Mai Yu-Jie, Qiu Lu-Gui, Li Zeng-Jun, Li Xin, Yu Zhen, Li Chang-Hong, Wang Ya-Fei, Li Qian
State Key Laboratory of Hematology, Institute of Hematology, Blood Disease Hospital, CAMS and PUMC, Tianjin 300020, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2008 Jun;30(3):290-5.
To inhibit the expression of beta-catenin and investigate the effect of the beta-catenin gene on Jurkat and K562 cells.
siRNA specifically knocking down the expression of beta-catenin was used to testify the function of beta-catenin in Jurkat and K562 cells. Real time polymerase chain reaction and Western blot were performed respectively to testify the mRNA level and protein level of beta-catenin. Growth curve was determined by counting viable cells using trypan blue refusal-dyed method. The proliferation of cells was assayed by clonogenic counting and MTT method. The apoptotic cells were measured by Annexin V/PI staining. The cell cycle analysis was performed based on propidium iodide staining.
Compared with the control group (transfected with siRNA directed against scramble gene), the survival, colonogenicity, and proliferation of the Jurkat and K562 cells were significantly decreased in experimental group transfected with beta-catenin siRNA. The colonogenicity was decreased from 31.9 +/- 5.55 (siRNA) to 25.0 +/- 5.13 (control) in Jurkat cells, and from 47.33 +/- 8.52 (siRNA) to 39.33 +/- 6.26 (control) in K562 cells (both P <0.05). The inhibition rate was (49.3 +/- 9.86)% (siRNA) and (15.1 +/- 6.55)% (control) respectively in Jurkat cells, and (39.4 +/- 7.56)% (siRNA) and (10.1 +/- 6.89)% (control) in K562 cells (both P <0.05). In addition, the apoptotic rate increased from (23.5 +/- 2.82)% (control group) to (55.9 +/- 2.22)% (experiment group) in Jurkat cells and from (14.9 +/- 8.54)% (control group) to (27.9 +/- 15.3)% (experiment group) in K562 cells. However, cell cycle analysis revealed no obvious phases change both in Jurkat and in K562 cells.
Knock-down of beta-catenin gene may decrease the proliferation, survival, and clonogenicity in Jurkat cells and K562 cells.
抑制β-连环蛋白的表达，研究β-连环蛋白基因对Jurkat细胞和K562细胞的影响。
使用特异性敲低β-连环蛋白表达的小干扰RNA（siRNA）来验证β-连环蛋白在Jurkat细胞和K562细胞中的功能。分别采用实时聚合酶链反应和蛋白质免疫印迹法来验证β-连环蛋白的mRNA水平和蛋白质水平。采用台盼蓝拒染法通过计数活细胞来测定生长曲线。通过克隆形成计数和MTT法检测细胞增殖。采用膜联蛋白V/碘化丙啶染色法检测凋亡细胞。基于碘化丙啶染色进行细胞周期分析。
与对照组（转染针对乱序基因的siRNA）相比，转染β-连环蛋白siRNA的实验组中Jurkat细胞和K562细胞的存活率、克隆形成能力和增殖能力均显著降低。Jurkat细胞的克隆形成率从31.9±5.55（siRNA组）降至25.0±5.13（对照组），K562细胞的克隆形成率从47.33±8.52（siRNA组）降至39.33±6.26（对照组）（均P<0.05）。Jurkat细胞的抑制率分别为(49.3±9.86)%（siRNA组）和(15.1±6.55)%（对照组），K562细胞的抑制率分别为(39.4±7.56)%（siRNA组）和(10.1±6.89)%（对照组）（均P<0.05）。此外，Jurkat细胞的凋亡率从(23.5±2.82)%（对照组）升至(55.9±2.22)%（实验组），K562细胞的凋亡率从(14.9±8.54)%（对照组）升至(27.9±15.3)%（实验组）。然而，细胞周期分析显示Jurkat细胞和K562细胞均未出现明显的时相变化。
敲低β-连环蛋白基因可能会降低Jurkat细胞和K562细胞的增殖、存活及克隆形成能力。
