miRNA omics reveal neferine induces apoptosis through Ca2+mediated endoplasmic reticulum stress pathway in human endometrial cancer
Feifei Ma, Run‐Hui Ma, Kiran Thakur, Jian‐Guo Zhang, Hui Cao, Zhao‐Jun Wei, Jesús Simal‐Gándara
Abstract
• Neferine exhibited a strong inhibitory effect on Ishikawa cells (IC 50 = 28.09 μM). • Neferine blocked the cell cycle in the G2/M phase. • Neferine activated Ca 2+ release to trigger the mitochondrial apoptotic pathway. • Neferine induced cell apoptosis by inhibiting the PI3K/AKT pathway. Endometrial cancer (EC) as one of the most prevalent malignancies in the female reproductive system, usually has a poor diagnosis and unfavorable health effects. Neferine (Nef), derived from the edible and medicinal lotus seed, has been known for its functional activity; however, its anti-cancer mechanism for EC remains elusive. We explored the potential anti-cancer effects and underlying molecular mechanisms of Nef on EC. The cytotoxicity was tested using MTT, and the cell cycle, apoptosis, Ca 2+ levels, and the mitochondrial membrane potential (MMP) were observed through flow cytometry. After Nef treatment, differences in miRNA expression were identified using miRNA-seq data. Furthermore, western blot and immunohistochemistry (IHC) were employed to identify the proteins associated with apoptosis in both mice and cells. Nef treatment led to Ishikawa cell apoptosis and blocked cell proliferation in the G2/M phase. In total, 101 significantly different miRNA (p 〈 0.05 and |logFC| 〉 1) were obtained and subjected to GO and KEGG enrichment analysis, which revealed the Ca 2+ and PI3K/AKT signaling pathways pertaining to apoptosis. Nef treatment significantly changed intracellular Ca 2+ levels and MMP, activating the endoplasmic reticulum stress (ERS) pathway and the expression of key proteins in the mitochondrial pathway. In addition, Nef also inhibited the expression of key proteins in the PI3K/AKT pathway, causing cell apoptosis. Moreover, in mouse tumor tissues, the expression of CHOP, Bcl-2, Caspase 3, Cyto-c, and p-AKT was also consistent with the results in vitro . Nef could block the cell cycle and induce the activation of the mitochondrial apoptotic pathway involving the Ca 2+ -mediated ERS pathway and the PI3K/AKT pathway, thereby inducing apoptosis in EC cells, confirming the potential role of Nef in the prevention and treatment of EC.