The Mechanism Of CircHIPK3 Contributes To Cisplatin Resistance In Gastric Cancer By Blocking Autophagy-Dependent Ferroptosis

Objective:Gastric cancer(GC)is one of the most common malignant tumors in the world.Despite advances in early diagnosis and clinical treatment,the prognosis for gastric cancer patients remains poor due to resistance to chemotherapy such as cisplatin.Circular RNAs(CircRNAs)are a new class of non-coding RNAs with highly conserved closed-loop structures.Previous reports have shown that circRNA plays an important role in drug resistance of gastric cancer.CircRNA could competitively bind with microRNA(miRNA),and further regulate target genes of miRNA to exert the malignant biological function of cells.Previous studies reported that circHIPK3 promoted proliferation,migration and invasion of gastric cancer cells.However,whether circHIPK3 regulates cisplatin resistance in gastric cancer remains unclear.Therefore,this study investigated the effect of circHIPK3 on cisplatin resistance of gastric cancer cells,and explored the mechanism of circHIPK3/miR/508-3p/Bcl-2/Beclin1/SlC7A11 axis and autophagy-dependent ferroptosis in cisplatin resistance of gastric cancer.Methods:The cisplatin-resistant gastric cancer cells(AGS/DDP,HGC-27/DDP)were used to overexpress or knock down circHIPK3.1.The levels of expression of circHIPK3 in cisplatin-resistant GC tumors and cells were analyzed by RT-qPCR.CCK-8,EdU,and colony formation assays were performed to investigated the proliferative capacity of GC/DDP cells.The IC50 value of cisplatin was detected.To further investigate the biological functions of circHIPK3 in vivo,cells overexpressing circHIPK3 or circHIPK3 knockdown cells were subcutaneously injected into BALB/c nude mice.Nude mice were divided into NC+DDP group,circHIPK3+DDP group,si-Control+DDP group,and si-circHIPK3+DDP group.The tumor volume and weight were measured respectively.RT-qPCR detected the relative expression of circHIPK3 in tumors.2.Flow cytometry and TUNEL assay were used to detect apoptosis rate.The changes of LC3 Ⅱ/Ⅰ,Beclin1,and P62 were measured by western blot.The autophagic flux in GC/DDP cells were monitored by the reporter mRFP-GFP-LC3.The role of autophagy in circHIPK3 mediated cisplatin resistance was investigated by using the autophagy inhibitor 3-MA.3.After erastin and sorafenib treatment,we explored the correlation between circHIPK3 and ferroptosis in GC cells.The levels of reactive oxygen species(ROS),lipid peroxidation products--MDA,Fe2+ and glutathione(GSH)were detected in cells,and transmission electron microscopy detected the mitochondrial changes.The regulatory effect of autophagy on ferroptosis in cisplatin-resistant cells was investigated by rapamycin,an autophagy activator,and 3-MA,an inhibitor.4.RT-qPCR and FISH were used to locate circHIPK3 in cisplatin-resistant GC cells.The downstream target miR-508-3p of circHIPK3 and the downstream target gene Bcl-2 of miR-508-3p were predicted by Starbase,CircBank and Circular RNA Interactome.Dual luciferase assay was used to investigate the targeting relationship between circHIPK3 and miR-508-3p,miR-508-3p and Bcl-2.5.CircHlPK3,miR-508-3p mimics and si-circHIPK3,anti-miR-508-3p were transfected into AGS/DDP and HGC-27/DDP cells,respectively.The apoptosis and autophagy effects of circHIPK3/miR-508-3p axis was investigated by detecting the protein levels of caspase3,cleaved caspase3,Bcl-2,LC3 Ⅱ/Ⅰ,Beclin1,and P62.The ferroptotic effect of circHIPK3/miR-508-3p axis was studied by detecting ROS,MDA,Fe2+,and GSH levels.Obatoclax Mesylate,a Bcl-2 inhibitor,was used to study the autophagy function of miR-508-3p/Bcl-2 axis in cisplatin-resistant gastric cancer cells.6.Co-IP verified the binding between Beclinl and SLC7A11.The AGS/DDP and HGC-27/DDP cells overexpressing or knocking down circHIPK3 were transfected with BECN1.Western blot was used to detect LC3 Ⅱ/Ⅰ,Beclinl,and P62.Furthermore,we also detected the changes of ferroptosis in cells.7.Transmission electron microscopy,NTA and western blot were used to verified exosomes extracted from serum and cell supernatants.RT-qPCR detected exosomal circHIPK3 expression in GC patients’ cisplatin-resistant serum and cells.In addition,we measured exosomal circHIPK3 levels in GC patients’ serum treated with cisplatin for three months.Results:1.The expression level of circHIPK3 in AGS/DDP,HGC-27/DDP and MKN-28 cells was significantly higher than that of AGS,HGC-27 and MKN-28 cells.Elevated levels of circHIPK3 promoted cell proliferation and the IC50 value of AGS/DDP and HGC-27/DDP cells(P<0.05).Knocking down circHIPK3 reduced the IC50 value and inhibited the proliferation of GC/DDP cells(P<0.05).Compared with the control group,the tumor volume,weight and circHIPK3 expression level in the circHIPK3+DDP group were significantly increased(P<0.05),while the tumor volume,weight and circHIPK3 expression levels in the si-circHIPK3+DDP group were significantly reduced(P<0.05).2.Overexpression of circHIPK3 inhibited the apoptosis and autophagy of AGS/DDP and HGC-27/DDP(P<0.05).Knocking down circHIPK3 promoted the apoptosis and autophagy of AGS/DDP and HGC-27/DDP(P<0.05).When 3-MA was cocultured with circHIPK3 silenced cells,we observed that 3-MA eliminated the basic autophagic processes and reversed the effects of chemosensitivity initiated by circHIPK3 repression(P<0.05).3.After erastin,and sorafenib treatment,circHIPK3 inhibited ferroptosis P<0.05).While knocking down circHIPK3 promoted ferroptosis(P<0.05).Transmission electron microscopy showed that cells transfected with the empty vector exhibited typical morphological changes of ferroptosis,including mitochondria atrophy and decreased cristae,whereas overexpression of circHIPK3 abolished these characteristic features.After treatment with rapamycin,ferroptosis was further activated;while 3-MA inhibited the ferroptosis(P<0.05).4.FISH and RT-qPCR demonstrated that circHIPK3 was mainly enriched in the cytoplasm.Dual luciferase assay showed circHIPK3 could directly bind to miR-508-3p,and miR-508-3p could directly bind to Bcl-2(P<0.05).5.Compared with circHIPK3+miR-NC mimics group,apoptosis,autophagy and ferroptosis levels were increased(P<0.05)in the circHIPK3+miR-508-3p mimics group.Compared with si-circHIPK3+anti-miR-NC,apoptosis,autophagy and ferroptosis levels were decreased in si-circHIPK3+anti-miR-508-3p group(P<0.05).Obatoclax Mesylate,a Bcl-2 inhibitor,further facilitated the autophagy-promoting function of miR-508-3p.6.Co-IP confirmed Beclin1 could bind to SLC7A11.After transfection of BECN1,BECN1 reversed the inhibitory ferroptosis function of circHIPK3 in GC/DDP cells(P<0.05).7.Transmission electron microscopy,NTA and western blot verified exosomes,and circHIPK3 expression was shown to be higher in exosomes from GC/DDP cell lines than in their parental cells(P<0.05).Furthermore,exosomal circHIPK3 was upregulated in cisplatin-resistant serum compared with in cisplatin-sensitive serum(P<0.05).In addition,RT-qPCR showed that exosomal circHIPK3 levels were significantly reduced after cisplatin treatment(P<0.05).Conclusion:CircHIPK3 promoted proliferation and cisplatin resistance in gastric cancer.CircHIPK3 inhibited apoptosis,autophagy and ferroptosis.CircHIPK3 directly bound to miR-508-3p and enhanced the resistance of GC/DDP cells to cisplatin by inhibiting autophagy-dependent ferroptosis via the miR-508-3p/Bcl-2/Beclin 1/SLC7A11 axis.Importantly,the level of exosomal circHIPK3 in serum decreased after cisplatin treatment,suggesting that circHIPK3 in serum exosomes may be a noninvasive indicator for evaluating the efficacy of chemotherapy in gastric cancer.