摘要
目的:探讨蛋白激酶CK2B特异性小干扰RNA对肺腺癌细胞系A549生长增殖的影响。方法:构建CK2βsiRNA表达质粒稳定转染A549细胞,RT-PCR、免疫印迹、免疫细胞化学技术检测转染前后CK2β的mRNA和蛋白表达水平,同位素掺入法测定细胞内CK2活性,细胞计数法绘制细胞生长曲线,计算细胞倍增时间,流式细胞术分析细胞周期。结果:与空白组和阴性对照组相比,稳定转染pGPU6-CSNK2B质粒后,A549细胞中CK2β的mRNA水平、胞浆蛋白表达水平以及CK2活性均明显下降(P<0.05),但各组间细胞核中CK2β的蛋白表达水平无明显差异(P>0.05);A549细胞生长明显受到抑制,倍增时间增加;A549细胞阻滞于G0/G1期。结论:CK2β siRNA可以下调A549细胞中CK2β的表达及其激酶活性,并抑制细胞增殖;本研究为CK2特异性siRNA作为抗肿瘤药物的开发和CK2药靶的研究奠定了基础。
Objective： Protein kinase CK2 （formally known as casein kinase II） is a highly conserved and ubiquitous eukaryotic Ser/Thr protein kinase. It is composed of catalytic subunits and regulatory subunits; the CK2β subunit performs regulatory functions. Genetic, biochemical, and cell biological studies have indicated the involvement of this enzyme in the control of cell proliferation and in signal transduction. However, the regulation of CK2 is not well defined, and the effect of CK2β on the proliferation of lung adenocarcinoma cells is not fully understood. Accordingly, our objective was to assess the effect of small interfering RNA （siRNA） specific to protein kinase CK2β on proliferation in A549 lung adenocarcinoma cells. Methods： Protein kinase CK2β specific siRNA expression plasmid pGPU6-CSNK2B and non-specific siRNA expression plasmid pGPU6-NC were constructed and transfected into A549 cells. The mRNA level of CK2β in the transfected cells was detected by RT-PCR, and the protein level was assessed by Western blot and immunocytochemistry. The CK2 activity was assayed by detecting incorporation of 32P of [y-32P] ATP into the substrate. Doubling time of the transfected cells and cell cycle analysis were observed through cell counting and flow cytometry （FCM）. Results： The mRNA and protein levels of CK2β as well as CK2 activity in the cytoplasm were significantly decreased in the cells transfected with pGPU6-CSNK2B （P〈0.05）, while the CK2β protein level in the nucleus was slightly decreased （P〉0.05）. The A549 cells grew more slowly after transfection with pGPU6-CSNK2B （P〈0.05） and were blocked in G0/G1 stage. Doubling time of untreated cells was 39h, while that of the transfected cells was 56h. Conclusion： The expression and bioactivity of CK2β were suppressed by the expression of CK2β-specific siRNA from the pGPU6-CSNK2B plasmid. The proliferation of A549 host cells was also suppressed. CK2β may be a potential target for anti-cancer drug screening.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2009年第12期701-706,共6页
Chinese Journal of Clinical Oncology
基金
国家自然科学基金(编号:30672205、30871440)
广东省自然科学基金资助(编号:05011579)~~
