The Mechanism Of PK2 Affecting Endometrial Decidualization Via LUCAT1-MMP9/TRPM2 Pathway

Successful decidualization of endometrial stromal cells(ESC)is a prerequisite for embryo implantation and pregnancy establishment.Currently,it has been confirmed that impaired decidualization is the key cause of recurrent implantation failure(RIF),but its underlying mechanism remains unclear.Decidualization is a complex biological process involving many biological processes,such as cytoskeleton,extracellular matrix decomposition,cell cycle,angiogenesis,autophagy,inflammatory response,as well as transcriptome,proteome,and metabolome changes.Therefore,the causes and mechanisms that mediate the loss of decidualization are diverse and uncertain.At the moment,though,may cause of decidual damage factors in the targeted therapy to improve embryo implantation rate,but in recent years RIF infertility patients clinical pregnancy rate has not been significantly improved,therefore,looking for and the key control factor of decidual mediated anomaly reveals the in decidual process related molecular mechanism has very important clinical significance.Prokineticin 2(PK2)is a newly discovered multifunctional secreted protein.Studies have shown that PK2 is involved in physiological functions such as cell differentiation autophagy,angiogenesis,circadian rhythm,cell cycle,and inflammatory response.However,the role of PK2 in endometrium remains unclear.Our previous study found that patients with RIF middle luteal endometrium tissue PK2,PKR1 expression level significantly higher than the control group patients,cell experiments showed that overexpression of PK2 slow virus infection endometrial stromal cells in vitro induced decidual change compared to the air carrier decidua group,decidual cell morphology change is not obvious,and decidual markers PRL and IGFBP1 expression levels did not rise,has shown that increased PK2 expression can lead to endometrial decidual process.RNA Sequencing(RNA-sequencing,RNA-seq)is the transcriptome Sequencing technology,which can analyze mRNA,smallRNA,NONcoding RNA,etc.,or some of them by high-throughput Sequencing technology to reflect their expression level.Through RNA-seq,this research group obtained the lncRNA and mRNA expression profiles of PK2 overexpressed decidualization and normal decidualization stromal cells,aiming to study the molecular mechanism of PK2’s regulation of decidualization through transcriptome.This study based on the early stage of the research of RNA-seq technology to detect IncRNA and mRNA expression of the spectral changes,combined with bioinformatics analysis technology,selected to participate in decidual PK2 regulation process of target genes lncRNA and mRNA,validation of mRNA in primary endometrial cells and related protein expression level,and build the control air carrier+not decidualized chemical group,control group empty carrier+decidualized,overexpression PK2+decidualized group,PK2-siRNA+decidualized lentivirus transfection of cell model,further define the objective target genes and explore the molecular mechanism of PK2 regulating decidualization process.Objective:1.According to RNA-seq results and bioinformatics analysis techniques,the target genes involved in PK2’s regulation of decidualization process were screened out preliminarily;2.Verify the expression of target genes in primary cells,and the decidualization model of lentivirus transfection was constructed to further determine the target genes,and the molecular mechanism of PK2 regulating the decidualization process was exploredMethods:(1)pregnancy and met the standards of NF from January 2019 to January 2020 in Reproduction Medicine Center of the First Affiliated Hospital of Zhengzhou University were screened.Endometrium in the middle luteal phase 7 days after the LH peak,was collected.(2)According to the differential lncRNA and mRNA obtained by RNA-seq,combined with biological analysis techniques and existing studies,the genes that may be transcriptional regulation of PK2 were screened out.(3)RT-qPCR was used to analyze the expression levels of LUCAT1,RAMP2-AS1,AL034397.3,mir199a-5p,MMP9,TRPM2,CDKN1C,ULK1,and NLRP3 in the endometrial tissues of middle luteal phase in 2 groups.(4)The expression levels of MMP9,TRPM2,LC3B in the endometrium tissues of the middle luteal phase of the two groups were analyzed by Western-blotting(5)The expression levels of LUCAT1,MMP9,TRPM2,LC3B in normal decuvialization were analyzed by RT-qPCR and Western-blotting.(6)The decidualization models of lentivirus transfection were constructed by empty vector+non-decidualization group in control group,empty vector+decidualization group in control group,overexpression of PK2+decidualization group,and PK2-siRNA+decidualization group.RT-qPCR and Western-blotting were used to verify the influence of PK2 overexpression or knockdown on the expression levels of LUCAT1,MMP9,TRPM2,and autophagy markers LC3B.Results:1.Combined with informatics analysis techniques,PK2 was preliminarily screened out as a possible pathway of LUCAT1/RAMP2-AS1-MMP9,AL034397.3 mir199a-5p-CDKN1C,TRPM2-autophagy(ULK1)/NLRP3 pathway regulates decidualization;2.RT-qPCR results showed that the expression level of lncRNA-LUCAT1 in RIF group was significantly higher than that in Control group,while the expression levels of mRNA MMP9 and TRPM2 were significantly lower than that in Control group(P<0.05);3.Western-blotting showed that the protein expression levels of MMP9,TRPM2 and LC3B in RIF group were lower than those in control group;4.During normal decidualization,the mRNA expression of LUCAT1 decreased slightly.the mRNA and protein expression levels of MMP9 and TRPM2 increased,and the protein expression levels of LC3B increased;5.RT-qPCR and Western-blotting showed that the mRNA expression level of LUCAT1 in PK2+decidualized group and non-decidualized group was higher than that in PK2-siRNA+decidualized group and normal decidualized group,and the mRNA and protein expression levels of MMP9 and TRPM2 were significantly lower than those in the other two groups(P<0.05);6.Western-blotting showed that the expression level of LC3B protein in PK2+decidualized group and non-decidualized group was lower than that in PK2-siRNA+decidualized group and normal decidualized group(P<0.05).Conclusions:1.There was insufficient expression of MMP9 in endometrial stromal cells of RIF patients,and autophagy was impaired;2.During normal decidualization,TRPM2 and MMP9 showed an upward trend,while LUCAT1 showed a downward trend.Autophagy of stromal cells was enhanced.3.PK2 overexpression can inhibite autophagy or MMP9 expression;4.PK2-LUCAT1-MMP9 or PK2-TRPM2-autophagy pathway may be involved in the regulation of endometrial endometriosis in RIF patients.