利用基因表达谱芯片从外周血筛选直肠癌术前放化疗敏感标志物的研究

Study on screening of sensitive markers from peripheral blood by gene microarray for preoperative chemo-radiotherapy in rectal cancer patients

目的利用基因表达谱芯片在进展期直肠癌患者外周血中初步筛选直肠癌术前放化疗敏感的分子标志物。方法接受术前放化疗并进行直肠癌根治术患者30例,根据放化疗后原发肿瘤降期情况,分为敏感组(第1组)7例、中度敏感组(第2组)15例、耐受组(第3组)8例。采用基因表达谱PCR芯片,应用实时荧光PCR同时对6条经典的肿瘤信息通道标志基因进行表达分析,检测放化疗前(A1、A2、A3亚组)、放化疗后(B1、B2、B3组)外周血游离RNA中肿瘤信号通路的关键基因情况,分析外周血中与放化疗敏感性相关的标记物。结果放化疗前存在29个基因差异,随着放化疗敏感性增加,表达逐渐增加的是DKK1、CD24、CDKN1A、ENC1、FOS、IER3、IFI6、PLEK、PPP2R2D、RCAN1、VDR、ZFP36;放化疗后存在20个基因差异,随着放化疗敏感性增加,表达逐渐增加的是:CEBPB、ARF1、CXCL9、CCND1、DNAJB9、ESR1、IER3、IFI6、IRSI、SEMA3C、TXNIP、TM4SF1。随着放化疗敏感性增加,放化疗前后差异基因在3组中均呈递增表达的基因为IER3、IFI6、PPP2R2D,其中IER3、PPP2R2D在放化疗前后组内比较,差异均有统计学意义(P<0.05);随着放化疗敏感性增加,放化疗前呈递减基因为GADD45B、SOCS3(P<0.05),但放化疗后组间比较,差异无统计学意义(P>0.05)。IER3、PPP2R2D、SOCS3在敏感组与耐受组间基因表达差异在4倍以上。结论利用基因表达谱芯片检测外周血游离RNA,可初步为直肠癌术前放化疗敏感性提供预测指标,其中IER3、PPP2R2D、SOCS3基因有可能成为理想的预测标志物。

Objective To preliminarily screen the sensitive molecular markers from peripheral blood by gene microarray for preoperative chemo-radiotherapy in patients with advanced rectal cancer.Methods Thirty patients receiving preoperative chemo-radiotherapy and following radical resection of rectal cancer were divided into sensitive group（NO.1 group,n=7）,moderate sensitive group（NO.2 group,n=15） and tolerance group（ NO.3 group,n=8） according to the descent stage of tumors after chemo-radiotherapy.The marker genes of six classic tumor signaling pathways were analyzed with gene microarray and real-time fluorescent PCR.Before （ Subgroup A1, Subgroup A2 and Subgroup A3） and after chemo-radiotherapy（Subgroup B1,Subgroup B2 and Subgroup B3）,the key genes of tumor signal pathway in free RNA of peripheral blood were detected, and the markers in peripheral blood which were related to chemo-radiotherapy sensitivity were analyzed.Results The expression differences of 29 genes were observed before chemo-radiotherapy,and the expressions of DKK1,CD24, CDKN1A,ENC1, FOS, IER3, IFI6, PLEK, PPP2R2D, RCAN1, VDR and ZFP36 increased gradually with the increasing sensitivity of chemo-radiotherapy among three groups.While the expression differences of 20 genes were observed after chemo-radiotherapy, and the expressions of CEBPB,ARF1,CXCL9,CCND1,DNAJB9,ESR1,IER3,IFI6,IRSI,SEMA3C,TXNIP and TM4SF1 increased gradually with the increasing sensitivity of chemo-radiotherapy among three groups.Before and after chemo-radiotherapy,the expressions of IER3,IFI6 and PPP2R2D increased with the increasing of sensitivity of chemo-radiotherapy among three groups,and there were statistically significant differences in the expressions of IER3 and PPP2R2D in each group before and after chemo-radiotherapy（P〈0.05）.The expressions of GADD45B and SOCS3 decreased with the increasing of sensitivity of chemo-radiotherapy before chemo-radiotherapy（P〈0.05）,but they showed no statistically significant difference among three groups after chemo-radiotherapy（P〉0.05）.The expression differences of IER3, PPP2R2D and SOCS3 of sensitive group were four times more than those of tolerant group.Conclusion Dectection of free RNA in peripheral blood with gene microarray can preliminarily provide the predictive indicators for the sensitivity of preoperative chemo-radiotherapy in rectal cancer patients,and IER3,PPP2R2D and SOCS3 genes might become the ideal predictive markers.