安罗替尼在肝癌中耐药的差异mRNA筛选

谷俊谋; 王立博; 曾德俊; 陆勤伟; 董凯; 梁若鹏; 王维杰; 朱荣涛; 孙玉岭

doi:10.3969/j.issn.1001-5256.2021.02.022

安罗替尼在肝癌中耐药的差异mRNA筛选

DOI: 10.3969/j.issn.1001-5256.2021.02.022

谷俊谋¹,
王立博¹,
曾德俊¹,
陆勤伟¹,
董凯¹,
梁若鹏¹,
王维杰¹,
朱荣涛¹,
孙玉岭^{1, 2, 3, ,}

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

基金项目:

国家自然科学基金 (81870457);

国家自然科学基金 (81900558)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突，特此声明。

作者贡献声明：谷俊谋负责课题设计，资料分析，撰写论文；王立博、曾德俊、陆勤伟、董凯、梁若鹏、王维杰、朱荣涛参与收集数据，修改论文；孙玉岭负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

作者简介:
谷俊谋(1993—)，男，主要从事肝脏相关疾病的研究

通信作者:
孙玉岭，doctorssh@126.com

中图分类号: R735.7
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- 被引次数: 0
出版历程
- 收稿日期: 2020-09-29
- 录用日期: 2020-11-27
- 出版日期: 2021-02-20

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

1.
Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
2.
Institute of Hepatobiliary and Pancreatic Diseases, Zhengzhou University, Zhengzhou 450052, China
3.
Zhengzhou Municipal Key Laboratory of Basic and Clinical Research on Hepatobiliary and Pancreatic Diseases, Zhengzhou 450052, China

摘要

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0.001 4、0.001 2、0.011 8)，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0.000 4、0.000 1、0.000 1、0.044 8)。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0.022 0、0.005 6)。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。
- 肝肿瘤 /
- 安罗替尼 /
- 抗药性, 肿瘤 /
- RNA, 信使
Abstract: 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=0.001 4, 0.001 2, and 0.011 8), 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=0.000 4, 0.000 1, 0.000 1, and 0.044 8). The high expression of BIRC7 and the low expression of MAPK8 were associated with the significant reduction in survival time (P=0.022 0 and 0.005 6). 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.
- Liver Neoplasms /
- Anlotinib /
- Drug Resistance, Neoplasm /
- RNA, Messenger

HTML全文

图 1 CCK8法检测安罗替尼对Huh7和Huh7/R的抑制作用

下载: 全尺寸图片幻灯片

图 2 差异mRNA热图见二维码

下载: 全尺寸图片幻灯片

图 3 正常肝癌细胞与耐药肝癌细胞差异基因的GO富集分析图

下载: 全尺寸图片幻灯片

图 4 正常肝癌细胞与耐药肝癌细胞差异基因的KEGG pathway分析图

下载: 全尺寸图片幻灯片

注：a, BIRC2;b, ABCC2;c, MAPK8；d.BIRC7。

图 5 4个基因的mRNA在正常肝癌细胞和耐药肝癌细胞中的表达水平比较

下载: 全尺寸图片幻灯片

注：a, BIRC2; b，ABCC2; c, MAPK8；d, BIRC7。

图 6 BIRC2、ABCC2、MAPK8、BIRC7的生存分析

下载: 全尺寸图片幻灯片

参考文献(20)

[1]	Bureau of Medical Administration, National Health Commission of the People's Republic of China. Guidelines for diagnosis and treatment of primary liver cancer in China (2019 edition)[J]. J Clin Hepatol, 2020, 36(2): 277-292. (in Chinese) DOI: 10.3969/j.issn.1001-5256.2020.02.007 中华人民共和国国家卫生健康委员会医政医管局.原发性肝癌诊疗规范(2019年版)[J].临床肝胆病杂志, 2020, 36(2): 277-292. DOI: 10.3969/j.issn.1001-5256.2020.02.007
[2]	WANG S, WU QW, LI XK, et al. Interpretation of guidelines for diagnosis and treatment of primary liver cancer in China (2019 edition)[J]. J Clin Hepatol, 2020, 36(5): 996-999. (in Chinese) DOI: 10.3969/j.issn.1001-5256.2020.05.009 王姗, 吴庆旺, 李小科, 等. 《原发性肝癌诊疗规范(2019年版)》解读[J].临床肝胆病杂志, 2020, 36(5): 996-999. DOI: 10.3969/j.issn.1001-5256.2020.05.009
[3]	SUN B, YANG XZ, XIE F, et al. Effect of sorafenib combined with transcatheter arterial chemoembolization and radiofrequency ablation on the survival of patients with hepatocellular carcinoma[J]. J Clin Hepatol, 2019, 35(1): 104-108. (in Chinese) DOI: 10.3969/j.issn.1001-5256.2019.01.019 孙斌, 杨晓珍, 谢放, 等.索拉非尼联合经肝动脉化疗栓塞术和射频消融对肝细胞癌患者生存期的影响[J].临床肝胆病杂志, 2019, 35(1): 104-108. DOI: 10.3969/j.issn.1001-5256.2019.01.019
[4]	HE Z, YU Y, NONG Y, et al. Hepatitis B virus X protein promotes hepatocellular carcinoma invasion and metastasis via upregulating thioredoxin interacting protein[J]. Oncol Lett, 2017, 14(2): 1323-1332. DOI: 10.3892/ol.2017.6296
[5]	LIN B, SONG X, YANG D, et al. Anlotinib inhibits angiogenesis via suppressing the activation of VEGFR2, PDGFRβ and FGFR1[J]. Gene, 2018, 654: 77-86. DOI: 10.1016/j.gene.2018.02.026
[6]	YE SL. Downstaging and conversion treatment of intermediate and advanced liver cancer should be taken seri-ously[J]. J Clin Hepatol, 2020, 36(2): 249-251. (in Chinese) DOI: 10.3969/j.issn.1001-5256.2020.02.001 叶胜龙.重视中晚期肝癌的降期转化治疗[J].临床肝胆病杂志, 2020, 36(2): 249 -251. DOI: 10.3969/j.issn.1001-5256.2020.02.001
[7]	WANG YM, MENG Y, SHI XN, et al. Analysis of clinical efficacy, safety and prognosis of anlotinib hydrochloride in the treatment of advanced primary liver cancer[J]. Chin J Hepatol, 2020, 28(7): 619-624. (in Chinese) DOI: 10.3760/cma.j.cn501113-20191020-00385 王亚梦, 孟宇, 师霄楠, 等.盐酸安罗替尼治疗晚期原发性肝癌的临床疗效、安全性及预后分析[J].中华肝脏病杂志, 2020, 28(7): 619-624. DOI: 10.3760/cma.j.cn501113-20191020-00385
[8]	PAN YF, FENG XL, YU X, et al. The clinical efficacy of transarterial chemoembolization combined with anlotinib in the treatment of advanced hepatocellular carcinoma[J]. Chin J Exp Surg, 2020, 37(3): 531-533. (in Chinese) DOI: 10.3760/cma.j.issn.1001-9030.2020.03.040 潘延凤, 冯晓丽, 余雪, 等.肝动脉化疗栓塞联合安罗替尼治疗晚期肝癌的临床研究[J].中华实验外科杂志, 2020, 37(3): 531-533. DOI: 10.3760/cma.j.issn.1001-9030.2020.03.040
[9]	YANG B, XIE H, WANG CP, et al. Study on Inhibition of arotinib on human intrahepatic cholangiocarcinoma cell line HCC-9810[J]. Chin J Med Guide, 2017, 19(12): 1389-1391. (in Chinese) DOI: 10.3969/j.issn.1009-0959.2017.12.037 杨斌, 谢辉, 王春平, 等.安罗替尼对人肝内胆管细胞癌细胞系HCC-9810作用研究[J].中国医药导刊, 2017, 19(12): 1389-1391. DOI: 10.3969/j.issn.1009-0959.2017.12.037
[10]	LIU K, YU Q, LI H, et al. BIRC7 promotes epithelial-mesenchymal transition and metastasis in papillary thyroid carcinoma through restraining autophagy[J]. Am J Cancer Res, 2020, 10(1): 78-94.
[11]	DOU H, BUETOW L, SIBBET GJ, et al. BIRC7-E2 ubiquitin conjugate structure reveals the mechanism of ubiquitin transfer by a RING dimer[J]. Nat Struct Mol Biol, 2012, 19(9): 876-883. DOI: 10.1038/nsmb.2379
[12]	SAMANTA D, HUANG TY, SHAH R, et al. BIRC2 expression impairs anti-cancer immunity and immunotherapy efficacy[J]. Cell Rep, 2020, 32(8): 108073. DOI: 10.1016/j.celrep.2020.108073
[13]	GYRD-HANSEN M, MEIER P. IAPs: From caspase inhibitors to modulators of NF-kappaB, inflammation and cancer[J]. Nat Rev Cancer, 2010, 10(8): 561-574. DOI: 10.1038/nrc2889
[14]	MA O, CAI WW, ZENDER L, et al. MMP13, Birc2 (cIAP1), and Birc3 (cIAP2), amplified on chromosome 9, collaborate with p53 deficiency in mouse osteosarcoma progression[J]. Cancer Res, 2009, 69(6): 2559-2567. DOI: 10.1158/0008-5472.CAN-08-2929
[15]	CAMPBELL GR, BRUCKMAN RS, CHU YL, et al. SMAC mimetics induce autophagy-dependent apoptosis of HIV-1-infected resting memory CD4⁺ T cells[J]. Cell Host Microbe, 2018, 24(5): 689-702. DOI: 10.1016/j.chom.2018.09.007
[16]	CORPECHOT C, BARBU V, CHAZOUILLÈRES O, et al. Genetic contribution of ABCC2 to Dubin-Johnson syndrome and inherited cholestatic disorders[J]. Liver Int, 2020, 40(1): 163-174. DOI: 10.1111/liv.14260
[17]	RAZALI RH, NOORIZHAB M, JAMARI H, et al. Association of ABCC2 with levels and toxicity of methotrexate in malaysian childhood acute lymphoblastic leukemia (ALL)[J]. Pediatr Hematol Oncol, 2020, 37(3): 185-197. DOI: 10.1080/08880018.2019.1705949
[18]	WU L, LI Y, SONG Y, et al. A recurrent ABCC2 p.G693R mutation resulting in loss of function of MRP2 and hyperbilirubinemia in Dubin-Johnson syndrome in China[J]. Orphanet J Rare Dis, 2020, 15(1): 74. DOI: 10.1186/s13023-020-1346-4
[19]	NGAN HL, LIU Y, FONG AY, et al. MAPK pathway mutations in head and neck cancer affect immune microenvironments and ErbB3 signaling[J]. Life Sci Alliance, 2020, 3(6): e201900545. DOI: 10.26508/lsa.201900545
[20]	XU P, ZHANG G, HOU S, et al. MAPK8 mediates resistance to temozolomide and apoptosis of glioblastoma cells through MAPK signaling pathway[J]. Biomed Pharmacother, 2018, 106: 1419-1427. DOI: 10.1016/j.biopha.2018.06.084