Study On The Effect Of PR-M On The Proliferation And Apoptosis Of Uterine Fibroids By Regulating The Bcl-2/Beclin1 Pathway

BackgroundUterine fibroids are the most common benign uterine smooth muscle tumors in pregnant women,and the incidence rate is higher than other gynecological oncology.Although surgery has been reported as an effective treatment,it still causes economic loss to most patients.As a new type of progesterone receptor,progesterone receptor(PR-M)is found in the outer membrane of mitochondria in recent years.Previous studies has been found that progesterone could up-regulate mitochondrial membrane potential to promote abnormal proliferation of uterine smooth muscle cells through PR-M,but its mechanism is currently unclear.Progesterone and its receptor may participate in the development of cancer by regulating apoptosis-related factor Bcl-2 or autophagy-related factor Beclin1.Besides,progesterone can promote the proliferation of uterine leiomyoma cells and inhibit their apoptosis by up-regulating Bcl-2 and down-regulating the expression of Caspase-3 through PR-M.In contrast,progesterone receptor antagonists can hinder this effect.These studies indicate that PR-M and Bcl-2 are closely related to the development of uterine leiomyoma.Therefore,whether PR-M can affect the behavior of uterine leiomyoma cells by regulating the Bcl-2/Beclin1 axis is the focus of this study.Part 1 Expression characteristics of PR-M and Bcl-2 in clinical uterine fibroidsObjectiveThis study is aimed to investigate the changes in the expression of PR-M and Bcl-2 in the clinical samples of uterine fibroids after laparotomy,and analyze the correlation between the expression levels of the two factors and the volume of uterine fibroids or the age of patients,to confirm further the possible role of PR-M and Bcl-2 in the development of uterine fibroids.Methods1.Collection of uterine fibroids specimenTwenty patients who received open myomectomy for pure uterine fibroids in the Third Affiliated Hospital of Zhengzhou University(Zhengzhou,China)from July 2016 to January 2018 were selected to participate in this study.The included patients ranged in age from 31 to 50 years old,with an average age of 40.56±6.41.The hysteromyoma tissue was removed and stored for later use.2.Determination of mRNA expression levelReal-time PCR was used to detect the mRNA expression levels of PR-M and Bcl-2 in uterine fibroids and normal tissues,respectively.3.Immunohistochemical stainingThe expression of PR-M and Bcl-2 in uterine fibroids and normal tissues was detected by immunohistochemistry.Results1.The relative expression levels of PR-M gene(4.74±2.21)and Bcl-2 gene(5.70±3.18)in the uterine fibroids group were significantly higher than those in the normal control group(2.18±1.16 and 1.25±0.57 respectively,P<0.05)Moreover,the expression of PR-M was mainly distributed in the cytoplasm,while the expression of Bcl-2 was concentrated primarily on the nucleus.2.There was no significant correlation between PR-M and Bcl-2 in uterine fibroids and the age of the patient(P=0.6096 and 0.6922 respectively).Still,there was a significant correlation between the expression levels of PR-M and Bcl-2 and the tumor size in the patients with high expression of PR-M and Bcl-2.ConclusionBoth PR-M and Bcl-2 were highly expressed in uterine fibroids,and their expression levels were related to tumor size.Part 2 Effect of PR-M on autophagy and apoptosis of PR-M(+)cells and the regulation of Bcl-2/BeclinlObjectiveIn this study,PR-M(+)cell lines were transfected with overexpression of PR-M or plasmid knockout to detect changes in cell proliferation activity,autophagy ability and apoptosis level,and the role and mechanism of PR-M in the development of uterine fibroids were explored,which will provide a new research idea for the diagnosis and treatment of uterine fibroids.Methods1.Isolation,culture and identification of uterine fibroid cellsThe uterine fibroids were isolated,and the primary uterine leiomyoma cells were extracted by enzymatic hydrolysis,and then the primary uterine leiomyoma cells were isolated,cultured and identified.2.Primary PR-M(+)uterine fibroid cells were transfected and groupedLentivirus vectors were constructed and divided into the following categories according to different transfections:Blank control group(without transfection),pSIH1-H1-copGFP-sh-NC transfection group(sh-NC,lentivirus transfection unrelated shRNA sequence),pSIHl-Hl-copGFP-sh-PR-M transfection group(sh-PR-M),PLV-EGFP-N transfection group(oe-NC,transfected with empty vector),pLV-EGFP-PR-M transfection group(oe-PR-M,transfected with Lentivirus overexpression of PR-M),pLV-EGFP-Bcl2 transfection group(oe-Bcl2,Lentivirus transfection with overexpression of Bcl2).3.Determination of protein expression The expressions of autophagy-related proteins P62,LC3-I,LC3-II,Beclin-1 andapoptosis-related proteins Caspase-3 and Bcl-2 were detected by Western Blotting.4.Determination of Cell proliferation abilityThe proliferation of PR-M(+)uterine fibroid cells in the above groups was detected by CCK8 and EDU staining methods.5.Apoptosis detectionThe apoptosis of PR-M(+)myoma cells in the above groups was detected by flow cytometry and Hoechst33258 staining.6.Autophagy detectionThe autophagy of PR-M(+)myoma cells in the above groups was detected by Monodansylcadaverine(MDC)staining and transmission electron microscopy.7.Detection of co-immunoprecipitationThe Co-IP method was used to detect the interaction between Bcl-2 and Beclinl in PR-M(+)uterine fibroid cells in the above groups.Results1.24 h and 48 h after cell transfection,cell viability was significantly decreased in the sh-PR-M group(P<0.05),and cell viability was significantly increased in the oe-PR-M group(P<0.05).48 h after transfection,the sh-PR-M group inhibited cell proliferation,while the oe-PR-M group significantly promoted cell proliferation.2.Compared with blank group,the expression of autophagy-related protein p62 was significantly decreased(P<0.05)and the expression of LC3-II/LC3-I increased(P<0.05)when PR-M was knockout,while over expression of PR-M reversed the expression of P62 and LC3-II/LC3-I.3.The expression of apoptosis-related protein Caspase-3 was increased significantly in PR-M(+)uterine fibroid cells after PR-M was knock out(P<0.05),while the expression of Caspase-3 was reduced significantly when PR-M was overexpressed(P<0.05).4.The result of CO-IP showed that the interaction between Bcl-2 and Beclinl was enhanced in PR-M(+)cells transfected with PR-M shRNA but weakened in PR-M overexpressing plasmid.ConclusionPR-M could regulate the proliferation,apoptosis and autophagy of PR-M(+)uterine fibroid cells,which may play a role through the Bcl-2/Beclinl pathway.Part 3 PRA antagonizes the effect of PR-M on PR-M(+)cell behavior by regulating the Bcl-2/Beclin1 pathwayObjectiveIn this study,the progesterone receptor antagonist mifepristone was selected as the treatment drug for uterine fibroid cells to explore further its effective and safe concentration and its regulatory effect on uterine fibroid cells.Methods1.Experimental groupControl group,PR-M(+)cells without drug treatment;PR-M(+)uterine fibroid cells treated with different concentrations of the progesterone receptor antagonist Mifepristone(MIF)1×10-6 mol/L(L-MIF group);1×10-5 mol/L(M-MIF group)and 1×10-4 mol/L(H-MIF group).2.Determination of cell proliferation abilityThe method is the same as in Part 2.3.Apoptosis detectionThe method is the same as in Part 2.4.Autophagy detectionThe method is the same as in Part 2.Results1.MIF inhibited the proliferation of PR-M(+)cells significantly in a dose-dependent manner(P<0.05),promoted autophagy and apoptosis of cells(P<0.05),significantly reduced the protein levels of p62 and Bcl-2(P<0.05),increased the levels of Beclin-1,LC3-Ⅱ/LC3-Ⅰ and Caspase-3,and decreased the interaction between Bcl-2 and Beclinl.The effect of high dose MIF was the most obvious.2.Compared with the cells treated with H-MIF alone,the effect of cell proliferation was increased significantly(P<0.05),the level of autophagy and apoptosis were decreased,p62 and Bcl-2 protein levels were was increased significantly(P<0.05),while,(P<0.05),increased(P<0.05),Beclin-1,LC3-Ⅱ/LC3-Ⅰand Caspase-3 levels were significantly decreased(P<0.05)in PR-M(+)cells with overexpression of Bcl-2 after treated with H-MIF.Furthermore,the interaction between Bcl-2 and Beclinl was enhanced.ConclusionPRA antagonizes PR-M by regulating the Bcl-2/Beclinl pathway,thereby promoting the autophagy and apoptosis of primary uterine fibroid cells and inhibiting the proliferation of primary uterine fibroid cells.