The Function Of Hypoxia Mediated Cell Adhesion And Cell Cycle In The Pathogenesis Of Endometriosis

Endometriosis(EM)is defined as the presence of tissue resembling the endometrium(the gland or stroma)outside the uterine cavity,which characterized by adhesion,proliferation,repeated cyclical haemorrhage,and formation of cysts or nodules.Pelvic pain,dysmenorrhea,dyspareunia,and infertility are common symptoms in endometriosis that negatively impact woman's quality of life.Although endometriosis is a common disease in women of reproductive age,the pathogenesis of endometriosis is still unclear and contravention.To date,the most convincing theory is the retrograde menstruation-implantation hypothesis by Sampson,that is,the endometrium of uterine reflux to the pelvic cavity with menstrual blood and experiences adhesion,proliferation,angiogenesis and finally forming ectopic cysts or nodules.But the implant theory doesn't explain why menstrual reflux occurs in 90% of women,only 10% of reproductive aged women have endometriosis.This reminds us that endometriosis is not only related to menstrual reflux,but also associated with the pelvic microenvironment and the biological disorders in ectopic lesion.Due to the vasoconstriction of spiral arteries during menstrual period,the detached endometrium is ischemic and hypoxic.The existing studies have proved that the refluent endometrium debris is in a state of relative hypoxia before attach to ectopic tissues.The expression of hypoxia-inducible factor-1?(HIF-1?)is robust under hypoxia,which mediates the endometriotic cells to overcome hypoxic stress and initiate the ectopic adhesion,proliferation and infiltration.Adhesion is considered to be the first step in the formation of ectopic lesion,transforming growth factor-?1(TGF-?1)and the members of the Integrins family play important roles in the initiation of adhesion in endometriosis,but the pivotal Integrins is unclear and no study reported the specific relationship between HIF-1?,TGF-?1 and Integrins in endometriosis.After ectopic adhesion,endometriotic cells are still stressed by hypoxia for a long time,and the mechanism of their survival and replication in an excessive oxidative stressed environment is unclear.Micro RNA-210(miR-210)is the most significantly up-regulated HIF-1? associated micro RNA under hypoxia,which function in a variety of biological processes including cell cycle progression,cell division and proliferation under oxidative stress(OS),DNA damage repair,vascular neogenesis and energy metabolism via regulating its target genes.Though excessive oxidative stress is identified in the ectopic lesion of endometriosis,the mechanism of the oxidative/antioxidant imbalance in the ectopic lesion is unknown.Our previous study found that chronic hypoxia caused DNA damage in endometrial stromal cells(ESCs)and Ishikawa,and the expression of miR-210-3p was significantly increased in endometrial stromal cells and endometrial epithelial cells(EECs)of ectopic lesions compared with the healthy proliferative endometrium in control group.The mechanism of survival and growth of ectopic cells after their ectopic adhesion is ambiguous,we speculated that hypoxia-upregulated miR-210-3p was involved.By Target Scan,RNA-hybrid and miRanda,BARD1 was predicted to be the target gene of miR-210-3p.BARD1(BRCA1 associated RING domain 1)connects with BRCA1 through RING region to form heterodimer,which plays a key role in DNA damage repair and the activation of cell cycle checkpoints.Moreover,BARD1 protein has been proved to determine the stability of BRCA1 protein and the function of the BRCA1/BARD1 complex in cell cycle regulation,DNA damage response and cancer progression.However,the roles of miR-210-3p and its target genes in endometriosis has not been reported.Based on the retrograde menstruation-implantation theory,our study discussed the roles of hypoxia in ectopic adhesion and abnormal proliferation of endometriotic cells.We have proved that hypoxia not only initiates ectopic adhesion of endometrial stromal cell via activating the HIF-1?/TGF-?1/p-Smad2/p-Smad3/Smad4/Integrins signaling pathway,hypoxia upregulated miR-210-3p directly targets BARD1,leading to physiological and biochemical function impairment of the BRCA1 complex,which results in an inactivation of cell cycle arrest in response to DNA damage and finally promotes the proliferation of endometriotic cells.The purpose of this study was to elucidate the specific mechanism of hypoxia and its related molecules in the formation and growth of ectopic lesions,and to search for potential targets for treatment of endometriosis.ObjectiveTo identify the key Integrins involved in the formation of endometriotic tissues and their relationships with hypoxia.Methods(1)We collected 15 normal proliferative endometrium tissues from 15 patients without endometriosis,15 paired ectopic lesions and eutopic endometrium tissues from patients with endometriosis that undergoing combined operation of hysteroscopy and laparoscopy.Immunohistochemistry assay(IHC)was used to analyze the expression of HIF-1? and Integrins in these samples.(2)Thirteen eutopic endometrium tissues were collected from endometriosis patients and used for extract eutopic endometrial stromal cells(ESCs),we also established a stable system for the extraction,purification,identification and culture of primary ESCs.(3)Thirteen ESCs were treated under hypoxia(1% O2)and then cell adhesion and migration abilities were analyzed.(4)m RNA and protein of ESCs were extracted after hypoxia treatment for 0 h and 48 h,and then quantitative real-time PCR(RT-q PCR)and western blot(WB)were conducted to detect the effects of hypoxia on HIF-1? and Integrins expression.Results(1)HIF-1? was strongly expressed in ectopic tissues in both epithelial and stromal cells,weakly expressed in eutopic tissues,and not expressed in EECs and ESCs of normal endometrium,while Integrin-?5,Integrin-?V,Integrin-?3 and Integrin-?5 expression were only significantly upregulated in stromal cells of ectopic lesions and eutopic endometrium compared with normal endometrium,with ectopic lesions showing the highest staining.(2)Hypoxia significantly improved the adhesion and migration ability of ESCs.(3)Hypoxia upregulated the m RNA and protein expression of HIF-1?,Integrin-?5,Integrin-?V,Integrin-?3 and Integrin-?5 in ESCs.ConclusionHIF-1? was strongly expressed in ectopic tissues in both epithelial and stromal cells.Hypoxia promotes the ectopic adhesion of ESCs by upregulating the expression of Integrin-?5,Integrin-?V,Integrin-?3 and Integrin-?5,but the specific mechanism is unclear.ObjectiveTo explore the specific mechanism of hypoxia increased ectopic adhesion ability of ESCs and to explore the expression pattern of HIF-1?,TGF-?1 and Integrins in the initial stage of endometriotic cysts from mouse model.Methods(1)The expression of TGF-?1 and TGF-? receptor II in paired ectopic lesions,eutopic endometria and normal proliferative stage endometria were detected via immunohistochemistry assay.(2)Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of activated TGF-?1 in the supernatant of ESCs cultured under normoxia or hypoxia.(3)CCK-8 based adhesion assay and transwell migration assay were conducted to explore the effects of TGF-?1,normoxia,hypoxia,and hypoxia-SB431542 co-stimulation on cell adhesion and cell migration abilities.(4)RT-q PCR,WB and Immunofluorescence assay(IF)were used to detect the activation of the classical TGF-?1/p-Smad2/p-Smad3/Smad4 signaling pathway.(5)RT-q PCR and WB were used to verify the expression of Integrins after knockdown of HIF-1a under normoxic or hypoxic culture.(6)Eleven endometriosis mouse models were established by stitching eutopic endometria onto the peritoneum of C57BL/6 female mice and lesions were harvested after 0 and 48 hours of transplantation,the expression of HIF-1?,TGF-?1,Integrin-?5,Integrin-?V,Integrin-?3 and Integrin-?5 were examined by immunohistochemistry assay.Results(1)TGF-?1 expression was higher in both ESCs and EECs of eutopic tissues than that of normal tissues,with the highest staining in both ESCs and EECs of ectopic lesions.The expression level of TGF-? receptor II in ESCs was higher in eutopic tissues than normal tissues,with the highest staining in ectopic lesions.(2)ELISA of supernatant from 13 ESCs under normoxic and hypoxic culture showed a significantly high concentration of activated TGF-?1 under hypoxia culture.(3)Immunofluorescence assay showed that p-Smad2 protein was mainly localized in the cytoplasm of ESCs under normoxic conditions(hypoxia 0 hour),p-Smad2 started to enter nucleus at 4 hours of hypoxia treatment(hypoxia 4 hours),and then peaked at 8 hours of hypoxia.(4)Protein expression of p-Smad2,p-Smad3,and total Smad4 protein were remarkably increased under TGF-?1 co-stimulation and hypoxic culture with no influence on total Smad2 expression,whereas hypoxia-SB431542 co-stimulation decreased the protein expression of p-Smad2,p-Smad3,and total Smad4 compared with hypoxic culture alone.(5)Decreased protein expression of HIF-1?,TGF-?1,p-Smad2,Integrin-?5,Integrin-?V,Integrin-?3 and Integrin-?5 were detected in HIF-1? interference groups compared with the negative control group under hypoxic culture.(6)Immunohistochemistry assay showed higher expression of HIF-1?,TGF-?1,Integrin-?5,Integrin-?V,Integrin-?3 and Integrin-?5 in ESCs of in transplanted lesions(48 h),whereas only HIF-1? and TGF-?1 were overexpressed in EECs in ectopic lesions with decreased Integrin-?V expression.ConclusionHypoxia significantly increased the secretion of TGF-?1 in ESCs,which promoted the translocation of p-Smad2 from cytoplasm to nucleus via binding to TGF-? receptor II and then activated the classical p-Smad2/p-Smad3/Smad4 signaling pathway.The activation of TGF-?1/Smads signaling pathway results in the upregulation of Integrins and the ectopic adhesion of ESCs,additionally,this process depends on HIF-1?.ObjectiveTo evaluate the effects of hypoxia on DNA damage of endometriotic cells,and to identify the localization and expression of miR-210-3p in ectopic lesions of endometriosis.Methods(1)Immunohistochemistry assay of HIF-1? and 8-OHd G(an oxidative stress-induced DNA damage marker)in 27 normal endometria and 57 paired eutopic and ectopic tissues from endometriosis patients.(2)Five ESCs(ESC-14?ESC-15?ESC-16?ESC-17?ESC-18)and Ishikawa were cultured under normoxia or hypoxia for 28 days,alkaline comet assays were performed to detect the DNA damage.(3)ESCs were collected after hypoxic or normoxic culture for 48 h and then miRNA high-throughput sequencing were conducted to find the crucial hypoxia-associated miRNAs.(4)Fluorescence in situ hybridisation(FISH)analysis was performed in five normal endometria and five paired eutopic and ectopic tissues to determine the location of miR-210-3p,RT-q PCR was performed in another 15 normal endometria and 15 paired eutopic and ectopic tissues to determine the expression of miR-210-3p.Results(1)HIF-1? and 8-OHd G expression in ESCs and EECs were significantly higher in eutopic endometria and ectopic lesions than in normal endometria,with ectopic lesions showing the strongest staining.The same trend of HIF-1? and 8-OHd G expression in both stroma and gland implied that hypoxia may be involved in DNA damage of endometriotic cells.(2)Hypoxia cultured ESCs and Ishikawa showed significantly increased tail lengths,tail DNA percentage and tail olive moments compared with cells in normoxic culture,which indicated severe DNA damage caused by chronic hypoxia.(3)Mi R-210-3p expression was obviously upregulated after hypoxic treatment for 48 h,and q RT-PCR results also confirmed that it was markedly elevated in ESCs and Ishikawa cells cultured in hypoxia compared with normoxia,which was consistent with HIF-1? expression trend of change.Moreover,miR-210-3p was highly expressed in ectopic lesions and eutopic endometria compared with normal endometria,with the highest level in ectopic lesions.(4)FISH and immunofluorescence double staining revealed that miR-210-3p was located in both ESCs and EECs of endometriotic lesions.And miR-210-3p signal was much stronger in ectopic lesions than in eutopic endometria,with the weakest signal observed in normal endometria.ConclusionChronic hypoxia is one of the reasons of oxidative stress induced DNA damage in endometriotic cells.However,how endometriotic cells escape DNA damage and maintain proliferation under hypoxia is unclear.We speculated that the highly expressed miR-210-3p may be involved,but the specific mechanism remains to be explored.ObjectiveTo explore the roles and specific molecular mechanisms of miR-210-3p in oxidative stress induced DNA damage and ectopic proliferation of endometriotic cells.Methods(1)Flow cytometric analysis,cell proliferation assay and WB were conducted in ESCs and Ishikawa cells infected with LV-In-210 or LV-In-CN under normoxia or hypoxia.(2)RNA high-throughput sequencing,bioinformatics analysis and double luciferase reporter gene analysis were performed to explore and verify the targets of miR-210-3p.(3)Western blot and RT-q PCR were used to detect the m RNA and protein expression of BARD1 and BRCA1 in ESCs and Ishikawa infected with LV-CN,LV-210,LV-In-CN or LV-In-210 under normoxia.(4)RT-q PCR assay of BARD1 m RNA expression in 15 normal endometria,15 paired eutopic and ectopic tissues were conduct,and then the correlation between miR-210-3p and BARD1 m RNA in ectopic lesions were analyzed.(5)Immunohistochemistry was performed to examine BARD1 and BRCA1 expression in 27 normal endometria and 57 paired eutopic and ectopic tissues fromendometriosis patients.(6)FCM and WB were conducted to examine the cell cycle distribution and BRCA1,p-BRCA1,p53,p21,Cyclin B1,Cdc2 protein expression after infected with LV-CN,LV-210,LV-BARD1 or LV-In-210 under normoxia or hypoxia.Results(1)Hypoxia significantly increased cyclin B1 and Cdc2 expressions but decreased p53 and p21 expressions in ESCs and Ishikawa cells compared with cells cultured in normoxia.Knockdown of miR-210-3p under hypoxia resulted in G2/M phase arrest of ESCs and Ishikawa,and both the increase in cyclin B1 and Cdc2 expression and decrease of p53 and p21 levels were rescued compared with LV-In-CN treatment under hypoxia.(2)Gene Ontology(GO)term enrichment analysis showed that many differentially expressed genes in miR-210-3p-overexpressing h EM15 A participated in cell cycle regulation processes,such as mitosis,chromosome localization,cell division,microtubule-based movement and spindle and kinetochore microtubule assembly.(3)ESCs and Ishikawa cells infected with LV-210 showed reduced BARD1 m RNA expression,while cells infected with LV-In-210 showed increased BARD1 and BRCA1 protein expression.(4)BARD1 was moderately expressed in ESCs and EECs of normal endometria,while BARD1 expression was significantly lower in eutopic and ectopic stroma and gland,with the lowest expressions in stromal cells and glandular cells of ectopic lesions.The correlation between miR-210-3p levels and BARD1 levels in ectopic lesions was identified as negative by linear regression analysis with R = 0.727.(5)knockdown of both miR-210-3p and BARD1 in hypoxia could attenuate the increases of BRCA1,phosphor-BRCA1,p53 and p21 and reverse the decrease of cyclin B1 and Cdc2,compared with knocking down only miR-210-3p.ConclusionHypoxia upregulated miR-210-3p inhibited BRCA1 protein expression via directly targeting BARD1,and lead to the disfunction of BRCA1/BARD1 complex in DNA damage responses and cell cycle checkpoint,which help endometriotic cells escape oxidative stress and maintain proliferation outside the uterine.ObjectiveTo explore the in vivo function of miR-210-3p inhibitor and Vitamin C via mouse model of endometriosis.Methods(1)Endometriosis was induced in 50 C57BL/6 mice by mouse–mouse intraperitoneal implantation.(2)Mi R-210-3p inhibitor(In-210)or its negative control(NC)was intraperitoneally injected in endometriotic mice(n = 8,each group)using the in vivo-jet PEI delivery agent every other day.(3)For vitamin C experiments,eleven endometriotic mice were given 2.5 mg vitamin C in 1 m L purified water via oral gavage each day,eleven endometriotic mice received intraperitoneal injection of vitamin C(500 mg/kg)every other day and purified water were supplied,another 11 endometriotic mice received normal breeding and PBS injection every other day.Animals were sacrificed after 4 weeks,and endometriotic cysts were excised for IHC.Results(1)Four weeks after implantation,classical endometriosis-like lesions were formed in the pelvic cavity,mesentery or peritoneum of each mouse.(2)The lesion sizes were significantly smaller in In-210 mice than in NC animals.Immunohistochemistry showed that ESCs and EECs of endometriotic tissues from NC mice were positive for BARD1 and BRCA1,while In-210 treatment increased BARD1 and BRCA1 expressions in ESCs and EECs.(3)Lesion sizes were significantly smaller in vitamin C-treated mice than in PBS-injected mice,with the smallest average volume of cyst in vitamin C-injected mice.Moreover,HIF-1? and 8-OHd G expressions in ESCs and EECs of vitamin C-injected mice were dramatically reduced compared with those of PBS-injected mice,with BARD1 and BRCA1 expressions increased.ConclusionMi R-210-3p promotes endometriosis progression in vivo,while knockdown miR-210-3p inhibit the growth of endometriotic cysts via accelerate the accumulation of BRCA1/BARD1 protein and activate the DNA damage repair pathway.Vitamin C may protect endometriotic cells from oxidative stress and DNA damage via reducing HIF-1? and 8-OHd G expression and increasing BARD1 and BRCA1 expression to ultimately alleviate endometriosis progression.