摘要
目的探讨环状RNA蛋白酪氨酸激酶2(circPTK2)对结直肠癌(CRC)细胞顺铂(DDP)耐药性的影响。方法收集56例CRC样本(CRC组)及癌旁正常组织样本(对照组),体外培养人CRC细胞系(LoVo、HT-29、HCT116、SW480和SW620)和人正常结直肠粘膜细胞系(FHC),并建立DDP耐药的HCT116细胞(HCT116/DDP)。实时荧光定量PCR(qRT-PCR)检测组织/细胞中circPTK2、miR-761和细胞质多聚腺苷酸结合蛋白2(CPEB2)mRNA表达。取HCT116/DDP细胞分为对照(Control)组、si-NC组、si-circPTK2组、si-circPTK2+inhibitor-NC组、si-circPTK2+miR-761 inhibitor组。细胞计数试剂盒8(CCK-8)法测量各组细胞活力和DDP的半数抑制浓度(IC_(50))和增殖能力;流式细胞术检测细胞凋亡;Transwell检测细胞迁移或侵袭数;Western blot检测细胞中CPEB2蛋白表达;qRT-PCR检测circPTK2水平;starBase数据库(http://starbase.sysu.edu.cn/)预测miR-761与circPTK2或CPEB2的互补位点;将含有miR-761结合位点的circPTK2或CPEB23’UTR的野生型(WT)序列克隆到psiCHECK-2载体中以形成相应的荧光素酶报告载体(WT-circPTK2和WT-CPEB2),双荧光素酶报告基因分析系统检测荧光素酶活性;RNA结合蛋白免疫沉淀(RIP)测定Ago2或IgG RIP复合物中circPTK2、miR-761和CPEB2的富集水平。结果与对照组比较,CRC组患者癌组织中circPTK2、CPEB2 mRNA表达水平升高[(2.03±0.36)vs.(1.02±0.13)、(2.55±0.44)vs.(1.01±0.12)],而miR-761表达水平降低[(0.41±0.07)vs.(1.00±0.10)],差异均有统计学意义(P均<0.001)。Pearson相关性分析结果显示,CRC患者癌组织中circPTK2表达水平与miR-761水平成负相关(r=-0.766,P<0.001);miR-761表达水平与CPEB2 mRNA表达成负相关(r=-0.866,P<0.001),circPTK2的表达与CPEB2 mRNA表达成正相关(r=0.653,P<0.001)。DDP耐药患者癌组织中circPTK2、CPEB2 mRNA表达水平高于DDP敏感患者,而miR-761表达水平低于DDP敏感患者,差异均有统计学意义(t=12.983、10.352、12.459,P均<0.001)。与FHC细胞比较,CRC细胞系(LoVo、HT-29、HCT116、SW480和SW620)中circPTK2和CPEB2 mRNA的表达水平显著升高,miR-761的表达水平显著降低,差异均有统计学意义(P均<0.001);且HCT116细胞中circPTK2和CPEB2 mRNA的表达水平高于其他CRC细胞系,miR-761的表达水平低于其他CRC细胞系,因此,选择HCT116细胞进行后续实验。与Control组和siNC组比较,si-circPTK2组circPTK2、CPEB2 mRNA和蛋白水平显著降低,miR-761水平显著升高,差异均有统计学意义(P均<0.05);与si-circPTK2组、si-circPTK2+inhibitor-NC组比较,si-circPTK2+miR-761 inhibitor组CPEB2 mRNA和蛋白水平显著升高,miR-761水平显著降低,差异均有统计学意义(P均<0.05)。与Control组和si-NC组比较,sicircPTK2组HCT116/DDP细胞中DDP的IC_(50)、细胞增殖活力(48、72 h)、迁移细胞数和侵袭细胞数降低,差异均有统计学意义(P均<0.05);与si-circPTK2组、si-circPTK2+inhibitor-NC组比较,si-circPTK2+miR-761 inhibitor组HCT116/DDP细胞中DDP的IC_(50)、细胞增殖活力(48、72 h)、迁移细胞数和侵袭细胞数升高,差异均有统计学意义(P均<0.05)。与Control组和si-NC组比较,si-circPTK2组HCT116/DDP细胞凋亡率升高,差异均有统计学意义(P均<0.05);与si-circPTK2组、si-circPTK2+inhibitor-NC组比较,si-circPTK2+miR-761 inhibitor组HCT116/DDP细胞凋亡率降低,差异均有统计学意义(P均<0.05)。生物信息学分析显示,circPTK2和miR-761存在靶向结合序列;双荧光素酶检测结果显示,与转染miR-NC比较,转染miR-761 mimic后,含有WT-circPTK2质粒细胞的荧光素酶活性降低,差异有统计学意义(P<0.05);RIP检测结果显示,相对于lgG,circPTK2和miR-761在Ago2免疫沉淀中富集(P<0.05)。生物信息学分析显示,CPEB2的3’-UTR序列包含miR-761的结合位点;双荧光素酶检测结果显示,与转染miR-NC比较,转染miR-761 mimic后,含有WT-CPEB2质粒细胞的荧光素酶活性降低(P<0.05);RIP检测结果显示,相对于lgG,miR-761和CPEB2在Ago2免疫沉淀中富集(P<0.05)。结论circPTK2敲低可增强耐药CRC细胞对DDP的敏感性,其作用机制可能与调节miR-761/CPEB2轴有关。
Objective To investigate the influences of circular RNA protein tyrosine kinase 2(circPTK2)on resistance of colorectal cancer(CRC)cells to cisplatin(DDP).Methods Fifty-six CRC samples(CRC group)and adjacent normal tissue samples(control group)were collected,and human CRC cell lines(LoVo,HT-29,HCT116,SW480 and SW620)and human normal colorectal mucosa cell line(FHC)were cultured in vitro,and DDP-resistant HCT116 cells(HCT116/DDP)were established.Quantitative real time polymerase chain reaction(qRT-PCR)was used to detect the expression of circPTK2,miR-761 and cytoplasmic polyadenylation binding protein 2(CPEB2)mRNA in tissues/cells.HCT116/DDP cells were separated into Control group,si-NC group,si-circPTK2 group,si-circPTK2+inhibitor-NC group,and si-circPTK2+miR-761 inhibitor group.Cell counting kit-8(CCK-8)method was used to measure cell viability,DDP half-inhibitory concentration(IC_(50))and proliferation ability;flow cytometry was used to detect apoptosis.Transwell assay was used to detect the number of cell migration or invasion;the expression of CPEB2 protein was detected by Western blot;circPTK2 levels were detected by qRT-PCR;starBase database(http://starbase.sysu.edu.cn/)was used to predict miR-761 and complementary circPTK2 or CPEB2 sites;wild type(WT)sequences of circPTK2 or CPEB23'UTR containing miR-761 binding sites were cloned into psiCHECK 2 vector to form corresponding luciferase reporter vectors(WT-circPTK2 and WT-CPEB2);dual luciferase reporter gene analysis system was used to detect luciferase activity;RNA binding protein immunoprecipitation(RIP)was used to determine the enrichment levels of circPTK2,miR-761 and CPEB2 in Ago2 or IgG RIP complexes.Results Compared with the Control group,the mRNA expression levels of circPTK2 and CPEB2 in cancer tissues of CRC group were increased[(2.03±0.36)vs.(1.02±0.13),(2.55±0.44)vs.(1.01±0.12)],the expression level of miR-761 was decreased[(0.41±0.07)vs.(1.00±0.10)],and the differences were statistically significant(all P<0.001).The results of Pearson correlation analysis showed that the expression level of circPTK2 in cancer tissues of CRC patients was negatively correlated with the level of miR-761(r=-0.766,P<0.001);the expression level of miR-761 was negatively correlated with CPEB2 mRNA expression(r=-0.866,P<0.001),and the expression of circPTK2 was positively correlated with CPEB2 mRNA expression(r=0.653,P<0.001).The mRNA expression levels of circPTK2 and CPEB2 in cancer tissues of DDP-resistant patients were higher than those of DDP-sensitive patients,while the expression level of miR-761 was lower than that of DDP-sensitive patients(t=12.983,10.352,12.459;all P<0.001).Compared with FHC cells,the expression levels of circPTK2 and CPEB2 mRNA were significantly increased in CRC cell lines(LoVo,HT-29,HCT116,SW480 and SW620),while the expression levels of miR-761 were significantly decreased,with statistical significance(all P<0.001);moreover,the expression levels of circPTK2 and CPEB2 mRNA in HCT116 cells were higher than those of other CRC cell lines,and the expression level of miR-761 was lower than those of other CRC cell lines,therefore,HCT116 cells were selected for follow-up experiments.Compared with the Control group and the si-NC group,the circPTK2 and CPEB2 mRNA and protein levels in si-circPTK2 group were significantly decreased,and the miR-761 level was significantly increased,with statistical significance(all P<0.05);compared with si-circPTK2 group and si-circPTK2+inhibitor-NC group,CPEB2 mRNA and protein levels were significantly increased and miR-761 levels were significantly decreased in si-circPTK2+miR-761 inhibitor group,the differences were statistically significant(all P<0.05).Compared with the Control group and the si-NC group,IC_(50),cell proliferation activity(48,72 h),the number of migrating cells and invading cells in HCT116/DDP cells in the si-circPTK2 group were decreased,and the differences were statistically significant(all P<0.05);compared with si-circPTK2 group and si-circPTK2+inhibitor-NC group,IC_(50),cell proliferation activity(48,72 h),the number of migration cells and invasion cells in DDP cells in si-circPTK2+miR-761 inhibitor group were increased,and the differences were statistically significant(all P<0.05).Compared with the Control group and the si-NC group,the apoptosis rate of HCT116/DDP cells in the si-circPTK2 group was increased,with statistical significance(both P<0.05);compared with si-circPTK2 group and si-circPTK2+inhibitor-NC group,the apoptosis rate of HCT116/DDP cells in si-circPTK2+miR-761 inhibitor group was decreased,and the differences were statistically significant(both P<0.05).Bioinformatics analysis showed that circPTK2 and miR-761 had targeted binding sequences;dual luciferase assay results showed that after transfecting miR-761 mimic,the luciferase activity of granulocytes containing WT-circPTK2 was decreased compared with that of transfected miR-NC,and the difference was statistically significant(P<0.05);RIP detection results showed that circPTK2 and miR-761 were enriched in Ago2 immunoprecipitation compared with lgG(P<0.05).Bioinformatics analysis showed that the 3'-UTR sequence of CPEB2 contained the binding site of miR-761;dual luciferase assay results showed that after transfecting miR-761 mimic,the luciferase activity of granulocytes containing WT-CPEB2 was decreased compared with that of transfected miR-NC,and the difference was statistically significant(P<0.05);RIP detection results showed that compared with lgG,miR-761 and CPEB2 were enriched in Ago2 immunoprecipitation(P<0.05).Conclusion CircPTK2 knockdown could enhance the sensitivity of drug-resistant CRC cells to DDP,and its mechanism might be related to the regulation of miR-761/CPEB2 axis.
作者
徐晓忠
赵飞龙
XU Xiaozhong;ZHAO Feilong(Department of Generalsurgery,Zhangjiagang Hospital of Traditional Chinese Medicine,Zhangjiagang,Jiangsu 215600,China)
出处
《热带医学杂志》
CAS
2023年第12期1645-1653,1796,共10页
Journal of Tropical Medicine
基金
江苏省卫生健康委医学科研立项项目(BK20200233)
苏州市社会发展科技创新项目
