MicroRNA-99a, a tumor suppressor in oral squamous cell carcinoma regulates insulin-like growth factors I receptor in a double-negative feedback loop

[[abstract]]MicroRNAs (miRNAs), small non-coding RNA molecules regulate several biological processes and function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with poor five-year survival rate around 50%. Treatment failures mainly caused by loco-regional relapse or distant metastasis. Comparison of the miRNAs expression profiles of OSCC and non-tumor tissues revealed miR-99a as one of the most downregulated miRNAs. Using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, we found that the level of miR-99a was reduced in all tested OSCC cell lines when compared with that in normal oral keratinocytes. This data suggested that down-regulation of miR-99a may play a pathological role in OSCC. Indeed, ectopic miR-99a expression in OSCC cells dramatically reduced in vitro migration and invasion and affected the expression of epithelial-mesenchymal transition (EMT)-related proteins, such as N-cadherin and Snail. To evaluate the specific targets of miR-99a, we found that ectopic miR-99a expression could down-regulate insulin-like growth factor 1 receptor (IGF1R) protein by immunoblot assay and the level of miR-99a showed a significantly negative correlation with IGF1R protein in OSCC cells. Insertion of the 3_UTR of IGF1R mRNA into the 3_UTR of a reporter gene markedly reduced the luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a decreased luciferase activity by targeting the 3_UTR of IGF1R mRNA. To understand the mechanisms of down-regulated miR-99a, we demonstrated that miR-99a expression was up-regulated by serum starvation and repressed upon insulin-like growth factor (IGF1) stimulation. The IGF1-induced repression of miR-99a could be blocked by inhibitors of phosphatidylinositol 3-kinases (PI3K) and mitogen-activated protein kinase (MAPK) kinase, suggesting that miR-99a expression was negatively regulated by IGF1R signaling. Collectively, we herein demonstrated that miR-99a could function as a tumor suppressor in OSCC cells and mutually regulate IGF1R expression in a double-negative feedback loop