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Background: A standardized extract of cultured Lentinula edodes mycelia (ECLM), an extract from cultured Lentinula edodes , has been reported to suppress breast cancer stem cell proliferation by regulating microRNA (miR) expression. Natural antisense RNAs (ASs), a type of protein non-coding RNA, can regulate the expression of protein-coding genes by acting as a competing endogenous RNA (ceRNA) that adsorbs miRNAs, resulting in the prevention of mRNA degradation, and can also form a transient RNA duplex with mRNA. EphA2, a receptor tyrosine kinase, is typically expressed at low levels in normal epithelial cells, whereas its overexpression has been widely observed in numerous solid tumors and is associated with cell transformation, primary tumor initiation, and tumor progression. Objective: This study aimed to investigate the effect of ECLM on the expression of both EphA2 mRNA and endogenous AS to this mRNA, which could negatively affect human breast carcinoma cell proliferation. Methods: We used MCF7 and MDA-MB-231 human breast carcinoma cells, which were sub-cultured three times in the presence of optimized concentrations of ECLM. The effect of ECLM on the expression of EphA2 AS and mRNA was analyzed by RT-qPCR. miRNAs targeting both EphA2 AS and EphA2 mRNA and their RNA miR response elements (MREs) were predicted and analyzed by RT-qPCR and luciferase reporter assays. Results: ECLM suppressed the proliferation of MCF7 and MDA-MB-231 cells in a dose-dependent manner. In cells for which proliferation was negatively affected by ECLM, EphA2 AS and mRNA expression was also significantly inhibited by ECLM. Although neutralization of miR-335 led to the de-repression of both EphA2 AS and mRNA, results did not fully support the possibility that EphA2 AS might function as a ceRNA to regulate EphA2 mRNA levels. Conclusion: ECLM suppressed the proliferation of breast carcinoma cells in a specific dose-dependent manner. This suppressive effect was associated with a concordant reduction in both EphA2 AS and mRNA expression. These effects were not thought to occur via the reported ceRNA effect. These results thus suggest that ECLM could regulate EphA2 AS and mRNA expression by forming a transient RNA duplex formation, thereby stabilizing EphA2 mRNA. Keywords:  ECLM, regulatory RNA, antisense RNA, microRNA, EphA2

A standardized suppresses breast cancer cell proliferation by regulating the expression of EphA2 antisense RNA-mRNA axis independently of micro RNA