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Specificity of RNAi to selected target is challenged by off-target effects, both canonical and non-canonical. Notably, more than half of all human microRNAs are co-expressed with hosting them proteincoding genes. Here we dissect regulatory subnetwork centered on  IGFBP6  gene, which is associated with low proliferative state and high migratory activity of basal-like breast cancer. We inhibited expression of  IGFBP6  gene in a model cell line for basal-like breast carcinoma MDA-MB-231, then traced secondary and tertiary effects of this knockdown to  LAMA4 , a laminin encoding gene that contributes to the phenotype of triple-negative breast cancer.  LAMA4 -regulating miRNA miR-4274 and its host gene  SORCS2  were highlighted as intermediate regulators of the expression levels of  LAMA4 , which correlated in a basal-like breast carcinoma sample subset of TCGA to the levels of  SORCS2  negatively. Overall, our study points that the secondary and tertiary layers of regulatory interactions are certainly underappreciated. As these types of molecular event may significantly contribute to the formation of the cell phenotypes after RNA interference based knockdowns, further studies of multilayered molecular networks affected by RNAi are warranted.

LAMA4-Regulating miR-4274 and Its Host Gene SORCS2 Play a Role in IGFBP6-Dependent Effects on Phenotype of Basal-Like Breast Cancer