Table_2_LncRNA-42060 Regulates Tamoxifen Sensitivity and Tumor Development via Regulating the miR-204-5p/SOX4 Axis in Canine Mammary Gland Tumor Cells.docx

Tamoxifen is the drug of choice for endocrine therapy of breast cancer. Its clinical use is limited by the development of drug resistance. There is increasing evidence that long non-coding RNAs (lncRNAs) are associated with tumor drug resistance. Therefore, we established two TAM-resistant cell lines, CHMpTAM and CHMmTAM. The different expression levels of lncRNA and miRNA in CHMmTAM and CHMm were screened by RNA sequencing, and the lncRNA-miRNA interactions were analyzed. LncRNA ENSCAFG42060 (lnc-42060) was found to be significantly upregulated in drug-resistant cells and tumor tissues. Further functional validation revealed that the knockdown of lnc-42060 inhibited proliferation, migration, clone formation, restoration of TAM sensitivity, and reduction of stem cell formation in drug-resistant cells, whereas overexpression of lnc-4206 showed opposite results. Bioinformatics and dual-luciferase reporter gene assays confirmed that lnc-42060 could act as a sponge for miR-204-5p, further regulating SOX4 expression activity and thus influencing tumor cell progression. In conclusion, we screened lncRNAs and miRNAs associated with TAM resistance in canine mammary gland tumor cells for the first time. lnc-42060 served as a novel marker that may be used as an important biomarker for future diagnosis and treatment.

他莫昔芬（Tamoxifen）是乳腺癌内分泌治疗的首选药物，但其临床应用因耐药性的产生而受到限制。越来越多的证据表明，长链非编码RNA（long non-coding RNAs，lncRNAs）与肿瘤耐药性密切相关。因此，本研究构建了两株他莫昔芬耐药细胞系CHMpTAM与CHMmTAM。通过RNA测序（RNA sequencing）筛选出CHMmTAM与CHMm细胞中lncRNA及miRNA的差异表达水平，并分析了lncRNA与miRNA的相互作用关系。研究发现，lncRNA ENSCAFG42060（简称lnc-42060）在耐药细胞与肿瘤组织中均显著上调。进一步的功能验证实验显示，敲低lnc-42060可抑制耐药细胞的增殖、迁移与克隆形成能力，恢复其对他莫昔芬的敏感性，并减少干细胞形成；而过表达lnc-42060则会产生相反的效应。生物信息学分析与双荧光素酶报告基因实验（dual-luciferase reporter gene assays）证实，lnc-42060可作为miR-204-5p的分子海绵，通过调控SOX4的表达活性进而影响肿瘤细胞的进展。综上，本研究首次在犬乳腺肿瘤细胞中筛选出与他莫昔芬耐药相关的lncRNA与miRNA；lnc-42060可作为新型标志物，有望成为未来临床诊断与治疗的重要生物标记物。