HER2 signaling upregulation is a hallmark of breast cancer which is the second cause of cancer‐related death in women. Here, we were looking for the candidate microRNAs which is capable of regulating the HER2 receptor and the genes of its downstream. To this aim, preliminary bioinformatics analysis introduced hsa‐miR‐1254 (miR‐1254) as a potential common regulator of HER2, HER3, PIK3R2, and AKT1 genes. Then, reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR) analysis indicated a lower expression level of miR‐1254 in breast cancer specimens, compared to their normal pairs. Furthermore, overexpression of miR‐1254 resulted in HER2, HER3, PIK3R2, and AKT1 genes downregulation, detected by RT‐qPCR and confirmed by western blot analysis and enzyme‐linked immunosorbent assay test against AKT1, BAX, FADD, and HER2 protein levels in SKBR3 cells. Dual‐luciferase assay also supported direct interaction of miR‐1254 with MREs within 3' untranslated region sequences of HER2, HER3, PIK3R2, and AKT1 target genes. Overexpression of miR‐1254 in SKBR3 cells was followed by increased BAX/BCL2 expression ratio, detected by RT‐qPCR, and increased proportion of G1 cell population, detected by flow cytometry. Corroborated by cell cycle analysis, MTT, Annexin V‐FITC, and Live‐Dead cell staining assays, overexpression of miR‐1254 in MDA‐MB‐231 cells showed opposing results following the overexpression of miR‐1254. Taken together, results indicated that miR‐1254 acts as cell‐type‐specific tumor suppressor that targets HER2, HER3, PIK3R2, and AKT1 transcripts. These results suggest miR‐1254 as a potential therapeutic candidate for breast cancer subtypes.
