Epithelial‐to‐mesenchymal transition (EMT) is key to invasion and metastasis by oral squamous carcinoma (OSCC) cells. MicroRNAs (miRNAs) such as miRNA‐146a are known to be upregulated in OSCC. However, it is unclear whether they are involved in driving EMT. Here, we investigated the effect of miR‐146a overexpression on proliferation, migration, and EMT in OSCC cells. OSCC cells were transfected with a plasmid expressing miR‐146a precursor. Cell lines that stably overexpressed miRNA‐146a were assessed for proliferation, colony formation, and invasiveness in vitro. Expression of markers and regulators of EMT, cell motility, and invasion were measured by qRT‐PCR and western blot. Potential miRNA‐146a binding sites in the 3′UTR of ST8SIA4 were identified by bioinformatic analysis. To confirm that miRNA‐146a binds to and regulates ST8SIA4, we transfected OSCC cell lines with miRNA‐146a mimics and a luciferase reporter construct containing either the wild type or mutant 3′UTR of ST8SIA4. OSCC cell lines that overexpressed miR‐146a displayed higher proliferation, colony formation, invasion, and MMP‐2 activity than cells transfected with a control vector. Overexpression of miR‐146a also decreased expression of the epithelial cell marker E‐cadherin and increased expression of Twist1, a transcription factor that promotes EMT, as well as markers associated with mesenchymal cells (vimentin and N‐cadherin) and tumor invasion (p‐paxillin and p‐cortactin). Luciferase expression was lower in OSCC cells transfected with miRNA‐146a mimics or with luciferase constructs carrying the wild type, but not mutant, 3′UTR of ST8SIA4. Overexpression of miR‐146a promotes EMT phenotypes and may drive tumorigenesis and progression in OSCC, making it a useful target for future OSCC treatments.

中文翻译：

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miR-146a促进口腔鳞癌细胞增殖、侵袭和上皮间质转化

上皮间质转化（EMT）是口腔鳞癌（OSCC）细胞侵袭和转移的关键。已知微小 RNA (miRNA) 如 miRNA-146a 在 OSCC 中上调。但是，尚不清楚他们是否参与推动 EMT。在这里，我们研究了 miR-146a 过表达对 OSCC 细胞增殖、迁移和 EMT 的影响。用表达 miR-146a 前体的质粒转染 OSCC 细胞。在体外评估了稳定过表达 miRNA-146a 的细胞系的增殖、集落形成和侵袭性。通过 qRT-PCR 和蛋白质印迹测量 EMT、细胞运动和侵袭的标志物和调节剂的表达。通过生物信息学分析鉴定了 ST8SIA4 3'UTR 中潜在的 miRNA-146a 结合位点。为了确认 miRNA-146a 结合并调节 ST8SIA4，我们用 miRNA-146a 模拟物和含有 ST8SIA4 的野生型或突变型 3'UTR 的荧光素酶报告基因构建体转染 OSCC 细胞系。与用对照载体转染的细胞相比，过表达 miR-146a 的 OSCC 细胞系显示出更高的增殖、集落形成、侵袭和 MMP-2 活性。miR-146a 的过表达还降低了上皮细胞标志物 E-cadherin 的表达，并增加了 Twist1 的表达，Twist1 是一种促进 EMT 的转录因子，以及与间充质细胞（波形蛋白和 N-钙粘蛋白）和肿瘤侵袭相关的标志物（p-桩蛋白和 p-cortactin）。在转染了 miRNA-146a 模拟物或携带野生型而非突变型 ST8SIA4 3'UTR 的荧光素酶构建体的 OSCC 细胞中，荧光素酶的表达较低。