The Mechanism Of UPF1 Regulating The Occurrence And Development Of Endometriosis By Targeting LncRNA NEAT1

Objective:Endometriosis is one of the common diseases in women,with an incidence of about 11%.It often causes chronic pelvic pain,infertility and constipation.Because of its malignant biological behaviors such as recurrence,tissue invasion,and distant metastasis,it has attracted people's attention.At present,there is no specific diagnosis method for endometriosis,the postoperative recurrence rate of surgical treatment is extremely high,and the effect of drug-assisted treatment is poor.Therefore,in-depth study of the pathophysiological mechanism of its occurrence and development is the key to improving the diagnosis and treatment of endometriosis.The pathogenesis of endometriosis is unknown,and there are many hypotheses,among which menstrual blood reflux is a widely accepted theory.However,although most women have menstrual blood reflux,women with endometriosis account for only a small percentage.It is speculated that some differences between healthy women and women with endometriosis determine the occurrence and development of endometriosis.These factors are including genetics,endocrine,pelvic micro-environment and immunity,etc.Studies have found that compared with healthy women,patients with endometriosis have unique immune factors.These abnormal immune factors enable endometrial fragments to escape immune surveillance(immune escape)when they enter the peritoneal cavity.In addition,disrupted cellular immune responses and related cytokines drive inflammatory networks related to other pathophysiological processes,such as adhesion,invasion,angiogenesis,and proliferation associated with ectopic lesions.lncRNA(long non-coding RNA)is non-coding RNAs with a length of more than200 nucleotides.They participate in the regulation of protein-coding genes by regulating epigenetic,transcription and post-transcriptional levels,and play an important role in human physiological processes and pathological mechanisms.Certain specifically expressed lncRNA could also be used as potential diagnostic markers and new therapeutic targets for diseases.In endometriosis,lncRNA participated in hormone regulation network and signal pathway regulation,and could be used as a potential diagnostic marker,but whether it was involved in the immune disorder of endometriosis,so far,there was no relevant research.Methods:Part I:Differentially expressed lncRNAs in 6 paired of ecEM and euEM tissues in endometriosis were screened by using high-throughput sequencing technology.4 differentially expressed lncRNAs were randomly selected to verify the accuracy of high-throughput sequencing results by qRT-PCR on the enlarged tissue samples.According to the results of high-throughput sequencing,Immu Cell AI was used to analyze the difference of 24 immune cell components in ecEM and euEM tissues.To identify the differentially expressed immune-related lncRNAs,immune-related proteins obtained from IMMPORT were performed co-location(cis-acting)and co-expression(trans-acting)analysis.The main components of immune-related lncRNA in 6 pairs of ecEM and euEM tissues were analyzed.Finally,through bioinformatics analysis,the UPF1-IML-IMP network was constructed,and the UPF1-NEAT1 axis was finally selected for follow-up research.Part II:By using qRT-PCR,the expression level of UPF1 was detected in the ecEM and euEM.The primary euEM cells were extracted,and verified by immunofluorescence.By transfecting UPF1 overexpressing lentivirus and si-UPF1 to euEM cells,the expression of UPF1 was changed.Under different UPF1 expression levels,the proliferation,migration,invasion ability of euEM cells were detected by CCK8 assay,cell scratch assay and Transwell assay,IL-6 secreted by euEM cells was tested by ELISA assay.Part III:By using qRT-PCR,the expression level of NEAT1 was detected in the ecEM and euEM.The correlation with the expression of UPF1 and NEAT1 was also analyzed.Under overexpressing and silencing UPF1,the expression changes of NEAT1were detected by qRT-PCR on euEM cells.The RIP assay verified the combination of UPF1 and NEAT1.By transfecting of si-NEAT1 into euEM cells,the expression of NEAT1 was changed.Under different expression of NEAT1,the regulation of UPF1/NEAT1 axis on cell proliferation,migration,invasion was detected by CCK8assay,cell scratch assay and Transwell assay;ELISA assay was used to test effect of UPF/NEAT1 axis on IL-6 secreted by euEM cells.Under different expression of NEAT1,the effect of UPF1/NEAT1 axis on cell proliferation,migration,invasion were detected by CCK8 assay,cell scratch assay and Transwell assay;the effect of UPF/NEAT1 axis on IL-6 secretion in euEM cells was detected by ELISA assay.Results:Part I:Through high-throughput sequencing analysis of 6 pairs of euEM and ecEM tissues in endometriosis,a total of 952 differentially expressed lncRNAs(?log₂FC?1?and P_adj<0.05)were obtained,of which 475 were up-expressed and 477were down-expressed.qRT-PCR verified the differential expression of lncRNA UCA1,MIR202HG,LINC00261,and GAGA2-AS1 in euEM and ecEM tissues,and the results were consistent with the RNA-seq results and were statistically significant(all P<0.05).Immu Cell AI's analysis of 24 immune cell components showed that 13 types of immune cells euEM and ecEM were significant differences in abundance(down-regulation:CD8 naive,n Treg,Th17,Tcm,B cells,Tgd and CD8 T cells;Up-regulation:Tex,Tfh,DC,monocytes,macrophages and CD4 T cells)(all P<0.05).Through the screening of immune-related proteins obtained at IMMPORT,661 immune-related proteins were identified.Among them,475 immune-related proteins were up-regulated and 186immune-related proteins were down-regulated.The differentially expressed immune-related proteins were analyzed by co-location(cis-acting)and co-expression(trans-acting),and 427 differentially expressed immune-related lncRNAs were identified.Principal component analysis showed that the expression of immune-related lncRNA between euEM and ecEM was clearly differentiated,and the immune-related lncRNA components of the 6 samples in the euEM group were relatively stable,while the ecEM group showed inconsistent immune-related lncRNA components.UPF1 is negatively correlated with the expression of 3 dysregulated immune-related lncRNAs.Among them,NEAT1 has the highest expression abundance,so the UPF1-NEAT1 axis was chosen as the follow-up research pathway.Part II:By using qRT-PCR,it was verified the expression of UPF1 was lower in ecEM tissues than euEM tissues.The results of immunofluorescence showed that the extracted primary euEM cells had the mesenchymal cell marker Vimentin.CCK8 assay results showed that the proliferation rate of euEM cells was significantly reduced after transfection of UPF1 overexpressing lentivirus,while the proliferation rate of cells was significantly increased after transfection of si-UPF1.The results of the cell scratch assay showed that the cell migration ability of UPF1 overexpression lentivirus was reduced after transfection,while the cell migration ability was enhanced after si-UPF1transfection.The results of cell invasion assay also showed that the cell invasion ability decreased after UPF1 overexpression lentivirus transfection,while the cell invasion ability increased after si-UPF1 transfection.The results of ELISA assay showed that IL-6 in the cell supernatant was decreased after UPF1 overexpression lentivirus transfection,while IL-6 in the cell supernatant was increased after si-UPF1 transfection.Part III:qRT-PCR results showed that the expression of NEAT1 is higher in ecEM tissues than euEM tissues,and its expression is negatively correlated with UPF1.After transfection of UPF1 overexpression lentivirus,the expression of NEAT1 decreased,and after transfection of si-UPF1,the expression of NEAT1 increased.RIP assay confirmed that UPF1 combined with NEAT1.The results of CCK8 assay,cell scratch assay and cell invasion assay show that transfection of si-NEAT1 can inhibit the proliferation,migration and invasion behavior of euEM cells,and reduce the level of IL-6 in the cell supernatant;and it can reverse the regulation of UPF1 on the the biological behavior and IL-6 secretion of euEM cells.Conclusion:1.High-throughput sequencing analysis of euEM and ecEM tissues in endometriosis was performed,952 differentially expressed lncRNAs(?log₂FC?1?and P_adj<0.05)were obtained,of which 475 were up-expressed and 477 were down-expressed.The accuracy of RNA-seq results were verified through qRT-PCR.There are differences in the abundance of 13 types of immune cells in euEM and ecEM tissues.By co-location(cis-acting)and co-expression(trans-acting)analysis of lncRNAs,427immune-related lncRNAs were identified.2.UPF1 was low expressed in ecEM tissues.Overexpression of UPF1 inhibited the ability of proliferation,migration,invasion of euEM cells and decreased secretion of IL-6 on euEM cells,while silencing UPF1 promoted proliferation,migration,invasion and secretion of IL-6 on euEM cells.3.NEAT1 was highly expressed in ecEM tissues,and its expression was negatively correlated with UPF1.UPF1 targeted NEAT1 and negatively regulated its expression.Silencing NEAT1 inhibited the ability of proliferation,migration,invasion on euEM cells and decreased secretion of IL-6 on euEM cells,and also can reverse the effect of UPF1 on cell biological behavior and secretion of IL6.UPF1 as an RNA binding protein targeted NEAT1 to regulate the occurrence and development of endometriosis.