Dysregulated MicroRNA Affects Pathways and Targets of Biological Relevance in Nasal-Type Natural Killer/T-Cell Lymphoma

Extranodal nasal-type Natural Killer/T-cell lymphoma (NKTL) is a relatively rare but aggressive type of non-Hodgkins lymphoma that is more prevalent in Asia. The outcome of patients with disseminated stage is universally fatal. Progress in therapy has been slow and is based on combination of chemotherapy. MicroRNA are short non-coding RNA sequences that could regulate the expression of a large number of genes by inhibiting translation or leading to mRNA degradation. It has been implicated in tumorigenesis and has prognostic value across a wide range of malignancies including haematologic malignancies. We performed a comprehensive genome-wide miRNA expression profiling (MEP) of NKTL to identify deregulated miRNA and their potential role in NKTL biology.

MEP was performed using the Agilent human miRNA Microarray V2 (Agilent Technologies, Santa Clara, CA) on formalin fixed paraffin-embedded tissue (FFPE) (n=30) and NK cell lines (n=6) in comparison with normal NK cells. Differential expressed miRNA were identified using fold change and Significance Analysis of Microarray (SAM). Some of the differentially expressed miRNA were validated using quantitative polymerase chain reaction (q-PCR). The functional relevance of candidate miRNAs are assessed using miRNA mimics or inhibitors, and observing for apoptosis and growth arrest in the cell lines. Potential targets of candidate miRNAs are identified using predictive algorithms and significant negative correlation with gene expression data. The strongest candidate target genes are further verified using luciferase assay and q-PCR. miRNA and target gene relationship was further confirmed in the patients samples using immunohistochemistry for the protein expression on tissue microarray of NKTL.

Compared to normal NK cells, differentially expressed miRNAs in NKTL are predominantly downregulated. Re-expression of downregulated miRNAs, such as mir-101, mir-26a, mir26b, mir-28-5 and mir-363, reduced the growth of NK cell line and modulated the expression of their predicted target genes, suggesting the potential functional role of the deregulated miRNAs in the oncogenesis of NKTL. Taken together, the predicted targets whose expression are inversely correlated with the expression of deregulated miRNA in NKTL are significant enriched for genes involved in cell cycle-related, p53 and MAPK signaling pathways. We validated and confirmed the regulation of STMN1, and BLIMP1 by miR-101 and miR-30b respectively. In addition, miR-101, miR26a and miR-26b also affect the expression of BCL2 and IGF-1. We also performed immunohistochemical validation for selected target proteins and found over-expression of MUM1, BLIMP1 and STMN1 in NKTL, and notably, a corresponding increase in MYC expression.

miRNA are dysregulated in NKTL. Since MYC is known to cause repression of miRNA expression, it is possible that MYC activation in NKTL as we have shown previously may contribute to the suppression of the miRNAs. These suppressed miRNA in turn lead to increase and aberrant expression of proteins and pathways of biological relevance to NKTL including cell cycle related genes, genes involved in p53 and MAPK signaling pathways as well as MUM1, BLIMP1 and STMN1. Reintroduction of these suppressed miRNA lead to death of NKTL cells and may be a potential therapeutic strategy.

No relevant conflicts of interest to declare.