Effect of CCAR2 depletion on the gene expression profile of BJ-hTERT cells

CCAR2 is a nuclear protein recently emerged as a pivotal player of the DNA damage response since it has been found involved in both apoptosis induction and DNA repair. Differently, its role in tumorigenesis and cancer progression is still elusive. In our studies we found that CCAR2 depletion impairs the proliferation of human cancer cell lines, but leaves unaffected the growth of normal immortalized cells. To better investigate this point we performed a genome wide gene expression analyses in U2OS and BJ-hTERT depleted of CCAR2 and we found that loss of this protein causes the deregulation of genes implicated in the AKT pathway specifically in U2OS cells, but not in BJ-hTERT. In accordance with these results we found a reduction in AKT activation in all the tested cancer cell lines depleted of CCAR2, but not in the normal ones. The defective activation of AKT is caused by the upregulation of TRB3 gene in cancer cells depleted of CCAR2 and finally results in the reduction of GSK3β phosphorylation, prevention of G1/S transition and inhibition of cancer cell growth. The human normal fibroblast cell line BJ-hTERT was cultured in Dulbecco's Modified Eagle Medium (DMEM)/Medium 199 (4:1) supplemented with 10% of fetal calf serum and 10µg/ml hygromycin B as selection for hTERT transgene. To obtain a population of CCAR2-knock-out (KO) cells we used the CRISPR/Cas9 system as described in Magni et al, Oncotarget 2015. Cells were transfected with the Amaxa Nucleofector Device using the Basic Fibroblast Kit (VPI-1002) and U23 program. A population of 40% cells negative for CCAR2 was initially obtained. Then, after a second round of transfection, a mass culture with the 80% of cells KO for CCAR2 was produced. These cells were then collected in triplicates for RNA extraction and gene expression analyses.

CCAR2是一种核蛋白，近期被发现作为DNA损伤应答的关键调控因子，因其同时参与细胞凋亡诱导与DNA修复过程。与之不同的是，其在肿瘤发生及癌症进展中的作用仍尚不明确。本研究团队发现，敲低CCAR2会损害人类癌细胞系的增殖能力，但对永生化正常细胞的生长无显著影响。为深入探究这一现象，我们在CCAR2敲减的U2OS细胞与BJ-hTERT细胞中开展了全基因组基因表达分析，结果显示该蛋白的缺失仅在U2OS细胞中引发AKT通路相关基因的表达失调，而在BJ-hTERT细胞中未观察到此现象。与上述结果相符，我们在所有受试的CCAR2敲减癌细胞系中均检测到AKT激活水平降低，但正常细胞中并无该变化。AKT激活缺陷的成因是CCAR2敲减的癌细胞内TRB3基因的上调，该变化最终会降低GSK3β的磷酸化水平、阻断G1/S期转换并抑制癌细胞增殖。人类正常成纤维细胞系BJ-hTERT培养于高糖达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM）与199培养基按4:1比例混合的培养液中，添加10%胎牛血清及10μg/ml潮霉素B以筛选携带hTERT转基因的细胞。为构建CCAR2基因敲除（knock-out, KO）细胞群，我们采用CRISPR/Cas9系统，实验方法参照Magni等人2015年发表于《Oncotarget》的研究。使用Amaxa核转染仪（Amaxa Nucleofector Device）搭配基础成纤维细胞试剂盒（Basic Fibroblast Kit, VPI-1002）及U23程序完成细胞转染。初始获得CCAR2阴性率为40%的细胞群，经第二轮转染后，得到CCAR2敲除率达80%的混合细胞群体。随后将这些细胞分为三份进行RNA提取与基因表达分析。