Circadian Gene PER1 Senses Progesterone Signal During Human Endometrial Decidualization

PartⅠ The correlation between infertility induced by EMS and the expression levels of the circadian genes Objective Endometriosis（EMS）is a common and frequently-occurring disease that causes female infertility.It has been proved that infertility caused by EMS is related to biorhythm disorder such as long-term night shift.The regulation of biorhythm is mediated by a kind of protein called biological clock system,which can adapt to the diurnal variation and participate in many physiological processes.Therefore,we aim to clarify the correlation between infertility induced by EMS and the expression levels of core clock genes.Methods We compared the PER1 expression of midluteal endometrial biopsies obtained from 9 women with infertility diagnosed with EMS,a chronic syndrome characterized by infertility due to P₄ resistance and disrupted decidualization,and 10 women with normal fertility.Next,we measured PER1 expression levels in human endometrial biopsy samples from each phase of the menstrual cycle.Using an in vitro decidualization system,the mRNA and protein levels of prolactin（PRL）and insulin-like growth factor-binding protein-1（IGFBP1）,two reliable decidual markers,were measured.Then,we measured the expression levels of 7 core clock genes every 4 h for 32 h in immortalized HESCs in vitro.Results The correlation between infertility induced by EMS and the expression levels of the circadian genes:The endometrial PER1 mRNA expression in EMS patients was significantly lower than that in control women.The PER1 mRNA expression was minimal in the proliferative phase,increased gradually in the early secretory phase and significantly high in the mid-and late-secretory phase of the cycle.The mRNA and protein levels of PER1,PRL and IGFBP1 increased steadily in decidualized immortalized HESCs.Immunofluorescence staining showed that PER1 protein was weakly expressed in the cytoplasm and nuclei of stromal cells;however,it was predominantly expressed in the nuclei of stromal cells after differentiation,consistent with the patterns in decidua cells in early pregnancy in vivo.These core clock genes did not exhibit obvious rhythmic activity in undifferentiated culture,furthermore,they did not significantly flatten in decidualized culture.Conclusion Decreased fertility in EMS patients may be related to attenuated PER1 expression.The upregulation of PER1,rather than other clock genes,is a striking feature of decidualization and may also be involved in the maintenance of decidualization.Circadian gene PER1 may be involved in both the initiation and the maintenance of decidualization.PartⅡ The effect of decreased PER1 on decidualization of human uterine stromal cellsObjective It is well known that the principal change of the endometrium in mid-secretory phase is the decidualization of the stromal cells.Based on the down-regulation of PER1 expression in the endometrium during the mid-secretory phase of the EMS patient with infertility,we hypothesized that this change has resulted in abnormal decidualization of endometrial stromal cells.This part aims to test the hypothesis that the circadian clock gene PER1 could regulate decidua development.Methods Human endometrial samples were collected and stromal cells were isolated and cultured in vivo.To investigate the function of PER1 in decidualization,siRNA-mediated knockdown was performed on primary HESCs.Next,the mRNA and protein levels of PER1,PRL and IGFBP1 were measured,the volumes of fifty cells in siRNA group and control group were determined by using Image J software.In the meantime,a PER1 knockout（PER1-KO）immortalized HESC line was constructed using the CRISPR/Cas9 system,the protein levels of PER1 in control and PER1-KO cell lines were analyzed by western blot analysis.The protein levels of PRL and IGFBP1 in PER1-KO cells after decidualization treatments were analyzed by qRT-PCR.Results Successful establishment of primary stromal cell cultures was confirmed by positive staining for vimentin and negative staining for cytokeratin.The qRT-PCR and Western blot analysis results confirmed that PER1 mRNA and protein levels were significantly lower and these changes were accompanied by severely compromised induction of IGFBP1 and PRL at the mRNA levels,the decidualized cells were also significantly smaller in the knockdown group than in the control group.The loss of the PER1 protein was observed in PER1-KO cells,and the declines in the levels of the FOXO1,PRL,and IGFBP1 proteins were observed in PER1-KO cells,consistent with the results in knockdown cells.Conclusion PER1 deficiency leads to compromised decidualization in human endometrial stromal cells.Part Ⅲ The mechanism of PER1 deficiency attenuated human endometrial decidual transformationObjective Many kinds of growth factors,transcription factors,cytokines are involved in the process of decidualization to promote the decidual cell differentiation.It is not known yet whether these factors are the downstream molecules of the clock gene PER1.Because all the circadian clock proteins are transcription factors,whether PER1 affects the expression of downstream molecules through transcription regulation mechanism remains to be confirmed.Methods We assessed the FOXO1 mRNA and protein levels after PER1 knockdown in primary HESCs followed by decidualization treatments.The protein levels of Akt and phosphorylated Akt,the upstream regulator of FOXO1,were estimated in siCON and siPER1 primary decidualizing cells.PER1-KO and control immortalized HESCs were decidualized followed by treatment with cycloheximide（CHX）,a protein synthesis inhibitor,the protein level of PER1 was measured.The same treatments were implemented with the calpain inhibitor MG101,the proteasome inhibitor MG132,and the autophagy inhibitor 3-methyladenine（3-MA）,the protein level of PER1 was measured.In primary HESCs with PER1 knockdown and FOXO1 overexpression,the protein levels of FOXO1 and IGFBP1 were measured.Results PER1 knockdown in primary HESCs exerted no significant impact on the transcripts of FOXO1but inhibited FOXO1 protein expression.Furthermore,the immunofluorescence staining results showed that FOXO1 was strongly expressed in the nuclei and cytoplasm of decidual cells in the control group after decidualization,but distinctly decreased in both the nuclei and cytoplasm of cells in the siPER1group.The comparable level of Akt and p-Ser473 Akt in both siCON and siPER1groups.The half-life of FOXO1 protein was 1 h shorter in PER1-KO cells than in control cells.Treatment of cells with the calpain inhibitor MG101,the proteasome inhibitor MG132,the autophagy inhibitor 3-MA,or all three compounds revealed that MG101 could efficiently attenuate the rapid degradation of FOXO1 protein in PER1-KO cells,while the combination of the three compounds had no synergistic effect.In primary HESCs with PER1 knockdown and FOXO1 overexpression,the aberrant decidualization was attenuated,and IGFBP1expression levels were rescued.Conclusion PER1 deficiency leads to compromised decidualization at least partly by decreasing FOXO1 protein stability.PER1 regulates FOXO1 protein stability mainly through the calpain-mediated degradation pathway.PartⅣ The mechanism of P₄-PR signaling pathway regulates the expression of PER1Objective Progesterone（P₄）is an important hormone for the process of decidualization.Since the circadian clock system could sense the hormone dynamics for homeostatic regulation of physiological functions,we suspect that the circadian clock gene PER1 may be involved in the regulation of progesterone signaling on decidualization.In this part,we tested the hypothesis that the PER1 could respond to progesterone signaling during human endometrial decidual transformation.Methods To assess the impacts of P₄ and cAMP signaling on PER1 expression,we first treated immortalized HESCs with db-cAMP and MPA separately or in combination,the mRNA level of PER1was measured.Next,we examined whether RU486（a PR inhibitor）could diminish P₄-activated PER1expression.An immortalized PGR knockout（PGR-KO）HESC line was constructed with CRISPR/Cas9technology,the protein levels of PRA/PRB and PER1 in control and PER1-KO cell lines were analyzed by western blot analysis.The mRNA levels of PRL and IGFBP1 in PGR-KO cells after decidualization treatments were analyzed by qRT-PCR.The mRNA level of PER1 in control and PGR-KO cell lines after treatment with MPA were analyzed.The mRNA levels of PGR,DKK1 and HTBS1 in control and PER1-KO cell lines after treatment with MPA were analyzed by qRT-PCR.By using a PR antibody,chromatin immunoprecipitation（ChIP）-PCR analysis was conducted for finding the conspicuous binding of PR at the PER1 promoter sequence,the transcriptional activities of PR on PER1 promoter was performed by dual-luciferase reporter assay.Results The increase in PER1 expression was more pronounced with MPA（a progesterone analogue）than with db-cAMP（a cAMP analogue）,and combined treatment resulted in an additive effect.The PER1mRNA levels were significantly lower in RU486-treated cells than in control cells exposed to MPA alone or to a combination of db-cAMP and MPA.Decreased mRNA expression of PRL and IGFBP1 and a loss of PRA/PRB and PER1 protein in PGR-KO cells.Induction by MPA attenuated the increase in the PER1mRNA level in the MPA-treated group compared to the control group.The PGR mRNA level was not affected in PER1-KO cells after combined db-cAMP and MPA treatment,but the expression levels of the DKK1 and HTBS1,two PR downstream genes have been identified,were significantly decreased.The chromatin immunoprecipitation（ChIP）analysis using a PR antibody showed that decidualization was associated with conspicuous binding of PR at the locus-1780bp to-1644bp and-500bp to-350bp.The dual-luciferase reporter assay confirmed that the activity of the reporter with-500bp to-350bp insert was significantly higher than the control reporter and was actively induced by P₄-PR signaling.Conclusion The expression of PER1 mRNA is regulated mainly by P₄-PR signaling in human endometrial stromal cells.PER1 may be associated with decreased P₄-PR signaling responsiveness.The expression of PER1 is potentially regulated by direct binding of PR to the PER1 promoter sequence.