FK506结合蛋白52对人子宫内膜间质细胞增殖作用的影响
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摘要
目的探讨FK506结合蛋白52(FKBP52)对人子宫内膜间质细胞(HESCs)增殖的影响,为薄型子宫内膜的治疗提供新的依据和靶点。方法分离并培养原代HESCs,分别感染过表达FKBP52的慢病毒和转染靶向沉默FKBP52的小干扰RNA(siRNA),CCK8法检测细胞增殖活性,平板克隆形成实验检测细胞克隆形成能力,流式细胞分析法检测细胞周期的变化,Western blotting法检测FKBP52及细胞周期相关蛋白表达水平。结果过表达FKBP52后,子宫内膜间质细胞的增殖活性增强(P <0. 05),细胞克隆形成能力提高(P <0. 01),细胞周期进程加快(P <0. 01),细胞周期素依赖激酶4(CDK4)、细胞周期蛋白D1(Cyclin D1)、细胞周期素依赖激酶2(CDK2)及细胞周期蛋白E1(Cyclin E1)的蛋白表达水平均升高(P <0. 05);干扰FKBP52后,子宫内膜间质细胞的增殖能力减弱(P <0. 05),细胞克隆形成能力降低(P <0. 05),细胞周期进程受阻滞(P <0. 05),CDK4、Cyclin D1、CDK2、Cy-clin E1的表达均被抑制(P <0. 05)。结论 FKBP52可通过促进HESCs的细胞周期进程来促进HESCs的增殖,有可能作为薄型子宫内膜治疗的靶点。
Objective To explore the effects of FK506 binding protein 52( FKBP52) on proliferation of human endometrial stromal cells( HESCs),and provide the new basis and target for treatment of thin endometrium. Methods Normal HESCs were isolated and cultured. After infecting HESCs with lentiviruses contain GFP-FKBP52 or transfecting HESCs with FKBP52-targeting small interfering RNA( siRNA) to overexpress or knockdown FKBP52 respectively,the cells proliferation and colony-formation were compared with CCK8 assay and plate clone formation assay. Besides,changes in cell cycle were detected by flowcytometry and expression levels of FKBP52 and cell cycle-related proteins were examined by Western blotting. Results After overexpressing FKBP52,the proliferation activity of HESCs was enhanced( P< 0. 05),and the clone formation ability of cells was improved( P < 0. 01). The process of cell cycle was accelerated( P < 0. 01) and the expression levels of cell cycle dependent kinase 4( CDK4),CyclinD1,cell cycle dependent kinase2( CDK2) and CyclinE1 were increased( P < 0. 05). Affected by FKBP52 knockdown in transfected cell,the proliferation capacity of endometrial stromal cells was reduced( P < 0. 05),and the clone formation capacity was weakened( P< 0. 05). The cell cycle process was blocked( P < 0. 05) and expression levels of CDK4,CyclinD1,CDK2 and CyclinE1 were suppressed( P < 0. 05). Conclusion FKBP52 can control the proliferation of HESCs by regulating the cell cycle and can be considered as a target for the treatment of thin endometrium.
Objective To explore the effects of FK506 binding protein 52( FKBP52) on proliferation of human endometrial stromal cells( HESCs),and provide the new basis and target for treatment of thin endometrium. Methods Normal HESCs were isolated and cultured. After infecting HESCs with lentiviruses contain GFP-FKBP52 or transfecting HESCs with FKBP52-targeting small interfering RNA( siRNA) to overexpress or knockdown FKBP52 respectively,the cells proliferation and colony-formation were compared with CCK8 assay and plate clone formation assay. Besides,changes in cell cycle were detected by flowcytometry and expression levels of FKBP52 and cell cycle-related proteins were examined by Western blotting. Results After overexpressing FKBP52,the proliferation activity of HESCs was enhanced( P< 0. 05),and the clone formation ability of cells was improved( P < 0. 01). The process of cell cycle was accelerated( P < 0. 01) and the expression levels of cell cycle dependent kinase 4( CDK4),CyclinD1,cell cycle dependent kinase2( CDK2) and CyclinE1 were increased( P < 0. 05). Affected by FKBP52 knockdown in transfected cell,the proliferation capacity of endometrial stromal cells was reduced( P < 0. 05),and the clone formation capacity was weakened( P< 0. 05). The cell cycle process was blocked( P < 0. 05) and expression levels of CDK4,CyclinD1,CDK2 and CyclinE1 were suppressed( P < 0. 05). Conclusion FKBP52 can control the proliferation of HESCs by regulating the cell cycle and can be considered as a target for the treatment of thin endometrium.
引文
[1] Zhu H,Hou CC,Luo LF,et al. Endometrial stromalcells and decidualized stromal cells:origins,transforma-tion and functions[J]. Gene,2014,551(1):1-14.
[2]Salamonsen LA,Nie GY,Hannan NJ,et al. Society forReproductive Biology Founders'Lecture 2009. Preparingfertile soil:the importance of endometrial receptivity[J].Reprod Fertil Dev,2009,21(7):923-934.
[3]Kader MA,Abdelmeged A,Mahran A,et al. The use-fulness of endometrial thickness,morphology and vascu-lature by 2D Doppler ultrasound in prediction of pregnan-cy in IVF/ICSI cycles[J]. The Egyptian Journal of Radi-ology and Nuclear M edicine,2016,47(1):341-346.
[4]Zhang T,Li Z,Ren XL,et al. Endometrial thickness asa predictor of the reproductive outcomes in fresh and fro-zen embryo transfer cycles:a retrospective cohort studyof 1512 IVF cycles w ith morphologically good-qualityblastocyst[J]. M edicine(Baltimore),2018,97(4):e9689. doi:10. 1097/M D. 0000000000009689.
[5]Riad ON,Hak AA. Assessment of endometrial receptivityusing Doppler ultrasonography in infertile w omen under-going intrauterine insemination[J]. Gynecol Endocrinol,2014,30(1):70-73.
[6]Aydin T,Kara M,Nurettin T. Relationship between en-dometrial thickness and in vitro fertilization-intracytoplas-mic sperm injection outcome[J]. Int J Fertil Steril,2013,7(1):29-34.
[7]Liu SM,Zhou YZ,Wang HB,et al. Factors associatedw ith effectiveness of treatment and reproductive outcomesin patients w ith thin endometrium undergoing estrogentreatment[J]. Chin M ed J,2015,128(23):3173-3177.
[8]Eftekhar M,Tabibnejad N,Tabatabaie AA. The thin en-dometrium in assisted reproductive technology:an ongoingchallenge[J]. M iddle East Fertility Society Journal,2018,23(1):1-7.
[9]Bedaiwy MA,Abdelaleem MA,Hussein M,et al. Hor-monal,follicular and endometrial dynamics in letrozole-treated versus natural cycles in patients undergoing con-trolled ovarian stimulation[J]. Reprod Biol Endocrinol,2011,9:83. doi:10. 1186/1477-7827-9-83.
[10]Young SL. Oestrogen and progesterone action on endo-metrium:a translational approach to understanding endo-metrial receptivity[J]. Reprod Biomed Online,2013,27(5):497-505.
[11]俞凌,王淑芳,叶明侠,等.薄型子宫内膜治疗新进展[J].国际生殖健康/计划生育杂志,2016,35(2):165-169.YU Ling,WANG Shufang,YE M ingxia,et al. Treat-ment of thin endometrium:a brief review[J]. Journal ofInternational Reproductive Health/Family Planning,2016,35(2):165-169.
[12]Guy NC,Garcia YA,Cox MB. Therapeutic targeting ofthe FKBP52 co-chaperone in steroid hormone receptor-regulated physiology and disease[J]. Curr M ol Pharma-col,2015,9(2):109-125.
[13]Hong CQ,Li T,Zhang F,et al. Elevated FKBP52 ex-pression indicates a poor outcome in patients w ith breastcancer[J]. Oncol Lett,2017,14(5):5379-5385.
[14]Daikoku T,Tranguch S,Friedman DB,et al. Proteomicanalysis identifies immunophilin FK506 binding protein 4(FKBP52)as a downstream target of Hoxa10 in theperiimplantation mouse uterus[J]. M ol Endocrinol,2005,19(3):683-697.
[15]Chen JC,Roan NR. Isolation and culture of human en-dometrial epithelial cells and stromal fibroblasts[J]. BioProtoc,2015,5(20). pii:e1623.
[16]Matthews CJ,Redfern CP,Hirst BH,et al. Character-ization of human purified epithelial and stromal cellsfrom endometrium and endometriosis in tissue culture[J]. Fertil Steril,1992,57(5):990-997.
[17]Kim SM,Kim JS. A review of mechanisms of implanta-tion[J]. Dev Reprod,2017,21(4):351-359.
[18]Wang W,Taylor RN,Bagchi IC,et al. Regulation ofhuman endometrial stromal proliferation and differentia-tion by C/EBPβinvolves cyclin E-cdk2 and STAT3[J].M ol Endocrinol,2012,26(12):2016-2030.
[19]Bhowal A,Majumder S,Ghosh S,et al. Pathway-basedexpression profiling of benign prostatic hyperplasia andprostate cancer delineates an immunophilin molecule as-sociated w ith cancer progression[J]. Sci Rep,2017,7(1):9763. doi:10. 1038/s41598-017-10068-9.
[20]Wolf E,Lin CY,Eilers M,et al. Taming of the beast:shaping M yc-dependent amplification[J]. Trends CellBiol,2015,25(4):241-248.
[21]Zhang DD,Qi JP,Liu R,et al. c-Myc plays a key rolein TADs-induced apoptosis and cell cycle arrest in humanhepatocellular carcinoma cells[J]. Am J Cancer Res,2015,5(3):1076-1088.
[22]Storer Samaniego C,Suh JH,Chattopadhyay A,et al.The FKBP52 cochaperone acts in synergy w ithβ-cateninto potentiate androgen receptor signaling[J]. PLoS One,2015,10(7):e0134015. doi:10. 1371/journal. pone.0134015.
[23]Kumar P,Mark PJ,Ward BK,et al. Estradiol-regulatedexpression of the immunophilins cyclophilin 40 and FK-BP52 in M CF-7 breast cancer cells[J]. Biochem Bio-phys Res Commun,2001,284(1):219-225.
[24]Jeong YY,Her J,Oh SY,et al. Hsp90-binding immu-nophilin FKBP52 modulates telomerase activity by pro-moting the cytoplasmic retrotransport of h TERT[J].Biochem J,2016,473(20):3517-3532.
[25] Fu XJ,Li HX,Yang K,et al. The important tumorsuppressor role of PER1 in regulating the cyclin-CDK-CKI netw ork in SCC15 human oral squamous cell carci-noma cells[J]. Onco Targets Ther,2016,9:2237-2245. doi:10. 2147/OTT. S100952.
[26]Havens CG,Ho A,Yoshioka N,et al. Regulation oflate G1/S phase transition and APC Cdh1 by reactive ox-ygen species[J]. M ol Cell Biol,2006,26(12):4701-4711.
[27]陈希彦,王琪,顾伟亭,等.干扰TAZ基因对舌鳞癌细胞CAL27增殖凋亡的影响及其机制[J].山东大学学报(医学版),2018,56(10):79-85.CHEN Xiyan,WANG Qi,GU Weiting,et al. Effectsof TAZ knockdow n on the proliferation and apoptosis ofTSCC cell line CAL27 and the molecular mechanism[J]. Journal of Shandong University(Health Science),2018,56(10):79-85.
[28]Satoh T,Kaida D. Upregulation of p27 cyclin-dependentkinase inhibitor and a C-terminus truncated form of p27contributes to G1 phase arrest[J]. Sci Rep,2016,6:27829. doi:10. 1038/srep27829.
[2]Salamonsen LA,Nie GY,Hannan NJ,et al. Society forReproductive Biology Founders'Lecture 2009. Preparingfertile soil:the importance of endometrial receptivity[J].Reprod Fertil Dev,2009,21(7):923-934.
[3]Kader MA,Abdelmeged A,Mahran A,et al. The use-fulness of endometrial thickness,morphology and vascu-lature by 2D Doppler ultrasound in prediction of pregnan-cy in IVF/ICSI cycles[J]. The Egyptian Journal of Radi-ology and Nuclear M edicine,2016,47(1):341-346.
[4]Zhang T,Li Z,Ren XL,et al. Endometrial thickness asa predictor of the reproductive outcomes in fresh and fro-zen embryo transfer cycles:a retrospective cohort studyof 1512 IVF cycles w ith morphologically good-qualityblastocyst[J]. M edicine(Baltimore),2018,97(4):e9689. doi:10. 1097/M D. 0000000000009689.
[5]Riad ON,Hak AA. Assessment of endometrial receptivityusing Doppler ultrasonography in infertile w omen under-going intrauterine insemination[J]. Gynecol Endocrinol,2014,30(1):70-73.
[6]Aydin T,Kara M,Nurettin T. Relationship between en-dometrial thickness and in vitro fertilization-intracytoplas-mic sperm injection outcome[J]. Int J Fertil Steril,2013,7(1):29-34.
[7]Liu SM,Zhou YZ,Wang HB,et al. Factors associatedw ith effectiveness of treatment and reproductive outcomesin patients w ith thin endometrium undergoing estrogentreatment[J]. Chin M ed J,2015,128(23):3173-3177.
[8]Eftekhar M,Tabibnejad N,Tabatabaie AA. The thin en-dometrium in assisted reproductive technology:an ongoingchallenge[J]. M iddle East Fertility Society Journal,2018,23(1):1-7.
[9]Bedaiwy MA,Abdelaleem MA,Hussein M,et al. Hor-monal,follicular and endometrial dynamics in letrozole-treated versus natural cycles in patients undergoing con-trolled ovarian stimulation[J]. Reprod Biol Endocrinol,2011,9:83. doi:10. 1186/1477-7827-9-83.
[10]Young SL. Oestrogen and progesterone action on endo-metrium:a translational approach to understanding endo-metrial receptivity[J]. Reprod Biomed Online,2013,27(5):497-505.
[11]俞凌,王淑芳,叶明侠,等.薄型子宫内膜治疗新进展[J].国际生殖健康/计划生育杂志,2016,35(2):165-169.YU Ling,WANG Shufang,YE M ingxia,et al. Treat-ment of thin endometrium:a brief review[J]. Journal ofInternational Reproductive Health/Family Planning,2016,35(2):165-169.
[12]Guy NC,Garcia YA,Cox MB. Therapeutic targeting ofthe FKBP52 co-chaperone in steroid hormone receptor-regulated physiology and disease[J]. Curr M ol Pharma-col,2015,9(2):109-125.
[13]Hong CQ,Li T,Zhang F,et al. Elevated FKBP52 ex-pression indicates a poor outcome in patients w ith breastcancer[J]. Oncol Lett,2017,14(5):5379-5385.
[14]Daikoku T,Tranguch S,Friedman DB,et al. Proteomicanalysis identifies immunophilin FK506 binding protein 4(FKBP52)as a downstream target of Hoxa10 in theperiimplantation mouse uterus[J]. M ol Endocrinol,2005,19(3):683-697.
[15]Chen JC,Roan NR. Isolation and culture of human en-dometrial epithelial cells and stromal fibroblasts[J]. BioProtoc,2015,5(20). pii:e1623.
[16]Matthews CJ,Redfern CP,Hirst BH,et al. Character-ization of human purified epithelial and stromal cellsfrom endometrium and endometriosis in tissue culture[J]. Fertil Steril,1992,57(5):990-997.
[17]Kim SM,Kim JS. A review of mechanisms of implanta-tion[J]. Dev Reprod,2017,21(4):351-359.
[18]Wang W,Taylor RN,Bagchi IC,et al. Regulation ofhuman endometrial stromal proliferation and differentia-tion by C/EBPβinvolves cyclin E-cdk2 and STAT3[J].M ol Endocrinol,2012,26(12):2016-2030.
[19]Bhowal A,Majumder S,Ghosh S,et al. Pathway-basedexpression profiling of benign prostatic hyperplasia andprostate cancer delineates an immunophilin molecule as-sociated w ith cancer progression[J]. Sci Rep,2017,7(1):9763. doi:10. 1038/s41598-017-10068-9.
[20]Wolf E,Lin CY,Eilers M,et al. Taming of the beast:shaping M yc-dependent amplification[J]. Trends CellBiol,2015,25(4):241-248.
[21]Zhang DD,Qi JP,Liu R,et al. c-Myc plays a key rolein TADs-induced apoptosis and cell cycle arrest in humanhepatocellular carcinoma cells[J]. Am J Cancer Res,2015,5(3):1076-1088.
[22]Storer Samaniego C,Suh JH,Chattopadhyay A,et al.The FKBP52 cochaperone acts in synergy w ithβ-cateninto potentiate androgen receptor signaling[J]. PLoS One,2015,10(7):e0134015. doi:10. 1371/journal. pone.0134015.
[23]Kumar P,Mark PJ,Ward BK,et al. Estradiol-regulatedexpression of the immunophilins cyclophilin 40 and FK-BP52 in M CF-7 breast cancer cells[J]. Biochem Bio-phys Res Commun,2001,284(1):219-225.
[24]Jeong YY,Her J,Oh SY,et al. Hsp90-binding immu-nophilin FKBP52 modulates telomerase activity by pro-moting the cytoplasmic retrotransport of h TERT[J].Biochem J,2016,473(20):3517-3532.
[25] Fu XJ,Li HX,Yang K,et al. The important tumorsuppressor role of PER1 in regulating the cyclin-CDK-CKI netw ork in SCC15 human oral squamous cell carci-noma cells[J]. Onco Targets Ther,2016,9:2237-2245. doi:10. 2147/OTT. S100952.
[26]Havens CG,Ho A,Yoshioka N,et al. Regulation oflate G1/S phase transition and APC Cdh1 by reactive ox-ygen species[J]. M ol Cell Biol,2006,26(12):4701-4711.
[27]陈希彦,王琪,顾伟亭,等.干扰TAZ基因对舌鳞癌细胞CAL27增殖凋亡的影响及其机制[J].山东大学学报(医学版),2018,56(10):79-85.CHEN Xiyan,WANG Qi,GU Weiting,et al. Effectsof TAZ knockdow n on the proliferation and apoptosis ofTSCC cell line CAL27 and the molecular mechanism[J]. Journal of Shandong University(Health Science),2018,56(10):79-85.
[28]Satoh T,Kaida D. Upregulation of p27 cyclin-dependentkinase inhibitor and a C-terminus truncated form of p27contributes to G1 phase arrest[J]. Sci Rep,2016,6:27829. doi:10. 1038/srep27829.