长链非编码RNA CRART16通过海绵吸附miR-193b-5p赋予结肠癌细胞5-氟尿嘧啶抗性。
Long noncoding RNA CRART16 confers 5-FU resistance in colorectal cancer cells by sponging miR-193b-5p.
作者信息
Wang Jingui, Zhang Xiaoqian, Zhang Junling, Chen Shangwen, Zhu Jing, Wang Xin
机构信息
Department of General Surgery, Peking University First Hospital, NO. 8 Xishiku Street, Xicheng, Beijing, 100034, People's Republic of China.
Department of Colorectal Surgery, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences, No. 17, Panjiayuan Nanli, Chaoyang, Beijing, 100021, People's Republic of China.
出版信息
Cancer Cell Int. 2021 Nov 29;21(1):638. doi: 10.1186/s12935-021-02353-5.
BACKGROUND
The emergence of chemoresistance to 5-fluorouracil (5-FU)-based chemotherapy is the main cause of treatment failure in advanced and metastatic colorectal cancer (CRC) patients. Long noncoding RNAs (lncRNAs) have been reported to be involved in 5-FU resistance. Previously, we first detected that lncRNA cetuximab resistance-associated RNA transcript 16 (CRART16) could contribute to cetuximab resistance by upregulating V-Erb-B2 erythroblastic leukemia viral oncogene homologue 3 (ERBB3) expression by sponging miR-371a-5p in CRC cells. The current study aimed to explore the role of CRART16 in acquired 5-FU resistance in CRC cells and its possible mechanism.
METHODS
Quantitative real-time PCR (RT-qPCR) was used to measure the expression levels of CRART16 in a 5-FU-resistant CRC cell subline (SW620/5-FU) and the parent cell line. Lentivirus transduction was performed to establish SW620 and Caco-2 cells stably overexpressing CRART16. Cell Counting Kit-8 (CCK-8) assays and colony formation assays were applied to measure cell chemosensitivity to 5-FU. Flow cytometric and immunofluorescence staining were adopted to assess cell apoptosis induced by 5-FU. The dual-luciferase reporter assay was used to validate the direct interactions between CRART16 and miR-193b-5p and between miR-193b-5p and high-mobility group AT-hook-2 (HMGA2). The expression levels of HMGA2, apoptosis-associated proteins and p-ERK were examined by western blotting. The statistical differences within any two groups were used Student's t test.
RESULTS
CRART16 was upregulated in SW620/5-FU cells. Overexpression of CRART16 reduced the sensitivity of CRC cells to 5-FU by attenuating apoptosis. In addition, CRART16 promoted 5-FU resistance by suppressing the expression of miR-193b-5p. Furthermore, CRART16 modulated the expression of HMGA2 by inhibiting miR-193b-5p and activated the MAPK signaling pathway.
CONCLUSIONS
CRART16 confers 5-FU resistance in CRC cells through the CRART16/miR-193b-5p/HMGA2/MAPK pathway.
背景
对基于5-氟尿嘧啶（5-FU）的化疗产生耐药性是晚期和转移性结直肠癌（CRC）患者治疗失败的主要原因。据报道，长链非编码RNA（lncRNA）参与了5-FU耐药。此前，我们首次检测到lncRNA西妥昔单抗耐药相关RNA转录本16（CRART16）可通过在CRC细胞中海绵化miR-371a-5p上调V-Erb-B2红白血病病毒癌基因同源物3（ERBB3）的表达，从而导致西妥昔单抗耐药。本研究旨在探讨CRART16在CRC细胞获得性5-FU耐药中的作用及其可能机制。
方法
采用定量实时PCR（RT-qPCR）检测5-FU耐药CRC细胞亚系（SW620/5-FU）和亲本细胞系中CRART16的表达水平。进行慢病毒转导以建立稳定过表达CRART16的SW620和Caco-2细胞。应用细胞计数试剂盒-8（CCK-8）试验和集落形成试验检测细胞对5-FU的化学敏感性。采用流式细胞术和免疫荧光染色评估5-FU诱导的细胞凋亡。双荧光素酶报告基因试验用于验证CRART16与miR-193b-5p以及miR-193b-5p与高迁移率族AT钩蛋白2（HMGA2）之间的直接相互作用。通过蛋白质印迹法检测HMGA2、凋亡相关蛋白和p-ERK的表达水平。任意两组之间的统计学差异采用Student's t检验。
结果
CRART16在SW620/5-FU细胞中上调。CRART16的过表达通过减弱凋亡降低了CRC细胞对5-FU的敏感性。此外，CRART16通过抑制miR-193b-5p的表达促进5-FU耐药。此外，CRART16通过抑制miR-193b-5p调节HMGA2的表达并激活MAPK信号通路。
结论
CRART16通过CRART16/miR-193b-5p/HMGA2/MAPK途径赋予CRC细胞5-FU耐药性。
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