Analysis of MicroRNA-21 in Cancer Cells and Tissue Specimens by Using a Highly Sensitive In Situ Hybridization Assay

MicroRNAs(miRNA) are 18 to 25 nucleotides long non-coding RNA that regulate the translation of their target mRNA in cells. miRNA plays an important role in human tumorigenesis, metastasis and angiogenesis. In situ hybridization (ISH) is an advanced method that combines molecular biological techniques with histological and cytological analysis to display gene expression status at the cellular level. In this study, a microRNA-21(miR-21) ISH assay with improved sensitivity was developed. The signal amplification using tyramide was assessed by a FISH quantitative test. The result demonstrated that the application of tyramide signal amplification significantly increased the CISH and FISH sensitivity. Other elements of the ISH assay including sample pretreatment and the use of LNA probe were also extensively tested and optimized. The miR-21 expression in cell lines HEK-293, MCF-7, MDA-BM-231, AGS and HeLa were analyzed by improved ISH assays. The results of both CISH and FISH were correlated with the miR-21 quantification by TaqMan miRNA real-time PCR. In all tests the miR-21 expression of cancer cell lines MCF-7, MDA-MB-231, AGS and HeLa were significantly higher than expressions in non-cancer cell line HEK-293. The CISH and FISH assays also successfully detected cells with high levels of miR-21 expression in tumor islands in clinical a breast cancer FFPE specimen.