PDCD10表达下调导致人胶质母细胞瘤细胞系耐替莫唑胺治疗的机制研究

Mechanism of downregulated PDCD10 expression promoting temozolomide resistance in human glioblastoma cell lines

目的探讨程序性细胞死亡分子10(PDCD10)表达下调介导胶质母细胞瘤(GBM)细胞系耐替莫唑胺(TMZ)治疗的机制。方法使用PDCD10小干扰RNA或阴性序列小干扰RNA转染U87、LN229和T98g细胞系,分别应用300µmol/L TMZ(处理转染后的T98g细胞系)及150µmol/L TMZ(处理转染后的U87和LN229细胞系)以构建TMZ耐药细胞系,即PDCD10小干扰RNA转染及TMZ耐药的U87细胞系(shPDCD10-U87-RG)、阴性序列小干扰RNA转染及TMZ耐药的U87细胞系(EV-U87-RG)、shPDCD10-T98g-RG、EV-T98g-RG、shPDCD10-LN229-RG、EV-LN229-RG。采用流式细胞术和实时荧光定量PCR(qRT-PCR)实验检验TMZ耐药细胞系的转染效率和PDCD10表达水平,通过MTT实验和克隆形成实验验证TMZ耐药细胞系的耐药能力。进一步通过生物信息学分析检验PDCD10与碱基错配修复系统中关键基因MSH6、PMS2的相关性,同时通过检测耐药细胞的细胞周期和神经球形成能力来研究耐药细胞系的耐药机制。结果(1)qRT-PCR检测结果显示,与EV-U87-RG比较,shPDCD10-U87-RG细胞PDCD10表达下调了(32.85±1.14)%,差异有统计学意义(t=2.925,P=0.049);与EV-T98g-RG比较,shPDCD10-T98g-RG细胞PDCD10表达下调了(57.17±1.81)%,差异有统计学意义(t=3.179,P=0.043);与EV-LN229-RG比较,shPDCD10-LN229-RG细胞PDCD10表达下调了(33.68±1.34)%,差异有统计学意义(t=3.085,P=0.045)。(2)MTT实验结果显示,与EV-U87-RG比较,shPDCD10-U87-RG细胞活力明显增强,差异有统计学意义(P<0.05);与EV-T98g-RG比较,shPDCD10-T98g-RG细胞活力明显增强,差异有统计学意义(P<0.05)。同一种细胞之间,与TMZ处理72 h后比较,TMZ洗出后3 d细胞活力亦显著提高,差异有统计学意义(P<0.05)。克隆形成实验显示PDCD10表达下调的细胞系成瘤能力更高。(3)与EV-U87-RG细胞、EV-T98g-RG细胞比较,PDCD10表达下调的细胞(shPDCD10-U87-RG细胞、shPDCD10-T98g-RG细胞)逃逸TMZ诱导的细胞周期G2/M阻滞,导致出现耐TMZ治疗。(4)生物信息学分析发现PDCD10的表达与MSH6(r=0.262,P<0.001)和PMS2(r=0.327,P<0.001)的表达呈正相关关系;qRT-PCR实验发现PDCD10下调引起MSH6和PMS2表达下调,破坏碱基错配修复系统。(5)与EV-U87细胞比较,shPDCD10-U87细胞形成的神经球数量明显增多,差异有统计学意义(P<0.05);与EV-U87-RG细胞比较,shPDCD10-U87-RG细胞形成的神经球数量明显增多,差异有统计学意义(P<0.05)。结论PDCD10通过阻滞细胞周期进程、破坏碱基错配修复系统及增加细胞干性影响GBM对TMZ的治疗敏感性。

Objective To investigate the mechanism of downregulated programmed cell death 10(PDCD10)expression mediating glioblastoma multiforme(GBM)resistance to temozolomide(TMZ).Methods U87,LN229 and T98g cell lines were transfected with PDCD10 small interfering RNA or negative small interfering RNA.TMZ-resistant cell lines were constructed using 300µmol/L TMZ(transfected T98g cell line)and 150µmol/L TMZ(transfected U87 and LN229 cell lines),respectively:TMZ-resistant U87 cell line transfected with PDCD10 small interfering RNA(shPDCD10-U87-RG cells),TMZ-resistant U87 cell line transfected with negative small interfering RNA(EV-U87-RG cells),shPDCD10-T98g-RG cells,EV-T98g-RG cells,shPDCD10-LN229-RG cells and EV-LN229-RG cells.Flow cytometry and real-time quantitative polymerase chain reaction(qRT-PCR)were used to detect the transfection efficiency of TMZ-resistant cell lines and PDCD10 expressions;MTT assay and colony formation assay were used to verify the drug-resistant ability of TMZ-resistant cell lines.Bioinformatics analysis was performed to detect the correlations of PDCD10 with key genes(MSH6 and PMS2)in mismatch repair(MMR)system,and drug resistant mechanism was explored by detecting the cell cycle and neurosphere formation ability of drug-resistant cells.Results (1)qRT-PCR showed that compared with that in EV-U87-RG cells,the PDCD10 expression in shPDCD10-U87-RG cells was statistically down-regulated by(32.85±1.14)%(t=2.925,P=0.049);compared with that in EV-T98g-RG cells,the PDCD10 expression in shPDCD10-T98g-RG cells was significantly down-regulated by(57.17±1.81)%(t=3.179,P=0.043);compared with that in EV-LN229-RG cells,the PDCD10 expression in shPDCD10-LN229-RG cells was significantly down-regulated by(33.68±1.34)%(t=3.085,P=0.045).(2)MTT assay showed that compared with the EV-U87-RG cells,the shPDCD10-U87-RG cells had significantly increased viability(P<0.05);compared with the EV-T98g-RG cells,the shPDCD10-T98g-RG cells had significantly increased viability(P<0.05).Among the same kind of cells,the viability 3 d after wash-out was significantly increased compared with that at 72 h after TMZ treatment(P<0.05).Colony formation assay showed that cell lines with down-regulated PDCD10 expression had higher tumorigenic ability.(3)Compared with EV-U87-RG cells and EV-T98g-RG cells,cells with down-regulated PDCD10 expression(shPDCD10-U87-RG cells and shPDCD10-T98g-RG cells)escaped from TMZ-induced G2/M arrest,resulting in TMZ resistance.(4)Bioinformatics analysis revealed that the PDCD10 expression was positively correlated with MSH6 and PMS2 expressions(r=0.262,P<0.001;r=0.327,P<0.001);qRT-PCR indicated that downregulated PDCD10 expression caused decreased MSH6 and PMS2 expressions,which disrupted the MMR system.(5)Compared with that by EV-U87 cells,number of neurospheres formed by shPDCD10-U87 cells was significantly increased(P<0.05);compared with that by EV-U87-RG cells,number of neurospheres formed by shPDCD10-U87-RG cells was significantly increased(P<0.05).Conclusion PDCD10 affects the therapeutic sensitivity of GBM to TMZ by arresting cell cycle,disrupting MMR system,and increasing cell stemness.