XLSX file - 841K, Supplementary Table 4. miRNA array analysis of discovery cohort. Supplementary Table 5. miRNA array significant call signals. Supplementary Table 6. RT-qPCR validation of miRNA array.
ARTICLE ABSTRACTPurpose: MicroRNA-21 (miRNA-21) has proto-oncogenic properties, although no miRNA-21–specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology.Experimental Design: miRNA expression profiles of 10 HNSCCs and 10 normal mucosa samples were investigated using a custom miRNA microarray. Thirteen HNSCCs and five normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of 16 HNSCCs and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets.Results: miRNA-21 was upregulated in HNSCCs and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3′-untranslated region (UTR) of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCCs and correlated with miRNA-21 overexpression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCCs and showed growth suppression function that was reversed by miRNA-21 overexpression.Conclusions: CLU is a specific, functional target of oncogenic miRNA-21 in HNSCCs. CLU-1 isoform is the predominant growth-suppressive variant targeted by miRNA-21. Clin Cancer Res; 20(4); 868–77. ©2013 AACR.