Chen Xitao, Sun Xiaodi, Guan Jingqian, Gai Junda, Xing Jilin, Fu Lin, Liu Shuli, Shen Feifei, Chen Keyan, Li Wenya, Han Libo, Li Qingchang
Department of Pathology, College of Basic Medical Sciences, China Medical University, Shenyang, China.
Department of Thoracic Surgery, the First Affiliated Hospital, China Medical University, Shenyang, China.
Cell Physiol Biochem. 2017;44(6):2322-2336. doi: 10.1159/000486116. Epub 2017 Dec 15.
BACKGROUND/AIMS: The therapeutic efficacy of paclitaxel is hampered by chemotherapeutic resistance in non-small cell lung cancer (NSCLC). Rsf-1 enhanced paclitaxel resistance via nuclear factor-κB (NF-κB) in ovarian cancer cells and nasopharyngeal carcinoma. This study assessed the function of Rsf-1 in the modulation of the sensitivity of NSCLC to paclitaxel via the NF-κB pathway.
The mRNA and protein levels of the related genes were quantified by RT-PCR and Western blotting. Rsf-1 silencing was achieved with CRISPR/Cas9 gene editing. Cell cycle, migration and proliferation were tested with flow cytometry, transwell test and CCK8 test. Cell apoptosis was analyzed with flow cytometry and quantification of C-capase3. The parameters of the tumors were measured in H460 cell xenograft mice.
Rsf-1 was highly expressed in H460 and H1299 cells. Rsf-1 knockout caused cell arrest at the G1 phase, increased cell apoptosis, and decreased migration and cell proliferation. Rsf-1 knockout increased the inhibition of cell proliferation, the reduction in cell migration and the augment in cell apoptosis in paclitaxel treated H460 and H1299 cells. Rsf-1 knockout further enhanced the paclitaxel-mediated decrease in the volume and weight of the tumors in H460 cell xenograft mice. Helenalin and Rsf-1 knockout decreased the protein levels of p-P65, BcL2, CFLAR, and XIAP; hSNF2H knockout decreased the protein level of NF-κB p-P65 without altering Rsf-1 and p65 protein levels, while Rsf-1 and hSNF2H double knockout decreased the level of NF-κB p-P65, in H1299 and H460 cells.
These results demonstrate that Rsf-1 influences the sensitivity of NSCLC to paclitaxel via regulation of the NF-κB pathway and its downstream genes.
背景/目的：非小细胞肺癌（NSCLC）中的化疗耐药性阻碍了紫杉醇的治疗效果。在卵巢癌细胞和鼻咽癌中，Rsf-1通过核因子κB（NF-κB）增强了紫杉醇耐药性。本研究评估了Rsf-1通过NF-κB途径调节NSCLC对紫杉醇敏感性的功能。
通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法对相关基因的mRNA和蛋白质水平进行定量分析。利用CRISPR/Cas9基因编辑技术使Rsf-1沉默。采用流式细胞术、Transwell实验和CCK8实验检测细胞周期、迁移和增殖情况。通过流式细胞术和Caspase3定量分析细胞凋亡情况。在H460细胞异种移植小鼠中测量肿瘤参数。
Rsf-1在H460和H1299细胞中高表达。Rsf-1基因敲除导致细胞停滞于G1期，细胞凋亡增加，迁移和细胞增殖减少。Rsf-1基因敲除增强了紫杉醇对H460和H1299细胞增殖的抑制作用、细胞迁移的减少以及细胞凋亡的增加。Rsf-1基因敲除进一步增强了紫杉醇介导的H460细胞异种移植小鼠肿瘤体积和重量的减小。在H1299和H460细胞中，海伦alin和Rsf-1基因敲除降低了p-P65、BcL2、CFLAR和XIAP的蛋白质水平；hSNF2H基因敲除降低了NF-κB p-P65的蛋白质水平，而未改变Rsf-1和p65的蛋白质水平，而Rsf-1和hSNF2H双基因敲除降低了H1299和H460细胞中NF-κB p-P65的水平。
这些结果表明，Rsf-1通过调节NF-κB途径及其下游基因影响NSCLC对紫杉醇的敏感性。
