Investigating The Mechanisms Underlying ALKBH5 Mediated M~6A Modification Of LncRNA UBOX5-AS1 Promotes Autophagy In Endometriosis

Objective1.To examine the expression of N~6-methyladenosine(m~6A)demethylase Human Alk B Homolog H5(ALKBH5)in ectopic,eutopic endometrium of patients with endometriosis(EMs)and normal endometrium,and analyze its relationship with autophagy of endometriosis.2.To investigate the role of ALKBH5-mediated m~6A modification in regulating the abnormal autophagy activation of LncRNA UBOX5-AS1 in endometriosis3.To elucidate the molecular mechanism of ALKBH5-mediated m~6A modification of LncRNA UBOX5-AS1.4.To analyze the expression of m~6A modifiation-related long non-coding RNA(LncRNA)by methylated RNA immunoprecipitation(Me RIP)sequencing combined with transcriptome sequencing in endometriosis.Methods1.32 cases of both ectopic and eutopic endometrial and 34 cases of normal endometrial tissues were collected to identify histological morphology by hematoxylin-eosin(HE)staining and to detect the expressions of m~6A demethylase ALKBH5 and autophagy bio-markers autophagy related gene 12(ATG12)and microtubule-associated protein light chain3(LC3)by q RT-PCR,Western Blot and immunohistochemistry(IHC).2.Primary human endometrial stromal cells(ESCs)were isolated from 3 types of endometrium.The purity was identified by immunocytochemistry staining.The level of ALKBH5,ATG12 and LC3 in ectopic,eutopic and normal ESCs were detected by q RT-PCR and Western Blot assay.3.After transfection of ALKBH5 overexpression plasmid or ALKBH5 small interfering RNA(si RNA)and corresponding negative control si RNA into the human endometrial stromal cells(THESCs),the levels of autophagy bio-markers were detected by Western Blot and q RT-PCR.Transmission electron microscope(TEM)and GFP-m RFP-LC3 adenovirusfluorescence test was used to detect autophagy flux.Invasion and migration ability of THESCs was detected by cell scratch test and Transwell assay.The ALKBH5-overexpressing THESCs were treated with autophagy inhibitor 3-Metyladenine(3-MA)and the ALKBH5-sliencing THESCs were treated with autophagy activator rapamycin,then THESCs invasion and migration ability was detected by Transwell assay and cell scratch test.4.The expression of LncRNA UBOX5-AS1 in ectopic,eutopic,normal endometrium and corresponding primary ESCs was detected by q RT-PCR.The intracellular localization of LncRNA UBOX5-AS1 was detected by nuclear cytoplasmicRNA isolation assay and RNA fluorescence in situ hybridization(FISH).RNA Immunoprecipitation(RIP)assay was used to detect the combination of LncRNA UBOX5-AS1 with ALKBH5 and ATG12.ALKBH5 was overexpressed or silenced in THESCs,and q RT-PCR was used to detect the expression of LncRNA UBOX5-AS1,the stability of LncRNA UBOX5-AS1 was detected by actinomycin D assay.After overexpressed or silenced LncRNA UBOX5-AS1,or the LncRNA UBOX5-AS1 was silenced based on the overexpression of ALKBH5 in THESCs,the autophagy bio-markers level,autophagosome and autophagy lysosome formation and THESCs invasion and migration ability was detected.5.The possible m~6A modification site on LncRNA UBOX5-AS1 was predicted online by SRAMP.The dual-luciferase wild type plasmid of LncRNA UBOX5-AS1 and the mutant plasmid of LncRNA UBOX5-AS1 with mutant m~6A modification site were constructed respectively,and co-transfected into THESCs with ALKBH5 overexpression or silencing,respectively.Dual-luciferase assay was used to detect the chemiluminescence intensity.Methylated RNA immunoprecipitation(Me RIP)was used to detect the m~6A level of LncRNA UBOX5-AS1 after overexpression or silencing of ALKBH5.6.C57 mice aged 6-8 weeks were selected to establish the EMs mouse model by allotransplantation and were divided into two groups,randomly.ALKBH5 si RNA and its corresponding control si RNA were injected intraperitoneally every other day.The abdominal wall lesions of mice were collected,and lesion size were compared.IHC,Western Blot and q RT-PCR were used to detect the level of ALKBH5 and autophagy bio-markers in the lesions.7.3 cases of both ectopic and normal endometrial tissues were collected,and the RNA was extracted for Me RIP sequencing and transcriptome sequencing(RNA sequencing).The transcripts with different m~6A methylation levels or different expression in endometriosis were detected and the general characteristics,common m~6A motif and functional enrichment were analyzed.8.The correlation between differentially expressed and differentially methylated LncRNA was analyzed.9.Differentially methylated LncRNAs were screened,and the expression levels of proliferative ectopic endometrium and normal endometrium were detected by q RT-PCR.10.LncRNA-mi RNA-m RNA and LncRNA-m RNA network of endometriosis were constructed.Results1.The expression of ALKBH5,ATG12 and LC3 in ectopic and eutopic endometrium of patients with EMs and their corresponding ESCs showed a statistical increasing compared to that in normal endometrium and ESCs.2.Overexpression of ALKBH5 could promoted autophagy,invasion and migration of THESCs,and autophagy inhibitor 3-MA could inhibit this effect.3.The expression of LncRNA UBOX5-AS1 in ectopic endometrium,eutopic endometrium and corresponding primary ESCs was higher than that in normal endometrium and its primary ESCs,and was mainly located in the nucleus of THESCs.4.LncRNA UBOX5-AS1 could bind to ALKBH5 and ATG12.5.Overexpression of ALKBH5 increased the expression of LncRNA UBOX5-AS1,while silencing ALKBH5 showed the opposite effect.6.Overexpression of LncRNA UBOX5-AS1 promoted autophagy,invasion and migration of THESCs,while silencing LncRNA UBOX5-AS1 showed the opposite effect.7.Silencing LncRNA UBOX5-AS1 could reverse the autophagy,invasion and migration of THESCs induced by overexpression of ALKBH5.8.ALKBH5 affects the stability and expression of LncRNA UBOX5-AS1 by changing the level of m~6A modification.9.The endometriosis mouse model was successfully constructed.Sliencing ALKBH5could inhibit the expression of ALKBH5,ATG12 and LC3 in ectopic lesions of endometriosis mice.10.m~6A modified LncRNA was detected in ectopic endometrium of endometriosis patients and normal endometrium.The most common motif of methylated transcripts was GGACU,and differentially methylated LncRNAs were mainly enriched in cell adhesion and other pathways.11.In OEM group,LncRNA DIO3OS was up-regulated,while the expression of LINC00665,LINC00937,FZD10-AS1 and GATA2-AS1 was decreased.12.LncRNA-mi RNA-m RNA ce RNA network and LncRNA-m RNA co-expression regulatory network were successfully constructed.Conclusion1.The levels of m~6A demethylase ALKBH5 and autophagy in ectopic and eutopic endometrium in patients with EMs were higher than those in normal endometrium.2.ALKBH5 regulates LncRNA UBOX5-AS1 by mediating m~6A modification to promote autophagy,invasion and migration of THESCs.3.Silencing the expression of ALKBH5 can alleviate the activation of autophagy and inhibit the progression of endometriosis.4.This study comprehensively describes the characteristics of LncRNA m~6A methylation in endometriosis and proves the potential relationship between the m~6A modification of LncRNA and the occurrence and development of endometriosis.