Zhang Xiaoqian, Wen Long, Chen Shanwen, Zhang Junling, Ma Yongchen, Hu Jianwen, Yue Taohua, Wang Jingui, Zhu Jing, Bu Dingfang, Wang Xin
1Department of General Surgery, Peking University First Hospital, Beijing, 100034 People's Republic of China.
2Department of Endoscopic Center, Peking University First Hospital, Beijing, 100034 People's Republic of China.
Cancer Cell Int. 2020 Mar 4;20:68. doi: 10.1186/s12935-020-1155-9. eCollection 2020.
Long noncoding RNAs (lncRNAs) have been shown to participate in multiple biological processes and confer drug resistance. However, it remains unclear whether lncRNAs are involved in conferring cetuximab resistance in colorectal cancer (CRC) cells.
Cell Counting Kit-8 (CCK-8) assays were performed to assess the sensitivity of CRC cell lines to cetuximab treatment. We incubated Caco-2 cells, which are partially responsive to cetuximab, with increasing concentrations of cetuximab for approximately 6 months to generate Caco-2 cetuximab-resistant (Caco-2 CR) cells. Microarray analysis comparing Caco-2 CR with Caco-2 cells was used to identify lncRNAs that were potentially related to cetuximab resistance. Caco-2 cells were stably transduced with cetuximab resistance-associated RNA transcript 16 (CRART16) or an empty vector using lentiviral infection; the cells were designated Caco-2-CRART16 and Caco-2-NC, respectively, and were analyzed with RNA sequencing (RNA-seq). Quantitative real-time PCR (qRT-PCR) was performed to investigate RNA expression. Flow cytometry and TUNEL assays were used to assess apoptosis levels induced by cetuximab. The cell cycle, stemness biomarkers and membrane proteins of CRC cells were assessed via flow cytometry. RNA fluorescence in situ hybridization (FISH) was used to examine CRART16 localization and expression. Bioinformatics analysis was performed to predict the potential mechanism of CRART16, which was further validated by a dual-luciferase reporter assay. Differences in measurement data were compared using Student's t test, one-way ANOVA followed by Dunnett's test and two-way ANOVA.
The novel lncRNA CRART16 was upregulated in Caco-2 CR cells. CRART16 overexpression reversed the effects of cetuximab on cell viability and reduced cetuximab-induced apoptosis. Meanwhile, CRART16 overexpression led to increases in the proportion of CD44/CD133 cells. In addition, CRART16 acts as a competing endogenous RNA (ceRNA) for miR-371a-5p to regulate V-Erb-B2 Erythroblastic Leukemia Viral Oncogene Homolog 3 (ERBB3) expression. MiR-371a-5p mimics counteracted the cetuximab resistance induced by CRART16 overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that after CRART16 was overexpressed, the resulting differentially expressed mRNAs were mainly enriched in the MAPK signaling pathway.
CRART16 overexpression may contribute to cetuximab resistance through the miR-371a-5p/ERBB3/MAPK pathway. Additionally, CRART16 contributes to the acquisition of stemness properties.
长链非编码RNA（lncRNA）已被证明参与多种生物学过程并赋予耐药性。然而，lncRNA是否参与结直肠癌（CRC）细胞中西妥昔单抗耐药性的产生仍不清楚。
采用细胞计数试剂盒-8（CCK-8）检测法评估CRC细胞系对西妥昔单抗治疗的敏感性。我们将对西妥昔单抗部分敏感的Caco-2细胞与浓度递增的西妥昔单抗孵育约6个月，以产生Caco-2西妥昔单抗耐药（Caco-2 CR）细胞。通过比较Caco-2 CR细胞与Caco-2细胞的微阵列分析来鉴定可能与西妥昔单抗耐药相关的lncRNA。使用慢病毒感染将西妥昔单抗耐药相关RNA转录本16（CRART16）或空载体稳定转导至Caco-2细胞；这些细胞分别命名为Caco-2-CRART16和Caco-2-NC，并进行RNA测序（RNA-seq）分析。采用定量实时PCR（qRT-PCR）研究RNA表达。使用流式细胞术和TUNEL检测法评估西妥昔单抗诱导的细胞凋亡水平。通过流式细胞术评估CRC细胞的细胞周期、干性生物标志物和膜蛋白。采用RNA荧光原位杂交（FISH）检测CRART16的定位和表达。进行生物信息学分析以预测CRART16的潜在机制，并通过双荧光素酶报告基因检测进一步验证。测量数据的差异采用Student's t检验、单因素方差分析后进行Dunnett检验和双因素方差分析进行比较。
新型lncRNA CRART16在Caco-2 CR细胞中上调。CRART16过表达逆转了西妥昔单抗对细胞活力的影响，并减少了西妥昔单抗诱导的细胞凋亡。同时，CRART16过表达导致CD44/CD133细胞比例增加。此外，CRART16作为miR-371a-5p的竞争性内源性RNA（ceRNA）来调节V-Erb-B2成红细胞白血病病毒癌基因同源物3（ERBB3）的表达。miR-371a-5p模拟物抵消了CRART16过表达诱导的西妥昔单抗耐药性。京都基因与基因组百科全书（KEGG）通路分析显示，CRART16过表达后，产生的差异表达mRNA主要富集于MAPK信号通路。
CRART16过表达可能通过miR-371a-5p/ERBB3/MAPK通路导致西妥昔单抗耐药。此外，CRART16有助于获得干性特性。
