CAJ | 학술논문

目的研究敲低带PDZ结合域的转录共激活子(transcriptional coactivator with PDZ-binding motif,TAZ)表达后对胶质瘤细胞株细胞生物学特性的影响.方法采用TAZ小干扰RNA(TAZsiRNA)敲低LN229和SNB19细胞株中TAZ表达,用实时定量聚合酶链式反应(real time-PCR) 及蛋白免疫印迹法(Western blot)分别鉴定转染后TAZ敲除效果,噻唑兰比色法检测两种细胞株的增殖能力变化;流式细胞术检测细胞凋亡的改变;划痕实验和Transwell实验检测细胞侵袭能力的变化;Western blot法检测凋亡、增殖及侵袭等相关蛋白的表达变化.结果转染TAZ siRNA可显著敲低LN229和SNB19细胞株中TAZ mRNA及蛋白表达水平;两种细胞株中TAZ被抑制后,细胞增殖率均下降且呈逐渐下降趋势,而细胞凋亡率分别增高至(12.05±0.65)％和(8.02 ±0.66)％(LN229:F=414.2,P=0.000;SNB19:F=156.8,P=0.000);两细胞株TAZ siRNA组细胞穿膜细胞数与对照组和无义序列组相比明显降低,划痕间隙融合能力也显著降低,差异均有统计学意义(P＜0.05);Western blot法检测显示转染后细胞增殖核抗原(Kiel 67 antigen,Ki-67)、B淋巴细胞瘤2(B-cell lymphoma-2,Bcl-2)、基质金属蛋白酶9(matrix metallopeptidase 9,MMP-9)等增殖、凋亡及侵袭相关蛋白表达明显下调.结论敲低TAZ可抑制胶质瘤细胞株LN229和SNB19增殖和侵袭能力,提示TAZ可能成为治疗胶质瘤的新靶点.
목적 연구고저대PDZ결합역적전록공격활자(transcriptional coactivator with PDZ-binding motif,TAZ)표체후대효질류세포주세포생물학특성적영향.방법 채용TAZ소간우RNA(TAZsiRNA)고저LN229화SNB19세포주중TAZ표체,용실시정량취합매련식반응(real time-PCR) 급단백면역인적법(Western blot)분별감정전염후TAZ고제효과,새서란비색법검측량충세포주적증식능력변화;류식세포술검측세포조망적개변;화흔실험화Transwell실험검측세포침습능력적변화;Western blot법검측조망、증식급침습등상관단백적표체변화.결과 전염TAZ siRNA가현저고저LN229화SNB19세포주중TAZ mRNA급단백표체수평;량충세포주중TAZ피억제후,세포증식솔균하강차정축점하강추세,이세포조망솔분별증고지(12.05±0.65)％화(8.02 ±0.66)％(LN229:F=414.2,P=0.000;SNB19:F=156.8,P=0.000);량세포주TAZ siRNA조세포천막세포수여대조조화무의서렬조상비명현강저,화흔간극융합능력야현저강저,차이균유통계학의의(P＜0.05);Western blot법검측현시전염후세포증식핵항원(Kiel 67 antigen,Ki-67)、B림파세포류2(B-cell lymphoma-2,Bcl-2)、기질금속단백매9(matrix metallopeptidase 9,MMP-9)등증식、조망급침습상관단백표체명현하조.결론 고저TAZ가억제효질류세포주LN229화SNB19증식화침습능력,제시TAZ가능성위치료효질류적신파점.
Objective To study the effect of knocking down the transcriptional coactivator with PDZ-binding motif (TAZ) expression on the biological characteristics of glioma cell lines.Methods The TAZ small interfering RNA (TAZ siRNA) was used to knock down TAZ expression in LN229 and SNB19 cells.Real-time quantitative polymerase chain reaction (RealTime-PCR) and Western blot were used to identify the effect of TAZ knockout after transfection.Thiazole blue (MTT) colorimetric assay was used to detect the changes of the proliferation ability of 2 kind cells.Flow cytometry was used to detect the changes of apoptosis.Scratch experiment and Transwell experiment were used to detect the changes of cell invasion ability.Western blot was used detect the expression changes of related proteins including apoptosis,proliferation and invasion,etc.Results The transfection of TAZ siRNA significantly knocked down the expression levels of TAZ mRNA and protein in LN229 and SNB19 cells ; after TAZ was inhibited in the 2 kinds of cells,the cell proliferation rates were decreased and showed a gradual downward trend,while the apoptosis rates were increased to 12.05％ ±0.65％ and 8.02％ ±0.66％ (LN229:F =414.2,P =0.000;SNB19:F =156.8,P =0.000).Compared with the control and nonsense sequence groups,the cell numbers of the cell-penetrating membrane per field were decreased significantly in the TAZ siRNA group in both cell lines.The fusion capability of scratch gap was also decreased significantly,and there was significant difference (P =0.05).Western blot for the proliferations of cellular proliferative nuclear antigen (Kiel67 antigen,Ki-67) B-cell lymphoma-2 (Bcl-2) and matrix metallopeptidase 9 (MMP-9),apoptosis and invasion-related protein expression were down-regulated significantly.Conclusion Knocking down TAZ inhibits proliferations and invasive abilities of LN229 and SNB19 of the glioma cell lines,suggesting TAZ may become a new target for the treatment of gliomas.