siRNA干扰wnt5a表达对Jurkat细胞生长的影响及机制研究

Influence of siRNA interfering the expression of wnt5a on the growth of Jurkat cells and investigation on its mechanism

目的:干扰wnt5a在Jurkat细胞中的表达,探讨wnt5a对Jurkat细胞增殖、细胞周期、凋亡的作用及其机制。方法:采用大肠杆菌BJ5183内同源重组的方法构建特异性干扰wnt5a的腺病毒Ad5-wnt5asi,感染Jurkat细胞。实验分为实验组(感染重组腺病毒的Jurkat细胞)、空载体组(感染腺病毒的Jurkat细胞)及空白对照组(Jurkat细胞)。采用MTT法检测细胞增殖,流式细胞仪检测细胞周期及凋亡,RT-PCR检测各组细胞wnt5a、β-catenin、钙调蛋白依赖性激酶Ⅱ(Calmodulin dependent proteinkinaseⅡ,CaMKⅡ)、cyclinD1、c-myc、survivin mRNA表达,Western blot检测干扰前后β-catenin、CaMKⅡ、cyclinD1蛋白表达。结果:(1)实验组细胞增殖与空载体组及空白对照组相比显著增高(P<0.05)。(2)实验组与其他两组相比,G1期细胞比例显著降低,S期细胞比例显著增多(P<0.05),G2期细胞比例无明显差异(P>0.05)。实验组与其他两组相比,细胞凋亡率显著降低(P<0.05)。(3)实验组细胞wnt5a mRNA表达降低(55.52±0.61)%,差异有统计学意义(P<0.05)。(4)实验组与其他两组相比,CaMKⅡmRNA表达明显降低,β-catenin、cyclinD1、c-myc、survivin表达增高,差异具有统计学意义(P<0.05)。(5)CaMKⅡ、β-catenin、cyclinD1蛋白与mRNA表达变化一致。结论:靶向沉默wnt5a基因促进Jurkat细胞恶性增殖,抑制凋亡。作用机制可能为:下调wnt5a后,解除了非经典wnt信号通路对β-catenin的抑制,从而下游cyclinD1、c-myc、survivin靶基因上调,改变细胞周期并抑制凋亡,导致Jurkat细胞进一步增殖。

Objective：To investigate the influence of siRNA targeting wnt5a on proliferation,apoptosis,and cell cycle of Jurkat cells and understand its mechanism.Methods：Targeting wnt5a siRNA recombinant adenovious vector Ad5-wnt5asi was constructed by homologous recombinated in E.coli BJ5183 and transfected into Jurkat cells.The cells were divided into experimental group（Jurkat cells infected with adenovirus）,empty vector group（Jurkat cells infected with adenovirus）,and blank control group（Jurkat cells）.Cell proliferation were tested by MTT.Apoptosis rate and cell cycle were detected by flow cytometry.mRNA expression of wnt5a,β-catenin,Calmodulin dependent protein kinase Ⅱ（CaMKⅡ） cyclinD1,c-myc,and survivin were detected by RT-PCR.The protein level of β-catenin,CaMK Ⅱ,and cyclinD1 were tested by Western blot.Results：（1）cell proliferation in the experimental group was significantly higher than that in the empty vector group and control group,the difference being statistically significant（P0.05）.（2）The percentage of G1 phase cells in the experimental group decreased,while S phase cells increased,as compared with other two groups,the difference being statistically significant（P0.05）,G2 phase cells showed no significant difference（P0.05）.The apoptosis rate in the experimental group was significantly lower than those in other two groups（P0.05）.（3）The mRNA expression of wnt5a in the experimental group decreased by（55.52±0.61）%,as compared with that in the control group（P0.05）.（4）The mRNA expression of CaMK Ⅱ in the experimental group significantly reduced,while β-catenin,cyclinD1,c-myc,and survivin significantly increased compared with other two groups（P0.05）.（5）The change of the protein level of CaMK Ⅱ,β-catenin,cyclinD1 had the same trend to that expression of mRNA in three groups.Conclusion：Targeted gene silencing wnt5a promotes malignant proliferation and inhibits apoptosis of Jurkat cells.The mechanism may be that the inhibition of non-classical wnt signaling pathway on β-catenin is relieved by down-regulating wnt5a and the downstream target genes of cyclinD1,c-myc,and survivin are up-regulated with the increase of β-catenin,which changes cell cycle and inhibits apoptosis,then leads to further proliferation of Jurkat cells.