安罗替尼在肝癌中耐药的差异mRNA筛选  

Differentially expressed mRNA involved in the resistance of liver cancer to anlotinib

作　　者：谷俊谋 王立博 曾德俊 陆勤伟 董凯 梁若鹏[1] 王维杰[1] 朱荣涛[1] 孙玉岭[1,2,3] GU Junmou;WANG Libo;ZENG Dejun;LU Qinwei;DONG Kai;LIANG Ruopeng;WANG Weijie;ZHU Rongtao;SUN Yuling(Department of Hepatobiliary and Pancreatic Surgery,The First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Institute of Hepatobiliary and Pancreatic Diseases,Zhengzhou University,Zhengzhou 450052,China;Zhengzhou Municipal Key Laboratory of Basic and Clinical Research on Hepatobiliary and Pancreatic Diseases,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院肝胆胰外科,郑州450052 [2]郑州大学肝胆胰疾病研究所,郑州450052 [3]郑州市肝胆胰疾病基础与临床研究重点实验室,郑州450052

出　　处：《临床肝胆病杂志》2021年第2期358-363,共6页Journal of Clinical Hepatology

基　　金：国家自然科学基金(81870457,81900558)。

摘　　要：目的运用ceRNA芯片筛选可能参与肝癌细胞对安罗替尼耐药过程的mRNA。方法利用大剂量冲击联合低剂量诱导的方法建立对安罗替尼耐药的肝癌细胞,用CCK8实验进行验证耐药细胞在安罗替尼作用下的细胞增殖差异;运用ceRNA芯片检测耐药肝癌细胞与正常肝癌细胞的基因表达差异;运用实时荧光定量PCR(real-time PCR)对部分芯片测出的部分基因差异进行验证。计量资料两组间比较采用独立样本t检验,Kaplan-Meier法对肝癌样本的总生存期进行生存分析,log-rank检验比较生存率差异。芯片筛选结果使用Fisher精确检验。结果耐药肝癌细胞与正常肝癌细胞基因表达差异较大,通过缩减范围筛选出差异最大的10个基因进行分析。与耐药和肿瘤生长相关的基因有4个,分别为BIRC2、BIRC7、ABCC2、MAPK8。其中BIRC2、ABCC2、MAPK8表达水平下降(P值分别为0.0014、0.0012、0.0118),BIRC7的表达水平增多(P<0.001)。real-time PCR的验证结论与芯片一致(t值分别为10.74、32.65、18.34、2.80,P值分别为0.0004、0.0001、0.0001、0.0448)。BIRC7的高表达与MAPK8的低表达对应显著减少的生存期(P值分别0.0220、0.0056)。结论BIRC2、BIRC7、ABCC2、MAPK8在对安罗替尼耐药的肝癌细胞中差异表达,可能参与了肝癌细胞对安罗替尼耐药的过程。Objective To screen out the mRNAs involved in the resistance of hepatoma cells to anlotinib using ceRNA microarray.Methods High-dose shock combined with low-dose induction was used to culture hepatoma cells resistant to anlotinib,and CCK8 assay was used to verify the difference in the proliferation of drug-resistant hepatoma cells treated by anlotinib.The ceRNA microarray was used to screen out the differentially expressed genes between drug-resistant hepatoma cells and normal hepatoma cells,and real-time PCR was used to verify the differentially expressed genes detected by some microarrays.the independent samples t-test was used for comparison of continuous data between two groups,and the Kaplan-Meier method was used to analyze the overall survival of hepatoma cells samples,and the log-rank test was used to compare survival rates.Fisher’s exact test was used for chip screening.Results There was a significant difference in gene expression between drug-resistant hepatoma cells and normal hepatoma cells,and 10 genes with the greatest difference were screened out for analysis by reducing the range.There were 4 genes associated with drug resistance and tumor growth,i.e.,BIRC2,BIRC7,ABCC2,and MAPK8.There were significant reductions in the expression levels of BIRC2,ABCC2,and MAPK8(P=0.0014,0.0012,and 0.0118),and there was a significant increase in the expression of BIRC7(P<0.001).The results of real-time PCR were consistent with those of microarray(t=10.74,32.65,18.34,and 2.80;P=0.0004,0.0001,0.0001,and 0.0448).The high expression of BIRC7 and the low expression of MAPK8 were associated with the significant reduction in survival time(P=0.0220 and 0.0056).Conclusion BIRC2,BIRC7,ABCC2,and MAPK8 are differentially expressed between anlotinib-resistant hepatoma cells and normal hepatoma cells and may be involved in the resistance of hepatoma cells to anlotinib.

关 键 词：肝肿瘤 安罗替尼 抗药性 肿瘤 RNA 信使 

分 类 号：R735.7[医药卫生—肿瘤]