The Regulation Of Luman On The Funactionality Of Granulosa Cell And Endometrial Stromal Cell

Luman(also called CREB3 or LZIP)is a member of CREB3(cAMP responsive element-binding)subfamily of the basic leucine-zipper(bZIP)transcription factors.The structure of Luman is considerably similar with ATF6,containing a transmembrane domain associating with the ER,a transcription activation domain,and a bZIP domain.Luman is widely distributed in various reproduction related tissues and organs including mouse ovary,mouse oviduct,mouse uterus,mouse Leydig cells and sperm.Previous studies proved the abundant expression of Luman in the mouse germinal tissues such as ovary and uterus during estrous cycle and peri-implantation.The Luman-deficient mice showed a maternal deficit evident by low pup survival.Thus,we hypothesized that Luman might play an important role in maintaining the functionality of ovary and uterus for reproduction.In this study,we applied RNAi,RT-qPCR,western blot,immunofluorescence,CCK-8,ELISA and flow cytometry techniques to study the role of Luman in the mouse folliculogenesis and decidualization.The following results were obtained:1.Three RNAi sequences targeting Luman gene were designed and used to construct three recombinant lentiviral plasmids named pCD513B-U6-Luman 1,2,and 3.The recombinant lentivirus was packaged and the tier was determined by well-by-dilution titer assay.The final titer of the recombinant shRNA lentivirus was 5-10×10~8 IU/mL.To identify the effect of shLuman lentivirus,NIH 3T3 cells were transduced with the lentiviruses.The western blot results showed that pCD513B-U6-Luman 3 has the highest knock down efficiency compared with other vectors.The expression of Luman in the shLuman group was80%lower compared to the control group.Thus,pCD513B-U6-Luman 3 was used for further experiments.2.We detected the regulatory role of Luman on the cell cycle,apoptosis and steroid hormone biosynthesis in mouse granulosa cells in vitro.After transduction,shLuman down-regulate the expression of the Luman gene about 80%.The concentration of E2 and P4in the granulosa cell culture medium significantly decreased in the shLuman 3 group compared with the shNC group.To further investigate the molecular mechanisms of the decreased E2 and P4,we analyzed the mRNA expression of steroidogenic enzymes and metabolic enzymes including Star,Cyp19a1,and Cyp1b1.Their mRNA expression was significantly decreased by Luman knockdown.We also found that the G1 phase of the cell cycle was significantly increased in the Luman knockdown group compared to that of control,which could be attribute to the increased mRNA expression of Cyclin A1,Cyclin B1,Cyclin D2 and Cyclin E.Apoptosis is the cellular mechanism underlying ovarian follicular atresia.our results showed that Luman knockdown has no significant effects on apoptosis in mouse GCs,as determined via flow cytometry.The key apoptosis related genes were not significantly affected by Luman knockdown at mRNA and protein level.Furthermore,Luman knockdown effects on the expression levels of Has2 and Ptgs2 that played important role in regulating the follicular growth and development.3.Function and regulation mechanism of Luman in the decidualization of endometrial stromal cell(ESC)were investigated by RNA-seq.ESCs were purified from day 4 of pregnant mouse,which were used to establish an in vitro decidualization model.Based on this model,an increased expression of Luman during ESC decidualization was observed.Moreover,Luman silencing significantly inhibits the expression of two decidualization markers,indicating an important role of Luman during decidualization.Knockdown of Luman causes a distinct transcriptome profile with endoplasmic reticulum-related protein processing being the most significantly affected pathway.The expression of bone morphology proteins(BMP),growth factors,as well as other decidulization related genes were altered by Luman knockdown.The expression of several cell cycle related genes was changed by Luman knockdown,and cells were consequently arrested at G1 phase.Gene enrichment and cell signal pathway analysis revealed that ECM-receptor interaction,focal adhesion,endocytosis,and PI3K-Akt signal pathway are the most enriched signal pathways.Moreover,we identified STMN4,CNN2,MLP3B,TRPA1,NPTX2,TRFL,and IOD3 as the potential downstream target gene of Luman.4.ESC were exposed to various concentrations of BPA.The cell viability was determined using a CCK-8 assay,which showed that low concentration of BPA(100 nM)didn't induce significant cell death.However,the expression of decidualization-related genes was affected by at low concentrations BPA.In addation,the upregulagtion of GRP78 protein indicated that ER stress was activated when ESC was exposed to low doses BPA.BPA-induced ERS and downstream effects might be mediated by Luman,since Luman knockdown can eliminate BPA-induced decidual gene expression changes.In conclusion,the above results showed that Luman plays an important role in female mammal reproduction.Our results deciphered the function and regulation mechanism of Luman in the development of follicles and uterine decidualization in mouse,which brings new insight and theretical basis of Luman for further study.