GC is one of the leading causes of cancer deaths worldwide, with 950,000 new cases being diagnosed yearly . Patients with GC have poor prognosis, with median survival of around 12 months . Ferroptosis, a freshly discovered subtype of nonapoptotic cell death, relates to overgeneration of iron-dependent ROS, ferroptosis can be activated in cancer cells by natural stimuli and synthetic agent . Some small molecules could induce ferroptosis in various tumor cells, such as GC , fibrosarcoma , kidney cancer  and prostate cancer cells .
Nrf2 is a transcription factor that initiates some transcriptions to safeguard cells from endogenous or exogenous injuries, such as oxidative stress and xenobiotic, via activating cellular antioxidant response . Overexpression of Nrf2 has been verified in many kinds of malignant tumors, such as breast, ovarian, and pancreatic cancers. In our experiment, we detected the expression of Nrf2 in 50 cases of gastric cancer samples by qPCR, compared to the precancerous tissues; Nrf2 mRNA was highly expressed in cancer samples. Patients whose tumors exhibit higher level of Nrf2 usually present poor prognosis . To reveal the influence of Nrf2 on the prognosis of gastric cancer patients, high- and low-Nrf2 expression group was divided according to expression of Nrf2 in the recruited gastric cancer specimens, and then Kaplan–Meier analyses were performed. There was prominent survival difference between Nrf2 low and Nrf2 high groups. These results indicated that Nrf2 plays an important role in occurrence and development of gastric cancer.
Constitutive activation of Nrf2-ARE shields malignant cells from oxidative damages [23, 24]. Therefore, it has recently been deemed to be a main defense mechanism and a cytoprotective transcription factor of tumor cells that are resistant to ferroptosis , Nrf2 overexpression rescues sorafenib-induced ferroptosis in hepatocellular carcinoma [5, 25], on the contrary, Nrf2 inhibition reverses the resistance of cisplatin-resistant neck and head cancer cells to artesunate-mediated ferroptosis .
Previous studies confirmed that miRNAs play an important role in the progression and development of various types of malignant tumors. miR-144 could target Nrf2 mRNA, leading to the downregulation of Nrf2 in cancer cells [14, 26]. miR-144-5p expression is downregulated in many kinds of human cancers, such as colon, liver, and bladder cancers . miR-144-5p can restrain the proliferation, migration, and invasion of malignant tumor cells through many mechanisms . In the experiment, we found that miR-144-5p was highly expressed in a panel of 62 cases of gastric cancer samples compared to the precancerous tissues; in addition, low expression of miR-144-5p involved in bad survival of gastric cancer patients. To reveal the influence of miR-144-5p on the biology of gastric cancer cells, gain and loss function experiment was performed. Overexpressing of miR-144-5p by transfecting a miR-144-5p mimic into GC cells inhibited the proliferation of GC cells; by contrast, anti miR-144-5p inhibitor promoted the proliferation of GC cells. Taken together, these data suggest that miR-144-5p is possibly involved in growth of gastric cancer cell and may worked as a role of tumor suppressor gene.
A meta-analysis was conducted on the basis of microarrays from the GEO database to explore the function of overexpression of miR-144-5p in malignant cell. Among the involved genes of miR-144-5p, some genes play a role on the anti-oxidative stress of cells, such as serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5), Carbonic Anhydrase IX (CA9) and Early growth response-1 (EGR1), indicating that miR-144-5p might play an important role in the oxidative stress. In our study, we found that exogenous miR-144-5p intensified the overproduction of ROS and proliferation inhibition of GC cells, highlighting that miR-144-5p participated in oxidation reaction.
Further, with help of MiRWALK2.0, TargetScan, PICTAR2 software, the targeted genes of miR-144-5p were predicated, among the targeted genes, Nrf2 was listed. On the contrary, when Nrf2 was put in, miR-144-5p was found. In consideration of both Nrf2 and miR-144-5p are associated with oxidative stress, Nrf2 was likely a target of miR-144-5p in gastric cancer. To further test this hypothesis, dual luciferase report assay was performed, overexpression of miR-144-5p could abrogated the luciferin intensity of wild type group, but not the mutant group.
In order to detect whether miR-144-5p could enhance the sensitization of gastric cancer cell to erastin-induced ferroptosis, exogenous miR-144-5p was introduced into gastric cancer cells. Results showed that cancer cell with overexpression of miR-144-5p were more sensitive to erastin with overgeneration of ROS and depletion of GSH. Exogenous Nrf2 rescued miR-144-5p promoted ferroptosis, indicating that miR-144-5p sensitized erastin-induced ferroptosis via modulating Nrf2.