周期蛋白依赖性激酶所介导的RBBP8磷酸化在同源性重组修复中的调控机制  

Study on the mechanism of cyclin-dependent kinases mediated RBBP8 phosphorylation in homologous recombination repair

作　　者：于洋[1] 王淑霞 尹艳华[3] 张澍田[1] YU Yang;WANG Shuxia;YIN Yanhua;ZHANG Shutian(Department of Gastroenterology,Beijing Friendship Hospital,Beijing 100032,China;Department of Oncology,Beijing Aviation General Hospital,Beijing 100032,China;Department of Pathology,Liaocheng People's Hospital,Liaocheng,Shandong 252000,China)

机构地区：[1]北京友谊医院消化内科,北京100032 [2]北京航空总医院肿瘤科,北京100032 [3]聊城市人民医院病理科,山东聊城252000

出　　处：《中华肿瘤防治杂志》2023年第19期1146-1153,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81970496)。

摘　　要：目的 阐明周期蛋白依赖性激酶(CDK)所介导的RBBP8磷酸化在同源重组(HR)修复中的调控机制,并初步验证CDK抑制剂与聚腺苷二磷酸核糖聚合酶(PARP)抑制剂在胃癌中的合成致死效应。方法 通过蛋白质印迹法、免疫共沉淀(Co-IP)验证CDK对RBBP8-BRCA1蛋白复合体的调控作用;免疫荧光实验探究CDK介导的RBBP8磷酸化在HR修复途径中调控机制;MTS实验研究CDK抑制剂与PARP抑制剂对胃癌细胞生存活力的影响;Compusyn计算联合指数以验证CDK抑制剂与PARP抑制剂的协同效应。结果 蛋白质印迹法结果显示,胃癌细胞系AGS和N87细胞中磷酸化的RBBP8蛋白327位丝氨酸(RBBP8-S327)蛋白表达水平分别为0.37±0.02和0.75±0.05,提示CDK抑制剂可降低AGS及N87中RBBP8-S327的磷酸化水平,t值分别为25.66和5.50,P值分别为<0.001和0.005。Co-IP结果显示,相对于NC组,CDK抑制剂组BRCA1蛋白水平为0.27±0.03,提示CDK抑制剂削弱了RBBP8和BRCA1之间的蛋白质相互作用,t=28.27,P<0.001。CDK抑制剂遏制了DNA损伤发生后的双链断裂修复蛋白51(RAD51)核内聚集效应及损伤位点的复制蛋白A(RPA)焦点形成。MTS实验结果提示,AGS与N87细胞中,单药使用PARP抑制剂浓度>1μmol/L可明显抑制胃癌细胞系的生存活力,差异均有统计学意义,F值分别为1 702.0和184.4,均P<0.001;PARP联合CDK抑制剂处理时,2μmol/L的CDK抑制剂可显著增强不同浓度(0.010、0.100、1.000、5.000和10.000μmol/L)PARP抑制剂对胃癌细胞活力的抑制作用,t值分别为7.27和10.11,均P<0.001。二者的联合指数<1。结论 CDK介导的RBBP8磷酸化及RBBP8-BRCA1蛋白复合体在HR修复中发挥了关键调控作用,CDK抑制剂联合PARP抑制剂在胃癌中具有潜在的合成致死效应。Objective To elucidate the regulatory mechanism of cyclin-dependent kinases(CDK)mediated RBBP8 phospho-rylation in homologous recombination(HR)repair,and preliminarily verify the"synthetic lethal"effect of CDK inhibitors combined with poly ADP-ribose polymerase(PARP)inhibitors in gastric cancer.Methods Western blot and Co-IP were performed to observe the regulatory effect of CDK on the RBBP8-BRCA1 protein complex.The regulatory mechanism of CDK mediated RBBP8 phosphorylation in the HR repair pathway was explored by immunofluorescence assay.The effects of CDK inhibitors and PARP inhibitors on the survival viability of gastric cancer cells was investigated through MTS experiments,and the synergistic effects of CDK and PARP inhibitors was verified by calculating the combination index using Compusyn software.Results The results of Western blot showed the protein expression levels of phosphorylated RBBP8 protein 327 serine(RBBP8-S327)in gastric cancer cell lines AGS and N87 were 0.37±0.02 and 0.75±0.05,respectively,which indicated CDK inhibitors could reduce the phosphorylation level of RBBP8 protein 327 serine in gastric cancer cell lines AGS and N87,the t-values were 25.66 and 5.50,and P-values were<0.001 and 0.005,respectively.The Co-IP results showed that compared to the NC group,the BRCAl protein level in the CDK inhibitor group were 0.27±0.03,this indicated CDK inhibitors treatment could weaken the protein interaction between RBBP8 and BRCA1(t=28.27,P<0.001).CDK inhibitor treatment inhibited the intranuclear aggregation of double-strand breaks repair protein RAD51(RAD51)and the formation of RPA focus in DNA damage site.The MTS experiment results indicated that in AGS and N87 cells,single drug use of PARP inhibitors with a concentration>1μmol/L could significantly inhibit the viability of gastric cancer cell lines,with statistically significant differences,the F-values were 1702.0 and 184.4,respectively,both P<0.001.When PARP inhibitors were combined with CDK inhibitors,CDK inhibitors(2μmol/L)could increase

关 键 词：胃癌 RBBP8 周期蛋白依赖性激酶抑制剂 同源重组修复 合成致死 

分 类 号：R735.2[医药卫生—肿瘤]