-catenin是腎母細胞瘤分子靶嚮治療的一箇潛在新靶點.
목적 채용RNAi기술억제Wnt/β-catenin신호통로중β-catenin기인(CTNNB1)적표체,관찰대신모세포류세포주SK-NEP-1세포증식적영향,병검측통로하유상관기인급β-catenin단백표체수평적변화급의의.방법 장량충불동서렬β-catenin특이성siRNA순시전염도SK-NEP-1세포중,실험분위4조:미전염조(UT)、음성대조조(NS)、siRNA1조(S1)、siRNA2조(S2),분별재전염후적0、24、48、72 h용CCK-8시제합검측비교각조광흡수도치(AV)；재전염24 h후검측각조중CTNNB1、CCND1、MYC기인적표체수평；전염48 h후면역형광법검측각조중β-catenin단백적표체수평.결과 전염우화실험표명,100 nmol/L적β-catenin특이siRNA1화siRNA2균가획득유효억제효응.순시전염24 h,UT조적AV치(0.62±0.03)분별시S1조(0.49±0.02)화S2조(0.50±0.02)적1.25배화1.23배(P＜0.01),48 h후UT조적AV치(1.03±0.07)분별시S1조(0.69±0.04)화S2조(0.72±0.03)적1.48배화1.43배(P＜0.01),전염조세포증식명현수도억제.전염24h후S1화S2조CTNNB1적mRNA수평분별피고감료67％화73％,S1、S2조여UT조비교,하유기인CC-ND1、MYC적mRNA표체수평야명현강저,CCND1조간비교P＜0.01；MYC조간비교P＜0.05,이UT조여NS조비교,CTNNB1、CCND1、MYC기인적P치균＞0.05.전염48 h후,적분광밀도/면적(IOD/Area)치분석비교각조β-catenin형광강도,S1조(0.50±0.36)、S2조(0.58±0.20)교UT조(2.06±0.30)、NS조(2.03±0.53)명현감소,P＜0.01.결론 순시전염β-catenin특이성siRNA능억제SK-N EP-1세포적증식,사세포중β-catenin단백적표체강저,Wnt/β-catenin신호통로하유상관기인MYC급CCND1표체하조.β-catenin시신모세포류분자파향치료적일개잠재신파점.
Objective To investigate the role of β-catenin siRNA on the proliferation of nephroblastoma cell line SK-NEP-1 in vitro and to detect the downstream genes and proteins of wnt/β-catenin signal pathway in each group by real time PCR and immunofluorescence.Methods Transient transfection of β-catenin siRNA was implemented by HiPerFect Transfection Reagant according to the manufacturer's instructions.One target sequence of -catenin siRNA (siRNA1) was 5′-ATGGGTAGGGTAAATCAGTAA-3′.Another target sequence of β-catenin siRNA (siRNA2) was 5′-TAAGAATTGAGTAATGGTGTA-3′.Cells were divided into four groups:no transfection group(UT),negative control group (NS),siRNA1 group(S1),siRNA2 group(S2).The effect of β-catenin siRNA on proliferation of SK-NEP-1 were tested by CCK-8 kit at 0、24、48、72hours after transfection.The expression change of genes in the wnt signal pathway (CTNNB1,CCND1 and MYC) was detected by real time PCR and immunofluorescence.Results Optimization of transfection indicated that growth inhibition was obvious when the working concentration of β-catenin siRNA reached 100 nM.24 hours after transfection,the absorbance value (AV) of UT was 1.25 times of S1 and 1.23 times of S2 respectively (0.62 ± 0.03 VS 0.49 ± 0.02/0.50 ± 0.02,P＜0.01) and after 48 hours it was 1.48 times of S1 and 1.43 times of S2 respectively (1.03 ± 0.07 VS 0.69 ± 0.04/0.72 ± 0.03,P＜0.01).100 nM siRNA1 and siRNA2 at 24hours inhibited the CTNNB1 mRNA by 67 ％ and 73 ％ (P＜0.05,Oneway,respectively).Real time PCR showed the down-regulated expression of CCND1 and MYC in S1 and S2 groups compared with UT group (P＜0.05).Immunofluorescence revealed the shutdown of β-catenin protein expression in siRNA groups.Conclusions The activation of β-catenin would result in the continuous proliferation of SK-NEP-1 cell.Growth inhibition and down-regulated expression of CCND1 and MYC are the result of β-catenin siRNA.The activation of β-catenin plays a key role in the development of nephroblastoma.
