| Objective:Endometriosis is a common gynecologic disorder,affecting 6 to 10%women of reproductive age and up to 35%-50%of women with pelvic pain and/or infertility.Although a benign condition,it shares malignant biological behaviors.Unfortunately,up to date there are still no optimal treatments for the disease due to the complexity of the pathogenesis and diversity of symptoms,and high rate of relapse,which seriously influences women's physical and mental health.Therefore,the key to improve the diagnosis and treatment is to study the pathophysiological mechanism of endometriosis.At present,there are several hypotheses about the pathogenesis of endometriosis,but the original molecular mechanism has not been elucidated.circRNA is a distinct class of non-coding RNA,which has been a research focus due to its circular-shaped structure,high sequence conservation among diverse species,and tissue-specific and space-specific expression patterns.The biological functions of circRNA manily include the followings:promote the expression of parental genes;regulate translation and even splicing process;translated into effective protein;and the most important one is to act as ceRNA,sponging miRNA to regulate downstream target genes expression.In recent years,a growing body of evidence has indicated that circRNAs,acting as miRNA“sponge”,play crucial regulatory roles in the pathogenesis,development and prognosis of various diseases like tumors,cardiovascular system diseases and neurodegenerative diseases.Some specific circRNAs can also be used as new diagnostic biomarkers and treatment targets for diseases.Although a benign condition,it shares malignant biological behaviors.However,there is little information available in literatures about the relationship between circRNAs and endometriosis,and there is still a huge gap in the expression and potential regulatory mechanism of circRNAs in endometriosis.As we know,ovarian endometrioma is the most common type of endometriosis,and it often affects the ovarian reserve function of women of reproductive age,leading to infertility.Therefore,we took ovarian endometriosis as a research focus in this study.We aimed to explore circRNA expression profiles using high throughput RNA sequencing in paired ecEM and euEM tissues,combined with the bioinformatics analysis,qRT-PCR,functional assays to find the key circRNAs which regulate the oncogenesis and development of endometriosis.This study may provide a new breakthrough point for etiology research and molecular targeted therapy of endometriosis.Methods:Part ?:High throughput RNA-Seq was used to detect the differentially expressed circRNAs in 6 paired ecEM and euEM tissues of ovarian endometriosis.Based on the fold change of differentially expressed circRNAs,Padj value,readcount,and sequence length,9 circRNAs were selected for qRT-PCR experiments were performed to verify the results of high throughput RNA-Seq.Combined with bioinformatics analysis,construct circRNA-miRNA-mRNA network for certified circRNAs and perform GO and KEGG analyses.Choose one upregulated circRNA,hsa?circ?0020093?named as circATRNL1?for further experiments.Part ?:The Ishikawa cell line was used to replace the primary endometrial epithelial cell line for cellular functional assays.FISH test was used to detect the distribution and subcellular location of circATRNL1 in Ishikawa cells.Construct lentivirus-mediated overexpression and interference sequences targeting circATRNL1 and transfect Ishikawa cells.The effects of circATRNL1 expression level on the biological function of Ishikawa cells were detected by CCK8 assay,cell invasion and migration assays.Western blot and Immunofluorescence assay were used to detect the changes of EMT protein markers.Part ?:miRNA targets of circATRNL1 and mRNA targets of miRNA were predicted by bioinformatics.The binding between circATRNL1 and mi R-141-3p/miR-200a-3p,and the binding between YAP1 and miR-141-3p/miR-200a-3p were verified by dual luciferase activity assays.Expression of miR-141-3p/miR-200a-3p and YAP1 in ecEM and euEM tissues were detected by qRT-PCR,and the correlation with the expression of circATRNL1 was analyzed.Mimics,inhibitors and YAP1 siRNA were constructed,transfected into Ishikawa cells,and the effect of circATRNL1 expression on miR-141-3p/miR-200a-3p and YAP1 was detected by qRT-PCR.The regulatory effect of the circATRNL1-miR-141-3p/miR-200a-3p-YAP1 axis on cell proliferation,invasion,metastasis was detected by CCK8 assay,cell migration and cell invasion assays.The effect of circATRNL1-miR-141-3p/miR-200a-3p-YAP1 axis on EMT protein markers was determined by Western blot.Results:Part ?:In this study,high-throughput RNA-Seq technology was used for the first time to analyze circRNA expression profiles in 6 paired euEM and ecEM tissues in ovarian endometriosis and obtained 294 differentially expressed circRNAs (?log2FC?1?and Padj<0.05),among which 146 upregulated and 148 downregulated.4 out of 9 selected upregulated circRNAs?hsa?circ?0003380,hsa?circ?0020093,hsa?circ?0008016 and hsa?circ?0077837?were confirmed to be significantly overexpressed in ecEM tissues by qRT-PCR?all P<0.05?,which was consistent with sequencing assays.CeRNA networks were constructed for 4 verified circRNAs,and a total of 48 miRNAs and 295 mRNAs were predicted,of which several miRNAs and mRNAs have been reported to regulate endometriosis.Further enrichment of GO and KEGG function analysis found that target genes mainly enriched with a variety of metabolic processes,apoptosis,cell differentiation,protease activity,etc.,and also constituted chromatin,cell membrane,cytoplasm and cell adhesive composition;mainly involved in signaling pathways including PI3K/AKT pathway,cancer pathway and MAPK pathway,microRNAs pathways in cancer,Ras pathway,EGFR tyrosine kinase inhibitors resistance pathway,etc.Finally,hsa?circ?0020093?circATRNL1?was selected as the follow-up research target.Part ?:FISH test results showed that circATRNL1 was widely distributed in Ishikawa cells and mainly located in the cytoplasm.CCK8 results showed that the proliferation rate of Ishikawa cells was significantly increased after transfection with pHBLV-ATRNL1,while the proliferation rate was significantly decreased after transfection with sh1-ATRNL1.The results of cell migration assay showed that pHBLV-ATRNL1transfection enhanced cell migration,while sh1-ATRNL1 significantly inhibited cell migration.Cell invasion results showed that cell invasion was enhanced after pHBLV-ATRNL1 transfection while sh1-ATRNL1 inhibited cell invasion.Western blot results showed that E-cadherin expression was down-regulated and ZEB1,N-cadherin and Vimentin expression were up-regulated in pHBLV-ATRNL1 transfection group.In contrast,E-cadherin expression was up-regulated after sh1-ATRNL1 transfection,while ZEB1,N-cadherin,and Vimentin expression were down-regulated.Cell immunofluorescence results depicted that the fluorescence intensity of E-cadherin was decreased in pHBLV-ATRNL1transfection group,indicating that the expression of E-cadherin was down-regulated,while the fluorescence intensity of N-cadherin was significantly increased,indicating that the up-expression of N-cadherin.The results of sh1-ATRNL1 transfection group were opposite.CircATRNL1 induces EMT phenotype of Ishikawa cells and promotes cell proliferation,migration and invasion.Part ?:Dual luciferase assays confirmed the targeted binding between circATRNL1and miR-141-3p/miR-200a-3p,miR-141-3p/miR-200a-3p and YAP1.The expression of circATRNL1 was negatively correlated with miR-141-3p/miR-200a-3p,while circATRNL1 was positively correlated with YAP1 in ecEM tissues.The results of CCK8 assay,cell migration and invasion assays showed that the overexpression of miR-141-3p/miR-200a-3p inhibited cell proliferation,migration and invasion.Western blot results showed that E-cadherin expression increased after overexpression of miR-141-3p/miR-200a-3p,while N-cadherin,Vimentin and ZEB1 expression decreased.After Ishikawa cells transfected with the corresponding vectors,qRT-PCR results showed that circATRNL1 negatively regulated the expression of miR-141-3p/miR-200a-3p,while indirectly positively regulated the expression of YAP1.Gain and loss of function experiments showed that up-regulation of miR-141-3p/miR-200a-3p expression or silencing of YAP1 expression could reverse the cell proliferation,migration and invasion induced by overexpression of circATRNL1,while the inhibitory effects of knockdown of circATRNL1 on cell biological behaviors were also reversed after transfection of miR-141-3p and mi R-200a-3p inhibitors.Western blot results showed that after overexpression of circATRNL1,E-cadherin expression was down-regulated,ZEB1,N-cadherin and Vimentin expression were up-regulated,and co-transfection of miR-141-3p mimics,miR-200a-3p mimics and YAP1-si#2 reversed the effect of overexpression of circATRNL1 on EMT protein markers.In contrast,E-cadherin expression was up-regulated after circATRNL1 knockdown,while ZEB1,N-cadherin and Vimentin expression were down-regulated,and the effect of miR-141-3p inhibitors and miR-200a-3p inhibitors were also reversed after co-transfection.Conclusion:1.In this study,high-throughput RNA-Seq technology was used for the first time to analyze circRNA expression profiles in 6 paired euEM and ecEM tissues in ovarian endometriosis and obtained 294 differentially expressed circRNAs(?log2FC?1?and Padj<0.05),among which 146 expressions were up-regulated and148 expressions were down-regulated.The high expression of hsa?circ?0003380,hsa?circ?0020093,hsa?circ?0008016 and hsa?circ?0077837 in ecEM tissues was verified by qRT-PCR,which was consistent with the sequencing results.2.Up-regulation of circATRNL1 expression promotes cell proliferation,migration and invasion of Ishikawa cells,inducing EMT phenotype,while knockdown of circATRNL1 inhibits the above biological behaviors and reverses EMT.3.This study confirmed the targeted binding relationship between circATRNL1 and miR-141-3p/miR-200a-3p,miR-141-3p/miR-200a-3p and YAP1.qRT-PCR results showed that circATRNL1 negatively regulated the expression of mi R-141-3p/miR-200a-3p,while indirectly positively regulated the expression of YAP1.Further gain and loss of function assays and Western blot results showed that circATRNL1,as ceRNA,negatively regulates the expression of miR-141-3p and mi R-200a-3p,thereby promoting the high expression of downstream target gene YAP1,promoting cell proliferation,migration,invasion,and inducing EMT. |
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