SW620细胞RACK1稳定RNA干扰细胞系的构建与鉴定

目的:为研究RACK1在结肠癌发生发展中的作用,构建结肠癌RACK1基因稳定RNA干扰(RNAi)细胞系。方法:根据人Gnb211 c DNA序列,运用干扰原则选择5个干扰位点并合成相应干扰片段,定向克隆入p Lentilox3.7干扰载体鼠U6启动子后并测序验证。用干扰及对照质粒分别转染HEK293T细胞48小时后,RT-PCR鉴定干扰效率,选出干扰效率较高的质粒包装慢病毒感染人结肠癌细胞SW620,流式无菌分选出荧光阳性的细胞扩增培养,RT-PCR及Western blotting鉴定慢病毒干扰效率。使用慢病毒构建的SW620 RACK1稳定RNAi细胞系及对照组进行MTT实验初步研究RACK1对SW620增殖的影响。结果:酶切和测序证实RACK1sh RNA质粒构建正确,产生能同时表达绿色荧光蛋白(EGFP)和RACK1 sh RNA的慢病毒载体质粒。慢病毒转导SW620并流式无菌分选扩增培养后,与空载体组相比,2个RNAi组均不同程度抑制RACK1表达,RACK1sh RNA5抑制作用最明显,RACK1干扰组细胞增殖得到了抑制。结论:SW620细胞RACK1稳定RNAi细胞系构建成功,为深入研究RACK1在结直肠癌发生发展中的作用奠定了基础。

Construction and Identification of Stable RACK1-RNAi SW620 Cell Lines

Objective:To construct stable RACK1-RNAi SW620 cell lines and lay a foundation for further research on the role of RACK1 in the occurrence and development of colon cancer.Methods:Following the principle of RNA interference, five interference sites were selected according to human Gnb211 cDNA sequence. These five interference fragments according to the five interference sites were synthesized and cloned into the site after mouse U6 promoter of pLentilox3.7 vector. After the plasmids were extracted and sequenced, they were subsequently transfected into HEK293T cells for 48 hours. The interference efficiency of the plasmids on the target gene was detected by RT-PCR. Two efficient plasmids were selected, then the control plasmid and the two selected plasmids were subsequently packaged into lentiviral viruses to infect human colon cancer cell line SW620. The interference efficiency of the lentiviral viruses was detected by RT-PCR and Western blotting. The impact of RACK1 RNAi on the proliferation of cell lines was assessed by MTT used the SW620 transfected with lentiviral virus.Results:The results of restriction enzyme analysis and DNA sequence analysis confirmed RACK1shRNA plasmids were constructed correctly, could express green fluorescent protein (EGFP) and RACK1 shRNA at the same time. SW620 cell lines transfected with lentiviral viruses were sorted by flow cytometry and amplified. Compared to control group, the RACK1 mRNA expression were suppressed in two experimental groups(RACK1Ri3,RACK1Ri5), especially RACK1Ri5 obviously suppressed the RACK1 mRNA expression. The proliferation of RACK1 RNAi group was inhibited.Conclusion:Stable RACK1-RNAi SW620 cell lines have been constructed successfully. This greatly facilitates the further studies on the role of RACK1 in the occurrence and development of colon cancer.