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.

中文翻译：

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新型长链非编码 RNA CRART16 通过 miR-371a-5p 增强 ERBB3 表达，使结直肠癌细胞对西妥昔单抗产生耐药性

长链非编码 RNA (lncRNA) 已被证明参与多种生物过程并赋予耐药性。然而，目前尚不清楚 lncRNA 是否与结直肠癌 (CRC) 细胞对西妥昔单抗的耐药有关。进行细胞计数 Kit-8 (CCK-8) 测定以评估 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响应的 Caco-2 细胞与增加浓度的西妥昔单抗一起孵育约 6 个月，以产生 Caco-2 西妥昔单抗抗性 (Caco-2 CR) 细胞。将 Caco-2 CR 与 Caco-2 细胞进行比较的微阵列分析用于鉴定可能与西妥昔单抗耐药相关的 lncRNA。使用慢病毒感染，用西妥昔单抗耐药相关 RNA 转录物 16 (CRART16) 或空载体稳定转导 Caco-2 细胞；这些细胞分别被命名为 Caco-2-CRART16 和 Caco-2-NC，并通过 RNA 测序 (RNA-seq) 进行分析。进行定量实时 PCR (qRT-PCR) 以研究 RNA 表达。流式细胞术和 TUNEL 测定用于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。RNA 荧光原位杂交 (FISH) 用于检查 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分析进一步验证。测量数据的差异使用学生 t 检验、单因素 ANOVA、随后的 Dunnett 检验和双向 ANOVA 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 红细胞白血病病毒癌基因同源物 3 (ERBB3) 的表达。MiR-371a-5p 模拟物抵消了由 CRART16 过表达诱导的西妥昔单抗耐药性。京都基因和基因组百科全书（KEGG）通路分析显示，CRART16过表达后，产生的差异表达mRNA主要富集在MAPK信号通路中。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，