DataSheet_2_BRAFV600E Mutation-Responsive miRNA-222-3p Promotes Metastasis of Papillary Thyroid Cancer Cells via Snail-Induced EMT.pdf

BRAF mutation accounts for 50% of the PTC (papillary thyroid carcinoma) and is closely associated with high-risk clinicopathological characteristics. Increasing evidence implied that dysregulation of miRNA participated in carcinogenesis and progression of cancer. Clinical data showed the significant up-regulation of miR-222-3p in PTC; however, the role of miR-222-3p and possible relationship with BRAF mutation remained unclear. Here, we identified significant up-regulation of miR-222-3p in PTC tissues harboring BRAFV600E mutation compared with BRAF wild type (BRAFWT) from collected PTC clinical samples. External validation performed with The Cancer Genome Atlas (TCGA) databases was consistent with the above result. Exogenous expression of BRAFV600E oncoprotein increased the expression of miR-222-3p in B-CPAP and TPC-1 cells. The treatment of BRAFV600E and MEK inhibitor, PLX4720 and PD0325901, decreased the expression of miR-222-3p in B-CPAP but not in TPC-1. Inhibition of miR-222-3p significantly suppressed the migration of B-CPAP and induced a mesenchymal-epithelial transition (MET) phenotype via the Snail transcription factor. Immunohistochemistry (IHC) analysis demonstrated the up-regulation of Snail correlated with lymph node metastasis and BRAFV600E mutation in PTC. Besides, in situ hybridization (ISH) and IHC analysis of PTC clinical samples confirmed the correlation between the expression of miR-222-3p and Snail. These results showed miR-222-3p conduced more aggressive clinical manifestation of PTC by promoting Snail-induced EMT.

BRAF突变在甲状腺乳头状癌（papillary thyroid carcinoma，PTC）中占比达50%，且与高危临床病理特征密切相关。越来越多的研究证据表明，微小RNA（microRNA，miRNA）的失调参与了癌症的发生与发展进程。临床数据显示，miR-222-3p在PTC组织中显著上调，但目前miR-222-3p的作用及其与BRAF突变的潜在关联仍不明确。本研究通过收集的PTC临床样本发现，相较于BRAF野生型（BRAF wild type，BRAFWT）组织，携带BRAFV600E突变的PTC组织中miR-222-3p的表达水平显著升高。通过癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库开展的外部验证结果与上述发现一致。在B-CPAP与TPC-1细胞中，外源性过表达BRAFV600E癌蛋白可上调miR-222-3p的表达。使用BRAFV600E抑制剂PLX4720与丝裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase，MEK）抑制剂PD0325901处理B-CPAP细胞，可下调其miR-222-3p的表达，但对TPC-1细胞无此效应。抑制miR-222-3p的表达可显著抑制B-CPAP细胞的迁移能力，并通过转录因子Snail诱导间质上皮转化（mesenchymal-epithelial transition，MET）表型。免疫组织化学（immunohistochemistry，IHC）分析显示，PTC组织中Snail的上调表达与淋巴结转移及BRAFV600E突变呈显著相关。此外，对PTC临床样本的原位杂交（in situ hybridization，ISH）与IHC分析证实，miR-222-3p的表达与Snail的表达存在相关性。上述结果表明，miR-222-3p可通过促进Snail介导的上皮间质转化（epithelial-mesenchymal transition，EMT），使PTC呈现更具侵袭性的临床表型。