| Background:The phenomenon of population aging is a major problem of the development of the world's population.Increasing the birth rate is one of the effective means to alleviate the aging of the population.In China,with the full liberalization of the two-child policy,the number of families with two children's reproductive claims has gradually increased,but according to statistics,one in six couples is infertile,and 25%of them are classified as unexplained infertile.Assistant reproductive technology?ART?is the main means of treating infertility.Although the development of human assisted reproductive technology is changed quickly,there are still some unresolved problems in the assisted reproductive cycle that plague clinicians and patients.Embryo implantation failure is still a major limiting factor:only about 25%of transplanted blastocysts can be successfully implanted[1].The process of implanting the blastocyst into the endometrium is called implantation or nidation.Embryo implantation is a key part of pregnancy success during human natural pregnancy cycles or assisted reproductive cycle.The two most important factors in embryo implantation are the quality of the embryo and the endometrial receptivity.Endometrial receptivity refer to a state that the endometrium,under the combined action of ovarian sex hormones and other various factors,exhibits a certain time to allow embryo positioning,adhesion,invasion and decidualization of endometrial stromal cells.Clinically,endometrial thickness and morphology are commonly used as non-invasive evaluation criteria for endometrial receptivity.The study found that endometrial thickness<7mm has a negative impact on pregnancy rate,the thinner the endometrial thickness,the lower the implantation rate and pregnancy rate,the higher the risk of miscarriage[2,3].It is generally believed that the thinner the endometrium,the lower the IVF-ICSI pregnancy rate.Endometrial receptivity changes in patients with thin endometrium due to decreased endometrial thickness are one of the important causes of embryo failure and reduced pregnancy rate in ART[2,5],therefore,improve those patient's endometrial thickness is important for achieving good pregnancy outcomes.Although there are many treatments for thin endometrium,there is no recognized effective treatment.Therefore,we need to find more effective and safe treatment methods on the basis of a large number of studies.FK506 binding protein 52?FKBP52?belongs to the immunophilin family and regulates the function of steroid hormones such as progesterone and androgen by participating in the formation of steroid hormone receptor complex.In addition,its expression is also affected by steroids's Feedback regulation of hormones.Studies have shown that FKBP52 plays a certain role in pregnancy.For example,mice that knock out FKBP52 gene have normal ovulation function,but blastocysts cannot be implanted[6];Compared with the normal control group,the serum level of FKBP52 in pregnant women with threatened abortion was significantly decreased,and the level of FKBP52 was significantly increased after placenta intervention;the expression of FKBP52 in the villus tissue of patients with recurrent spontaneous abortion was significantly reduced[7].The expression level of FKBP52 is also associated with cell proliferation such as breast cancer cells and prostate cancer cells.Endometrial stromal cells are the main components of endometrium.Their proliferation is regulated by sex hormones.Endometrial stromal cells also play a key role in and after embryo implantation.Compared with endometrial epithelial cells,endometrial stromal cells can maintain cell division activity in vitro for a long time and can be used for cell passage,which provides a possibility for further study.Therefore,human endometrial stromal cells were selected as subjects.However,the proliferation of human endometrial stromal cells?HESCs?by FKBP52 and its mechanism are still unclear.This study intends to provide a new experimental basis and ideas for the search for a new therapeutic target for thin endometrium by studying the role of FKBP52 in the proliferation of human normal endometrial stromal cells.Objective:To investigate the effect of FK506 binding protein 52?FKBP52?on the proliferation of Human endometrial stromal cells?HESCs?,aim to provide a basis and potentail target for the treatment of thin endometrium.Methods:1.Isolation and culture of primary cells:Collect human endometrial tissue scraped under hysteroscopy and extract HESCs and culture according to the method in reference[8,9].2.Immunocytochemistry:Immunocytochemistry was used to detect the expression of vimentin,cytokeratin 8+18?Cytokeratin 8+18,CK8+18?and identify HESCs.3.Lentivirus infection:HESCs were infected with FKBP52 upregulated lentivirus,and the mRNA level of FKBP52 and protein expression level of FKBP52 in cells was detected by real-time fluorescence quantitative PCR and Wetern blotting technology respectively,so as to determine the infection effect of lentivirus.4.Small interfering RNA?siRNA?transfection:HESCs were transfected with targeted silence FKBP52 small interference RNA,then the mRNA level and protein expression level of FKBP52 in cells was detected by real-time fluorescence quantitative PCR and Wetern blotting technology respectively,so as to determine the transfection effect of small interfering RNA transfection efficiency.5.CCK8 experiment:the proliferation ability of HESCs was detected by CCK8 experiment.6.Plate clone formation assay:The colony-formation ability of HESCs was detect by plate clone formation assay.7.Flow cytometry:flow cytometry was used to detect HESCs cell cycle.8.Western blotting:Western blotting was used to detect the protein expression levels of FKBP52and Cell cycle-related protein.Results:1.Identification of HESCs:The morphology of cells was consistent with HESCs under inverted phase contrast microscopy.The results of immunocytochemistry showed that the expression of interstitial cell marker protein vimentin was high?98.08%±0.95%?,while the glandular epithelial marker protein CK8+18 was almost not expressed.The HESCs were successfully separated.2.Establishment of HESCs FKBP52 overexpression and knockdown cell model:HESCs were infected with lentiviral vector overexpressing FKBP52.Real-time PCR showed that the level of FKBP52 mRNA in cells increased after up-regulation of FKBP52?P=0.009?.Western Blotting showed that the protein expression level of FKBP52 was significantly increased in cells?P<0.001?.After transfection of HESCs with siRNA targeting FKBP52,real-time PCR showed that the mRNA level of FKBP52 was decreased?P=0.001?,Western Blotting results showed that the protein expression level of FKBP52 was significantly decreased?p<0.001?.A cell model indicating that FKBP52 overexpression and interference was constructed was successful.3.The effect of FKBP52 expression on the proliferation of HESCs cells:CCK8 results showed that the proliferation of HESCs was significantly increased after up-regulated FKBP52?P<0.05?.After down-regulated FKBP52,the cell proliferation rate decreased significantly?P<0.05?.The results of plate clone formation assay showed that the ability of cell clonogenesis was significantly enhanced after up-regulated of FKBP52?P<0.001?.When FKBP52 was down-regulated,the ability of cell cloning was decreased?P<0.05?,indicating that FKBP52 could promote the proliferation of HESCs cells4.Effect of FKBP52 expression level on HESCs Cell Cycle:After overexpress FKBP52,cell cycle progression is accelerated?P<0.001?,cell cycle-dependent Kinase 4?CDK4?,and CyclinD1?CyclinD1?.Dependent Kinase 2?CDK2?and CyclinE1?CyclinE1?protein expression levels were all increased?P<0.05?.Affected by FKBP52 knockdown in transfected cell,cell cycle progression was blocked?P<0.05?,and expressions of all proteins,including CDK4,CyclinD1,CDK2 and CyclinE1,were suppressed?P<0.05?Conclusion:FKBP52 can promote the G1/S transition of HESCs by up-regulating the expression of cell cycle factor CDK4 cyclinD1 CDK2 CyclinE1,and then promote the proliferation of HESCs,which may be a potential target for the treatment of thin endometrium. |
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