The Study Of The Role And Related Mechanisms Of LncRNA MEG3-210 In Endometriosis

Endometriosis refers to the appearance of endometrial-like tissues outside the uterine cavity and myometrium,causing symptoms such as infertility,dysmenorrhea,and dyspareunia,which affects women's living quality severely.It has high incidence and recurrence in infertile women,which placed a huge burden on the social medical system.Although studies on endometriosis has lasted for decades,its specific pathogenesis is inconclusive.Currently,mainstream theories are"retrograde menstruation theory","metaplasia theory","immunology theory",etc.Among them,"retrograde menstruation theory"is widely accepted,but no one theory can explain all types of endometriosis perfectly.Based on the theory of menstruation reflux,the"determinant of eutopic endometrium"proposed by domestic scholar Professor Lang Jinghe points out that the biological characteristics of the endometrium determine the occurrence of endometriosis.With the development of sequencing and omics,emerging results suggested that epigenetics,RNAs and proteins are aberrantly expressed in eutopic endometrium of patients with endometriosis,which supports that the endometriotic lesions originate from abnormal endometrium.It is imperative to explore the pathogenesis of endometriosis for developing novel treatments and diagnostic approaches.Long noncoding RNA?lncRNA?is a class of non-coding RNA with over 200nucleotides.With the in-depth study of lncRNA,it was found that lncRNA can interact with DNA,RNA,and proteins,which regulates a variety of important physiological processes at transcription,translation and post-translation modification levels.Maternally expressed gene 3?MEG3?considered to be a"tumor suppressor"is widely expressed in various normal tissues and cells.Downregulation or deletion of MEG3can promote tumorigenesis.MEG3 has multiple transcripts,and a study have pointed out that the functional activity of different transcripts is different,so it is necessary to choose one or a type of transcripts for detailed and in-depth exploration and analysis.MEG3 has not yet been reported in endometriosis,and our previous sequencing results showed that MEG3-210 as a transcript of MEG3 expressed lower in eutopic endometrium from patients with endometriosis compared with control endometrium,suggesting that MEG3-210 may paly a role in the development of endometriosis.Galectin-1 binds to ?-galactose protein residues through the sugar recognition domain and participates in cell proliferation,migration,and apoptosis.The expression of Galectin-1 is increased in the eutopic endometrium of patients with endometriosis,and blocking Galectin-1 with antibody can reduce endometriotic lesions and inhibit angiogenesis in mouse models.The mitogen-activated protein kinases?MAPK?pathway is an important signal transduction pathway mediating the transmission of extracellular signals into cells.3?,5?-cyclic adenosine?3?,5?-cyclic adenosine,c AMP?-dependent protein kinase A?PKA?is the central molecule linking the calcium signal ?SERCA2?controls intracellular Ca²⁺concentration and maintains intracellular calcium balance by pumping intracellular Ca²⁺into the sarcoplasmic reticulum.The regulatory mechanism of MEG3-210 in endometriosis remains unknown,and study on its regulatory relationship may help to explore the pathogenesis of the disease.In this study,the differential expression of MEG3-210 in endometriosis was detected by reanalysis of sequencing data,and functional assays were used to explore the role of MEG3-210 in endometrial stromal cells?ESCs?after downregulation and overexpression.Bioinformatics analysis and molecular mechanism related experiments found Galectin-1 and p38 MAPK and PKA/SERCA2 signaling pathways were involved in the regulation of MEG3-210 on ESCs,and the schematic model of MEG3-210 regulatory network in endometrioisis plotted as the interaction between the pathways provided a molecular basis for new diagnosis and treatment of endometriosis.Part I The functions of MEG3-210 in endometrial stromal cellsObjective: To determine the differential expression levels of MEG3-210 in eutopic endometrial tissues and ESCs of patients with endometriosis or not.To explore the effects of MEG3-210 on the proliferation,migration,invasion and apoptosis of ESCs.Methods: The results of next generation sequencing of endometrium from 5 pairs of patients with endometriosis or not were reanalyzed.Tissue specimens came from eutopic endometrium of women with endometriosis?EU,n=40?or not?NE,n=39?.ESCs were isolated from eutopic endometrium of women with endometriosis?Eu ESCs,n=18?or not?Ne ESCs,n=17?,and identified by immunofluorescence.q RT-PCR was used to determine the expression levels of MEG3-210 in tissues and cells.MEG3-210 si RNA was transfected into Ne ESCs to inhibit MEG3-210 expression,MEG3-210 overexpression plasmid was transfected into Eu ESCs to promote MEG3-210 expression.And CCK-8,transwell and flow cytometry assays were performed to detect ESCs proliferation,migration,invasion and apoptosis.Results: 1.The results of next generation sequencing identified a total of 26 MEG3 transcripts.Among these transcripts,only MEG3-210 and MEG3-202 transcripts expressed differentially with statistical significance.2.MEG3-210 expressed lower in eutopic endometrium and ESCs in patients with endometriosis.3.After MEG3-210 downregulation,Ne ESCs enhanced migration,invasion and anti-apoptosis capabilities,but no significant effect on proliferation.4.After MEG3-210 overexpression,Eu ESCs weakened migration,invasion and anti-apoptosis capabilities,but no significant effect on proliferation.Conclusion: 1.The results of q RT-PCR are consistent with the sequencing results,indicating the sequencing results are reliable.2.MEG3-210 can regulate ESCs migration,invasion and apoptosis,but has no obvious effect on proliferation.3.MEG3-210 downregulation may be involved in the pathogenesis of endometriosis and promote the development of endometriosis.Part II MEG3-210 regulates ESCs migration,invasion and apoptosis through p38 MAPK and PKA/SERCA2 signaling pathwaysObjective: To explore the signaling pathways involved in the regulation of MEG3-210 in ESCs.Methods: The genes co-expressed with MEG3 were extracted and analyzed by bioinformatic analysis.Western blot assays were used to detect the changes in protein levels of related molecules.The rescue experiments were performed to confirm the role of pathways in the regulation of MEG3-210 in ESCs.Results: 1.The bioinformatics analysis showed that 26 signaling pathways were associated with MEG3,and in KEGG enrichment analysis,more genes were associated with c AMP signaling,calcium signaling and MAPK signaling pathways.2.After MEG3-210 downregulation,the protein levels of phosphorylated p38,and ATF2 increased,and the protein levels of Bcl-2 and MMP2 increased,but the protein levels of PKA and SERCA2 decreased.3.In combination with MEG3-210 si RNA transfection,p38 MAPK signaling inhibitor SB203580 inhibted Ne ESCs migration and invasion partially,and restored apoptosis.4.In combination with MEG3-210 si RNA transfection,SERCA2 overexpression inhibited Ne ESCs migration and invasion completely,and restored apoptosis.5.In combination with MEG3-210 overexpression plasmid,SERCA2 downregulation promoted Eu ESCs migration,invasion and anti-apoptosis.6.After SERCA2 downregulation,the protein levels of phosphorylated p38 and ATF2 increased.After the addition of SB203580,protein levels of SERCA2 decreased.Conclusion: 1.MEG3-210 downregulation can activate the p38 MAPK signaling and inhibit PKA/SERCA2 signaling.2.Activation of p38 MAPK signaling and inhibition of PKA/SERCA2 signaling can account for the regulation of MEG3-210 on ESCs migration,invasion and apoptosis partially.3.There is a crosstalk between the p38 MAPK and PKA/SERCA signaling pathways,which promotes the occurrence and development of endometriosis synergically.Part III Galectin-1 mediates the regulation of MEG3-210 in ESCsObjective: To identify the proteins interacting with MEG3-210 and explore the mechanism of MEG3-210 in endometriosis.Methods: The MEG3-210 RNA probe was constructed,and the interaction between Galectin-1 and MEG3-210 was determined by experiments such as RNA-pull down mass spectrometry,RIP,and co-localization.The regulation of Galectin-1 on p38 MAPK and PKA/SERCA2 signaling pathways was determined by western blot.The expression levesl of Galectin-1 were detected by western blot and ELISA.In combination with MEG3-210 si RNA and Galectin-1 overexpression plasmid,transwell and flow cytometry were used to detect Ne ESCs migration,invasion and apoptosis.Results: 1.RNA-pull down followed by mass spectrometry identified several proteins that interact with MEG3-210.2.MEG3-210 probes could capture MEG3-210 and Galectin-1.Galectin-1 antibody precipitated Galectin-1 and MEG3-210.3.The distribution of MEG3-210 and Galectin-1 was consistent in ESCs.4.After Galectin-1 overexpression,protein levels of phosphorylated p38 increased,and protein levels of PKA and SERCA2 decreased.5.Galectin-1 overexpressed in eutopic endometrial tissues,ESCs and serum from patients with endometriosis.6.In combination with MEG3-210 si RNA,Galectin-1 downregulation inhibited Ne ESCs migration,invasion and anti-apoptosis.Conclusion: 1.Galectin-1 can interact with MEG3-210 and mediate the regulation of MEG3-210 on ESCs migration,invasion and apoptosis.2.Galectin-1 overexpression can activate p38 MAPK signaling and inhibit PKA/SERCA2 signaling.3.In combination with other indicators,Galectin-1 can be used in the serological diagnostic model of endometriosis.Part IV The role of MEG3-210 and Galectin-1 in vivoObjective: To establish animal models of endometriosis and explore the effects of MEG3-210 and Galectin-1 in vivo.Methods: Human endometrium and nude mice were used to construct nude mice intraperitoneal and subcutaneous endometriotic lesions.Pathological examination was used to evaluate the vitality and structure of the lesions.Adenoviral particles contained sh-MEG3-210 were transfected into endometrium.Galectin-1 si RNA was injected in vivo to observe the effects of Galectin-1 downregulation on lesions.The expression levels of Galectin-1 in the lesions were detected by immunohistochemistry.Results: 1.Human endometrium could form ectopic lesions in the abdominal cavity and subcutaneous part of nude mice,but the subcutaneous lesions have better activity.2.The volume of lesions in the Ad-sh-MEG3-210 treatment group increased.3.In combination with Galectin-1 si RNA and Ad-sh-MEG3-210,lesion volume significantly decreased,compared with Ad-sh-MEG3-210 treatment group.Conclusion: 1.Adenovirus can transfect endometrium effectively.2.MEG3-210 downregulation can promote the growth of subcutaneous endometriotic lesions.3.Galectin-1 downregulation can reduce the increase in lesion volume caused by MEG3-210 downregulation.