高表达分泌颗粒蛋白Ⅱ增加结直肠癌细胞对奥沙利铂的耐药性

High expression of secretogranin II increases oxaliplatin resistance of colorectal cancer cells

目的 探究分泌颗粒蛋白Ⅱ(SCG2)在结直肠癌(CRC)组织中的表达特点,并分析其对奥沙利铂化疗敏感性的影响。方法 通过对组织芯片(包括96个癌组织和84个癌旁组织)进行免疫组化染色检测CRC患者癌组织及配对癌旁组织中SCG2的表达量并分析SCG2表达量与结直肠癌患者临床特征的关系。进一步采用蛋白免疫印迹Western blot和实时荧光定量PCR(qPCR)等方法检测化疗药物奥沙利铂(Oxa)处理结直肠癌细胞系后的SCG2的表达水平。利用CRISPR/Cas9技术建立SCG2敲除(SCG2-KO)的DLD1细胞株,采用CCK-8实验检测SCG2对药物敏感性的影响,采用Western blot和qPCR实验检测凋亡相关蛋白的表达水平。最后,用高浓度奥沙利铂持续处理DLD1细胞构建了对奥沙利铂耐药的DLD1细胞株,采用Western blot实验检测耐药株的SCG2的表达水平。结果 相较于癌旁组织,癌组织中SCG2表达量更高(P<0.0001),且更高的肿瘤N分期和临床分期患者结直肠癌组织SCG2阳性表达率更高。奥沙利铂处理结直肠癌细胞系可以显著增加其SCG2的表达量(P<0.05)。反过来,敲除SCG2降低了DLD1细胞对于奥沙利铂的药物敏感性(P<0.05)。机制研究发现SCG2敲除后凋亡相关分子表达量上调。并且,相较于亲本细胞株,SCG2在奥沙利铂耐药的DLD1细胞株(DLD1-OXAR)中显著高表达(P<0.05)。结论 SCG2是一个结直肠癌的危险性基因,且能够降低结直肠癌患者对奥沙利铂化疗药物的敏感性。

Objective To investigate the expression of secretogranin II(SCG2)in colorectal cancer(CRC)tissues and its impact on oxaliplatin resistance of CRC cells.Methods We performed immunohistochemistry to detect the expression level of SCG2 on a tissue microarray containing 96 CRC and 84 adjacent tissues and analyzed the association of SCG2 expression with the clinical features of the CRC patients.SCG2 expression was also measured in DLD1 cells treated with oxaliplatin using immunoblotting and RT-qPCR analyses.The effects of SCG2 expression on oxaliplatin sensitivity and cell viability were evaluated in a DLD1 cell model of SCG2 knockout established using CRISPR-cas9 technique,and the expressions of apoptosis-related proteins were detected using Western blotting and RT-qPCR.We further examined SCG2 expression levels in an oxaliplatin-resistant DLD1 cell line and its parental DLD1 cells.Results SCG2 expression was significantly increased in CRC tissues as compared with the adjacent tissues(1.932±0.816 vs 1),and the tumor tissues in advanced stages showed higher SCG2 expression levels.In DLD1 cells,treatment with oxaliplatin significantly increased SCG2 expression,and SCG2 knockout obviously increased oxaliplatin sensitivity of the cells and enhanced the expressions of apoptosis-related proteins.Compared with the parental cells,oxaliplatin-resistant DLD1 cells showed a significant increase of SCG2 expression by 3.901±0.471 folds.Conclusion SCG2 may serve as a risk gene in CRC,and its high expression increases oxaliplatin resistance of CRC cells.