title: Cell-Specific Mechanisms of Microrna Modulation of Anoctamin-1 (ANO1) in Oral Cancer
Creators: name: Hou, Daniel
Publisher: name: University of Nevada, Las Vegas
publisherIdentifier: https://ror.org/0406gha72
publisherIdentifierScheme: ROR
Year: 2023

description: Anoctamin-1 (ANO1) is a calcium-gated chloride ion channel with multiple physiological roles, including transepithelial ion transport, smooth muscle contraction, salivary secretion, and nociception. Recently, ANO1 has been identified to play a key role in cancer progression. ANO1 is overexpressed in different types of cancer, including oral cancer. ANO1 overexpression is commonly the result of ANO1 gene amplification on the chromosome 11q13 locus. In cancer cells, ANO1 upregulation has been reported to promote cell proliferation and migration of tumor cells, whereas ANO1 downregulation induces apoptosis through multiple signaling pathways. As such, ANO1 is now considered a useful biomarker for cancer progression and a prognostic indicator for poor outcomes. Reducing ANO1 expression is a therapeutic approach to suppress ANO1 activity in promoting tumor cell proliferation, invasion, and metastasis. Specific miRNAs that directly target ANO1 mRNA and negatively regulate ANO1 gene expression have been reported. The role of miRNA influencing the expression of ANO1 in oral cancer cells is not understood.The aims of this study were to determine if microRNAs that specifically target ANO1 are expressed in oral squamous cell carcinoma and to evaluate if the introduction of miRNA that target ANO1 to oral cancer cells will influence proliferation and apoptosis of oral cancer cells.
We performed quantitative PCR (qPCR) of miRNA expression in various cultured OSCC cell lines (SCC4, SCC15, SCC25, CAL27), and the non-malignant OKF4 cell line to examine endogenous microRNA expression. SCC9 and SCC15 cells were transfected with miR-144 or miR-381 mimics to determine the effect of upregulating miRNA that target ANO1 transcripts.  Experimental growth assays of SCC-9 and SCC-15 were performed to evaluate proliferation following miRNA targeting of ANO1 and changes in the expression of ANO1 and apoptotic caspase genes, CASP3, CASP8 and CASP9, were assessed by qPCR. 
We determined that miR-9, miR-132, and miR-381 that specifically target ANO1 transcripts for downregulation are expressed in SCC4, SCC9, SCC15, SCC25 and CAL27 cells. miR-144, which also targets ANO1 transcripts for downregulation, is only expressed in SCC9 and SCC15. Compared to the non-malignant OKF4 cells, miR-132, miR-144 and miR-381 appear to be dysregulated in oral cancer cells. The introduction and transfection of miR-144 and miR-381 mimics to SCC9 and SCC15 was confirmed by qPCR. miR-144 and miR-381 transfection in SCC9 cells resulted in a similar and significant reduction in SCC9 proliferation (p = 0.001 and p = 0.002, respectively). An anti-proliferative effect relative to controls was observed following miR-144 and miR-381 transfection in SCC15 cells. miR-144 significantly reduced SCC15 proliferation compared with non-transfected cells (p = 0.03). miR-381 did slow SCC15 proliferation, however, the reduction in proliferation was not significant. In both SCC9 and SCC15 cells transfected with miR-144, ANO1 transcripts were significantly decreased (p = 0.02; p = 0.007, respectively), compared to non-transfected controls. Transfection of miR-381 in SCC9 also decreased ANO1 transcripts significantly (p = 0.03), but no significant decrease in ANO1 expression was observed in SCC15 cells that were transfected with miR-381. Significant increases in CASP3, CASP8 and CASP9 apoptotic gene expression (2.8 to 9.2-fold) were found when miR-144 and miR-381 targeting ANO1 were transfected in SCC9 and SCC15 cells.
miR-9, miR-132, miR-144, miR-381 that specifically target ANO1 transcripts for downregulation are expressed in oral squamous cell carcinoma. Transfecting miR-144 and miR-381 in oral squamous cell carcinoma reduced ANO1 expression and influenced proliferation and apoptotic gene expression in these oral cancer cells. This study provides novel information regarding the cellular mechanisms through which ANO1 channel function may modulated in oral cancer.