The Role And Mechanism Of LNCRNA AC079944.2 In Regulating Endometrial Receptivity Through MIR-149-5P/EFNA1

Background:Endometrial receptivity(endometrial receptivity,ER)refers to the ability of endometrium to accept embryo implantation,which is the key factor for successful embryo implantation,and about 2% of embryo implantation failure is related to the decrease of ER,so accurate evaluation and improvement of ER is very important to increase the rate of embryo implantation.The role and mechanism of long noncoding RNA(long noncoding RNA,lnc RNA)in regulating endometrial receptivity has become a new focus of assisted reproductive technology.This project intends to screen and identify lnc RNA closely related to ER by combining bioinformatics and molecular biology experiments,and further analyze its role in regulating endometrial receptivity and its specific mechanism,in order to find a new marker of endometrial receptivity.And clarify the molecular mechanism of its regulation of endometrial receptivity,and explore new strategies to improve endometrial receptivity to increase the clinical pregnancy rate.Methods:1.The original data of transcriptome sequencing of 71 endometrial tissue samples provided by GSE106602 and GSE98386 were downloaded from GEO database,and the differentially expressed m RNA and lnc RNA were screened.Through WGCNA analysis,the gene co-expression module was constructed,the relationship between the module and sample characteristics was analyzed,and the key modules related to traits were selected for GO and KEGG analysis.2.Identify the hub gene of the key module,construct a co-expression network,identify the hub m RNA related to endometrial receptivity in the co-expression network,and screen the co-expressed hub lnc RNA for further study.3.q RT-PCR and Western blot were used to detect the expression of lnc AC079944.2 and EFNA1 in endometrial tissue and endometrial cells(ishikawa cells).4.The subcellular localization of lnc AC079944.2 was detected by nuclear and cytoplasmic separation test.5.Double luciferase assay confirmed the targeted binding of hsa-miR-149-5p to lnc AC079944.2 and EFNA1.6.The effects of lnc AC079944.2/miR-149-5p/EFNA1 molecular regulatory network on the proliferation,migration,invasion and adhesion of endometrial cells were evaluated by EDU test,scratch test,invasion test and adhesion test to JAR cells.Results:1.Through WGCNA analysis,it was determined that the turquoise module was closely related to the clinical characteristics of endometrium in the mid-secretory phase.2 GO analysis of turquoise module showed that it was enriched in angiogenesis and fine cell adhesion,while KEGG analysis found that it was mainly enriched in PI3K-Akt signal pathway,MAPK signal pathway and so on.3.The lnc RNA-m RNA-Pathway network of hub gene in turquoise module was constructed,and lnc AC079944.2 and EFNA1 were determined as the core genes in this module.4.In the mid-secretory endometrium and ishikawa cells treated with estradiol(E2)and progesterone(P4),the expression of lnc AC079944.2 and EFNA1 was upregulated by q RT-PCR and Western blot experiments.5.Nuclear-cytoplasmic separation experiment showed that lnc AC079944.2 was expressed in both cytoplasm and cells.6.Double luciferase assay confirmed the targeted binding of miR-149-5p to lnc AC079944.2 and EFNA1.7.Cell experiments show that lnc AC079944.2 regulates the expression of EFNA1 through ce RNA mechanism and competitive binding to miR149-5p.8.Functional experiments confirmed that lnc AC079944.2 promoted the migration,invasion and proliferation of ishikawa cells and adhesion to JAR cells,while miR-149-5p inhibited the migration,invasion and proliferation of ishikawa cells and adhesion to JAR cells.Conclusion:The expression of lnc AC079944.2 and EFNA1 is up-regulated in endometrial tissue at the middle secretory stage and ishikawa cells treated with E2+P4.lnc AC079944.2 can up-regulate the expression of EFNA1 through competitive binding to miR-149-5p,promote the migration,invasion and proliferation of endometrial cells,and promote the adhesion to JAR cells,which is beneficial to improve endometrial receptivity and promote embryo implantation.