2.1 circPDSS1 is differentially expressed in peripheral blood and GC tissues
First, to explore the expression of circPDSS1 in GC tissues and peripheral blood, the expression pattern of circPDSS1 in GC patients’ needs to be analyzed. It can be seen from the results that circPDSS1 presents a high expression pattern, while miR-1278 presents a low expression pattern, both in peripheral blood and tissues (Fig. 1A-D). Next, the subcellular localization of circPDSS1 in GC cells by FISH fluorescence, which showed that circPDSS1 was mainly expressed in the cytoplasm (Fig. 1E). At the same time, we also found that circPDSS1 was highly expressed in the nucleus (Fig. 1F). In addition, correlation analysis showed that circPDSS1 was negatively correlated with the expression of miR-1278 in GC (Fig. 1G and H). These results suggest that circPDSS1 and miR-1278 may have a certain regulatory relationship and play a role in the development of GC.
2.2 GC cells secrete circPDSS1 by exosomes
Previous studies have reported that circRNAs can be transferred from one cell to another via exosomes(26). We isolated exosomes from GC cell supernatants and evaluated them by TEM and Western blot. Firstly, we observed that exosomes were spherical in shape, with diameters ranging from 50-150nm, and expressions of these exosome markers, such as CD63, Alix, and TSG101, were detected, confirming that exosomes were successfully isolated from GC cells (Fig. 2A-C). Next, we detected the higher expression of circPDSS1 in GC cells and exosomes by RT-qPCR (Fig. 2D). It is thus clear that GC cells secrete circPDSS1 by means of exosomes. Next, we obtained the characterization of circPDSS1 by Circular RNA Interactome (circinteractome. Nia. NIH. Gov/) database analysis, which showed that circPDSS1 was circular and 799 BP in size (Fig. 2E). We then blocked RNA synthesis in GC cells by the transcriptional inhibitor actinomycin D, and RT-qPCR results showed that circPDSS1 was highly consistency and had a s obvious longer half-life than linear PDSS1 (Fig. 2F). In addition, circPDSS1 and PDSS1 showed better stability and depletion in RNase R digestion, respectively, while the relative concentration of circPDSS1 was almost unchanged (Fig. 2G), indicating that circPDSs1 had a cyclic structure.
2.3 GC cells promote NK cell ferroptosis via exosomal circPDSS1
To determine whether the GC cell-derived exosomal circPDSS1 would induce NK cell death, we cocultured BGC-823 cell-extracted exosomes with NK cells for 24 h, cell apoptosis was then detected. According to the results, the number of living cells of NK cells decreased significantly after co-culture with exosomes, while the number of non-apoptotic cells was on the contrary, and the number of apoptotic cells remained unchanged (Fig. 3A), indicating that GC cell-derived exosomes induced NK cell death in a non-apoptotic form. To explore the way NK cells die, we used CQ (Chloroquine, inhibitor of autophagy), Z-VAD (Z-VAD-FMK, inhibitor of apoptosis), Nec-1 (Necrostatin-1, inhibitor of necrosis), Fer-1 (Ferrostatin-1, ferroptosis inhibitor), Lip-1 (Liproxstatin-1, ferroptosis inhibitor), the CCK-8 assay results showed that Fer-1 and Lip-1 obviously inhibited NK cell death (Fig. 3B), indicating that NK cells died in the form of ferroptosis after co-culture with GC cell exosomes. To understand the effect of the GC cell exosomal circPDSS1 on the ferroptosis of NK cells, we knocked down circPDSS1 in GC cells and the expression of circPDSS1 in exosomes was also detected. Compared with the negative control (sh-NC) group, Exosomal circPDSS1 expression was significantly reduced in circPDSS1 knockdown (sh-circPDSS1) group (Fig. 3C). Subsequently, we collected the exosomes of knockdown circPDSS1 (Exo-sh-circPDSS1) and the corresponding negative control (Exo-sh-NC) to co-cultured with NK cells, and observed the effect of exosomes after knockdown of circPDSS1 on NK cells. Lipid ROS accumulation is a hallmark feature of ferroptosis(27). According to the results of immunofluorescence detection, the expression of ROS in NK cells in the circPDSS1 shRNA group was lower than that in the NC shRNA group (Fig. 3D). Lipid peroxidation is the key link of ferroptosis, and MDA can directly reflect the level of lipid peroxidation in cells(28). Therefore, we also examined the level of MDA in NK cells, and the results were identical to those of the ROS test (Fig. 3E). Ferroptosis depends on elevated levels of intracellular lipid peroxides and Fe2+(29), after examining the intracellular Fe2+ level, the intracellular Fe2+ level was significantly increased in circPDSS1 shRNA group (Fig. 3F). GPX4 and SLC7A11 are important regulators of ferroptosis(30, 31). The transferrin protein receptor is a specific marker of ferroptosis(32). its expression level can directly reflect the level of ferroptosis. The protein expression of lGPX4, SLC7A11 and Transferrin were detected by WB, and the expression levels of GPX4 and SLC7A11 were obviously increased in the shcircPDSS1 group. Transferrin expression levels were clearly reduced (Fig. 3G). In a word, the above results indicate that GC cells promote NK cell ferroptosis via the exosomal circPDSS1.
2.4 circPDSS1 may act in NK cells in the spongy form of miR-1278
Previous studies have reported that circRNA mainly acts as a miRNA sponge to bind miRNA, thereby promoting the expression of miRNA-targeted genes. To elucidate the underlying mechanism by which exosomal circPDSS1 secreted by GC cells regulates NK cell ferroptosis, We used StarBase v3.0 to predict that circPDSS1 and miR-1278 have binding sites, so it can be inferred that miR-1278 may play a role as the target of circPDSS1 (Fig. 4A). To further confirm the relationship between circPDSS1 and miR-1278, miR-1278 mimic was first transfected into NK cells, and then double luciferase reporter analysis and RNA pull-down assay were performed. The detection results showed that miR-1278 mimic was successfully transfected into NK cells (Fig. 4B). The luciferase activity of circPDSS1 WT was decreased (Fig. 4C). circPDSS1 was enriched in NK cells (Fig. 4D). Finally, we used RT-qPCR to examine the effect of knockdown of circPDSS1 in NK cells on the expression of circPDSS1 and miR-1278. It is not difficult to find that the knockdown expression of circPDSS1 shRNA instead increased the expression of miR-1278 in NK cells (Fig. 4E and F). Thus, the relationship between circPDSS1 and miR-1278 was confirmed
2.5 circPDSS1 up-regulates GOT1 expression by inhibiting miR-1278
On the basis of the above studies, we further predicted the possible targets of miR-1278 by StarBase v3.0. The results showed that there was a binding site between the GOT1 gene and miR-1278 (Fig. 5A). Next, we used the same method to verify the relationship between circPDSS1 and miR-1278 to verify the relationship between miR-1278 and GOT1. As expected, luciferase activity of GOT1 3’UTR WT was decreased in NK cells transfected with miR-1278 mimic, but there was no significant change in the activity of GOT1 3’UTR MUT (Fig. 5B). Subsequently, WB was used to detect the specific regulatory relationship between miR-1278 and GOT1, and the final results showed that the expression of GOT1 decreased after the over-expression of miR-1278. Finally, after over-expression of circPDSS on the basis of over-expression of miR-1278, the inhibitory effect of miR-1278 on GOT1 was reversed (Fig. 5C). Our results suggest that circPDSS1 up-regulates the expression of GOT1 by inhibiting the activity of miR-1278.
2.6 The exosomal circPDSS1 from GC cells regulates NK cell ferroptosis through miR-1278/GOT1 axis
circPDSS1 might regulate NK cell ferroptosis through miR-1278/GOT1 axis. We transfected miR-1278 inhibitor, GOT1 short hairpin RNA (sh-GOT1) and their corresponding negative controls NC inhibitor and sh-NC into NK cells, and then used the exosomes of knockdown circPDSS1 to co-culture with NK cells to observe whether the exosomal circPDSS1 regulated the ferroptosis of NK cells through the miR-1278/GOT1 axis. We used immunofluorescence to detect the level of ROS in NK cells, and the results showed that Exo-sh-circPDSS1 group led to a decrease in intracellular ROS levels compared with Exo-sh-NC group, and inhibition of miR-1278 expression increased intracellular ROS levels, while knockdown of GOT1 reversed this result (Fig. 6A). The detection result of the MDA level shows that Exo-sh-circPDSS1 group obviously reduces the MDA level in the NK cell, and inhibition of miR-1278 obviously reverses the effect of Exo-sh-circPDSS1 group, namely the increase of the MDA level in the NK cell. On the basis of miR-1278 inhibitor and sh-GOT1 transfection, co-culture with the exosomes of knockdown circPDSS1 led to the decrease of MDA level in NK cells(Fig. 6B). The level of Fe2+ in NK cells was decreased after co-culture with exosomes of knockdown circPDSS1; After co-culture with exosomes of knockdown circPDSS1 on the basis of transfection miR-1278 inhibitor, the level of Fe2+ in NK cells increased again. After co-culture with exosomes of knockdown circPDSS1 on the basis of transfection miR-1278 inhibitor and sh-GOT1, the level of Fe2+ in NK cells decreased again (Fig. 6C). In addition, the expression of GPX4, SLC7A11 and Transferrin were detected by WB. The protein expressions of GPX4 and SLC7A11 were significantly increased and the protein expression of Transferrin was significantly decreased after co-culture with exosomes of knockdown circPDSS1. The effect of Exo-sh-circPDSS1 group was reversed by co-culture with the exosomes of knockdown circPDSS1 after transfection miR-1278 inhibitor that the protein expression of GPX4 and SLC7A11 decreased, and the protein expression of Transferrin increased. However, co-culture with exosomes of knockdown circPDSS1 after transfection of miR-1278 inhibitor and sh-GOT1 further reversed this result that the protein expression of GPX4 and SLC7A11 increased, and the protein expression of Transferrin decreased (Fig. 6D). Taken together, our results indicate that the exosomal circPDSS1 from GC cells regulates NK cell ferroptosis through the miR-1278/GOT1 axis.
2.7 circPDSS1 induces GC progression in an NK cell ferroptosis dependent manner
To further explore whether circPDSS1 induces GC progression by promoting NK cell ferroptosis, we knocked down circPDSS1 in NK cells while adding the ferroptosis activator Erastin, and subsequently co-cultured with GC cells at a 1:1 ratio to observe the effects of NK cell ferroptosis on GC cell Behavioral characteristics. Eventually we found that circPDSS1 down-regulation decreased GC cell proliferation, which was reversed by erastin treatment (Fig. 7A); In addition, the migration ability of GC cells was also reduced after knockdown of circPDSS1, which was reversed after Erastin treatment (Fig. 7B). Finally, we detected the apoptosis of GC cells. In contrast to changes in proliferation and migration, reduced circPDSS1 expression increased the level of apoptosis in GC cells, while erastin treatment reduced the level of apoptosis in GC cells (Fig. 7C). So, that circPDSS1 induces GC progression in an NK cell ferroptosis dependent manner.
2.8 Knockdown of circPDSS1 in GC cells exosomes inhibits tumor growth
GC cells were transplanted into nude mice to establish tumor xenotransplantation model of nude mice, and the exosomes expressing circPDSS1 were knocked down by 3d posterior caudal injection. Tumor volume and weight were measured at day 7, 14, 21, and 28, respectively, and it was clear that both tumor size and volume were smaller in the nude mice injected with exosomes than in the normal group (Fig. 8A and B). Next, we examined the expression of circPDSS1 and miR-1278 in tumor tissues. Consistent with cell experiments, circPDSS1 expression was significantly down-regulated in the Exo-sh-circPDSS1 group, while miR-1278 expression was up-regulated (Fig. 8C). Finally, we also detected the expression of GOT1 protein, and again, the expression level of GOT1 protein was reduced in the Exo-sh-circPDSS1 group (Fig. 8D). This part of the experiment proved that circPDSS1 knockdown inhibited tumor growth in nude mice.