Impact of ALKBH5 on the progression of ovarian endometriosis cysts by inhibiting BECN1-mediated ferroptosis
Objective To explore the role and mechanism of ALKBH5 on ovarian endometriosis cysts.Methods The cyst tissue and normal endometrial tissue of 5 patients undergoing ovarian endometriosis cyst exfoliation from January 1,2022,to October 10,2022 at Qingdao Central Hospital were collected.The expression of ALKBH5 in mRNA and protein were detected by western blot and quantitative real-time polymerase chain reaction(qRT-PCR)assay.The ectopic endometriosis stromal cells(EESCs)were isolated and cultured.After knocking down ALKBH5 expression by transfecting small inter-fering RNA,the effects of ALKBH5 on the ability of proliferation,migration and invasion were verified by cell counting kit 8(CCK8),colony formation and transwell assays in EESCs.The effects of ALKBH5 on the ability of angiogenesis was verified by tube formation assay.Glutathione(GSH)and iron content were detected to verify the effect of ALKBH5 on ferroptosis in EESCs.The effect of ALKBH5 on subcellular structure of EESCs was observed by transmission electron microscopy.The effect of ALKBH5 on BECN1 expression was verified by qRT-PCR and western blot.RIP and MeRIP assays were used to verify whether ALKBH5 regulated BECN1 expression through m6A demethylation modification.The Student t test was used for statistical analysis.Results The results of western blot showed that the expression of ALK-BH5 protein in ovarian endometriosis cysts tissue was significantly higher than that in normal endometrial tissue.The re-sults of qRT-PCR showed that the relative expression level of ALKBH5 mRNA in normal endometrial tissue was 1.000±0.058,and that in ovarian endometriosis cysts tissue was 2.270±0.044,t=17.56,P＜0.001.The results of CCK8 as-say showed that the D(450 nm)value of siNC group and siALKBH5 group was 1.575±0.000 and 0.957±0.000,t=23.05,P＜0.001.The results of colony formation assay showed that the number of colony formed in siNC group and siALKBH5 group were 75.330±3.712 and 22.670±1.764,t=12.82,P＜0.001.The results of transwell assay showed that the number of migrated cells in siNC group and siALKBH5 group were 196.700±8.819 and 77.000±5.859(t=11.30,P＜0.001).The number of invaded cells in siNC group and siALKBH5 group were 62.000±2.309 and 18.330±2.028(t=14.21,P＜0.001).The results of tube formation assay showed that the number of tubes formed in siNC group and siALKBH5 group were 73.330±4.667 and 19.330±1.764,t=10.82,P＜0.001.Additionally,the results of GSH content detection showed that the relative content of GSH in siNC group and siALKBH5 group were 1.067±0.088 and 0.430±0.025,t=6.942,P＜0.001.The results of iron content detection showed that the relative content of iron in siNC group and siALKBH5 group were 1.000±0.058 and 2.773±0.059,t=21.43,P＜0.001.The results of transmis-sion electron microscopy showed that the mitochondrial and endoplasmic reticulum structures of siALKBH5 group were damaged and dissolved.The results of western blot showed that ALKBH5 knockdown promoted the expression of BECN1 protein.The results of qRT-PCR showed that the relative expression level of BECN1 mRNA in siNC group and siALK-BH5 group were 1.033±0.067 and 3.277±0.043,t=28.21,P＜0.001.The results of RIP assay showed that the rela-tive enrichment of BECN1 in IgG group and ALKBH5 group were 1.073±0.101 and 79.980±3.695,t=21.35,P＜0.001.The results of MeRIP assay showed that the relative enrichment of m6A+BECN1 in siNC group and siALKBH5 group were 1.007±0.052 and 2.453±0.098,t=13.01,P＜0.001.Conclusion ALKBH5 inhibits the ferroptosis of EESCs by mediating m6A demethylation to inhibit BECN1 expression,and promotes the progression of ovarian endome-triosis cysts.
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