Abstract: Objective To investigate the regulatory effect of miR⁃449a on the proliferation and apoptosis of non⁃small cell lung cancer cells and its molecular mechanism. Methods The miR⁃499a overexpression analogue Agomir⁃499a (Agomir⁃499a group) and its negative control (NC group) were transfected, respectively, into non⁃small cell lung cancer  NCI⁃H1975 cells. The expression level of miR⁃499a in transfected cells was detected by qRT⁃PCR, the cell proliferation was detected by CCK⁃8 and Ki67 staining, and the apoptosis was detected by Annexin V staining. NCI⁃H1975 cells transfected with Agomir⁃499a were treated with ferrostatin⁃1 (Fer⁃1) and glutathione (GSH), respectively (denoted as Agomir⁃499a + Fer⁃1 group and Agomir⁃499a+GSH group). The Fe2+ level and the content of GSH in cells was detected by the fluorescent probe and the optical colorimetry method, respectively. qRT⁃PCR was used to detect the mRNA expression of ferroptosis and iron homeostasis related genes, and Western blot was used to detect the expression of NOTCH1 signaling pathway related proteins. Results Compared with those in the NC group, the expression level of miR⁃499a in NCI⁃H1975 cells of the Agomir⁃449a group was significantly increased (P=0.001), the proliferation activity of NCI⁃H1975 cells was significantly decreased (P<0.05), and the proportion of cell apoptosis was significantly increased (P=0.004). The expression levels of ferroptosis⁃related factors PTGS2, ACSL4, SLC7A11 and COX2 , the proportion of Fe2+ positive cells, and the expressions levels of iron homeostasis⁃related genes TF, STEAP3, TFRC and DMT1 were significantly increased (all P<0.05). However, GSH content and expression levels of NOTCH1 signaling pathway related molecules NOTCH1, NICD, JAG1, JAG2, DLL1 and HES1 were significantly decreased (all P<0.05). Compared with the Agomir⁃449a group, the expression levels of ferroptosis⁃related factors PTGS2, ACSL4, SLC7A11 and COX2, the proportion of Fe2+ positive cells, and the expressions levels of iron homeostasis⁃related genes TF and STEAP3 in the Agomir⁃449a+Fer⁃1 group were significantly decreased (all P<0.05), while the GSH content and the expression levels of NOTCH1 signaling pathway related molecules NICD, JAG1 and DLL1 were significantly increased (all P<0.05). The expression levels of SLC7A11, COX2 and ACSL4 in the Agomir⁃449a+GSH group were also significantly lower than those in Agomir⁃449a group (all P<0.05). Conclusions The miR⁃449a can induce apoptosis and proliferation inhibition of non⁃small cell lung cancer NCI⁃H1975 cells, and its mechanism may be related to the inhibition of ferroptosis mediated by NOTCH1 signaling.

Key words: Non?small cell lung cancer, miR?449a, NOTCH1 signaling pathway, Ferroptosis, Proliferation, Apoptosis