CDKL1基因沉默逆转胃癌耐药细胞株SGC-7901/DDP对顺铂耐药

Silencing of CDKL1 gene reverses the resistance of gastric cancer resistant cell line SGC-7901/DDP to cisplatin

目的探讨细胞周期依赖性激酶样蛋白1(CDKL1)基因沉默对胃癌顺铂(DDP)耐药细胞株SGC-7901/DDP耐药性的影响及其机制。方法采用qRT-PCR法检测DDP化疗耐药胃癌患者癌组织及DDP耐药胃癌细胞株(SGC-7901/DDP)中CDKL1 mRNA表达水平。将CDKL1 siRNA干扰质粒(si-CDKL1)及其阴性对照质粒(si-NC)转染至SGC-7901/DDP中,MTT方法检测不同DDP处理24 h后细胞增殖活性,计算IC50值;qRT-PCR方法检测细胞中CDKL1 mRNA表达水平;流式细胞术检测细胞凋亡水平;Western blot检测细胞中CDKL1及Wnt/β-catenin信号通路相关蛋白β-catenin、c-myc、cyclin D1、MDR1表达水平。结果CDKL1在DDP化疗耐药胃癌患者癌组织及DDP耐药胃癌细胞株中高表达(P<0.01)。CDKL1基因沉默后,SGC-7901/DDP细胞中CDKL1 mRNA和蛋白表达水平显著降低(P<0.01)。不同浓度DDP作用24 h后,CDKL1基因沉默可降低SGC-7901/DDP细胞增殖活性及IC50值,并提高细胞凋亡水平(P<0.01)。同时,CDKL1基因沉默可降低SGC-7901/DDP细胞中β-catenin、c-myc、cyclin D1、MDR1等蛋白表达水平(P<0.01)。结论CDKL1基因沉默可逆转胃癌耐药细胞株SGC-7901/DDP对顺铂耐药,其作用机制可能与抑制Wnt/β-catenin信号通路活化有关。

Objective To investigate the effects of cyclin dependent kinase like 1(CDKL1)gene silencing on drug resistance of gastric cancer cisplatin(DDP)cell line SGC-7901/DDP and its mechanism.Methods The level of CDKL1 mRNA in the cancer tissues of gastric cancer patients with DDP chemotherapy-resistant and DDP-resistant gastric cancer cell lines(SGC-7901/DDP)was detected by qRT-PCR method.SGC-7901/DDP cells were respectively transfected with CDKL1 siRNA interference plasmid(si-CDKL1)and its negative control plasmid(si-NC),and the cell proliferation activity was detected by MTT method after 24 hours of different DDP treatment,and calculate the IC50 value.The level of CDKL1 mRNA in transfected cells was assessed by qRT-PCR method.The apoptosis level was evaluated by flow cytometry.The expressions of CDKL1 and Wnt/β-catenin signaling pathway related proteinsβ-catenin,c-myc,cyclin D1 and MDR1 were detected by Western blot method.Results CDKL1 was high expression in DDP chemotherapy-resistant gastric cancer tissues and DDP-resistant gastric cancer cell lines(P<0.01).The mRNA and protein expression levels of CDKL1in SGC-7901/DDP cells were significantly reduced after CDKL1 gene silencing(P<0.01).After 24 hours of different concentrations of DDP,silencing of CDKL1 gene reduced the proliferation activity of SGC-7901/DDP and IC_(50)value,and increased the level of apoptosis(P<0.01).In addition,silencing of CDKL1 gene down-regulated the protein expression levels ofβ-catenin,c-myc,cyclin D1 and MDR1 in SGC-7901/DDP cells(P<0.01).Conclusion Silencing of CDKL1 gene could reverse the resistance of gastric cancer DDP-resistant cell line SGC-7901/DDP to cisplatin,and its mechanism of action may be related to inhibition of Wnt/β-catenin signaling pathway activation.