Resveratrol protected against the development of endometriosis by promoting ferroptosis through miR-21-3p/p53/SLC7A11 signaling pathway.

Resveratrol is involved in regulating ferroptosis, but its role in Endometriosis (EMS) is not clear. In this study, we aim to investigate the effect of ferroptosis and resveratrol intervention in the pathogenesis of EMS cyst. Cell proliferation, migration, and oxidative stress level were analyzed. The interaction of miR-21-3p and p53 was analyzed by dual luciferase assay. The interaction between p53 and SLC7A11 were analyzed by chromatin immunoprecipitation (CHIP). The miR-21-3p, GPX4, ACSL4, FTH1, p53, SLC7A11, Ptgs2 and Chac1 expression were analyzed by RT-qPCR or Western blot. The Fe3+ deposition and miR-21-3p, GPX4, FTH1 and SLC7A11 expressions were increased, and ACSL4, p53, Ptgs2 and Chac1 expression were decreased in EMS patients. Resveratrol inhibited migration, induced Ptgs2 and Chac1 expression in EESCs. Overexpression of miR-21-3p inhibited p53, Ptgs2 and Chac1 expression, and promoted SLC7A11 expression, which was reversed by resveratrol. miR-21-3p bound to p53, which interacted with SLC7A11. Resveratrol promoted Ptgs2 and Chac1 expression in the sh-p53 EESCs. Resveratrol reduced miR-21-3p and SLC7A11 expressions, and increased p53, Ptgs2 and Chac1 expressions, and Fe3+ deposition in the lesion tissues of EMS mice, which were reversed by miR-21-3p mimics. Resveratrol activated p53/SLC7A11 pathway by down-regulating miR-21-3p to promote ferroptosis and prevent the development of EMS.

Hypoxia Enhances HIF1α Transcription Activity by Upregulating KDM4A and Mediating H3K9me3, Thus Inducing Ferroptosis Resistance in Cervical Cancer Cells.

Objective: Cervical cancer (CC) is a prevalent cancer in women. Hypoxia plays a critical role in CC cell ferroptosis resistance. This study explored the mechanism of hypoxia in CC cell ferroptosis resistance by regulating HIF1α/KDM4A/H3K9me3. Methods: Cultured SiHa and Hela cells were exposed to CoCl2 and treated with Erastin. Cell viability was detected by MTT assay, and concentrations of iron ion, MDA and GSH were determined using corresponding kits. Expressions of KDM4A, HIF1α, TfR1, DMT1, and H3k9me3 were detected by RT-qPCR, Western blot, and ChIP assay. The correlation of KDM4A and HIF1α was analyzed on Oncomine, UALCAN, and Starbase. CC cells were co-transfected with shKDM4A or/and pcDNA3.1-HIF1α. Iron uptake and release were assessed using the isotopic tracer method. The binding relationship between HIF1α and HRE sequence was verified by dual-luciferase assay. Results: Cell viability and GSH were decreased while iron concentration, MDA, KDM4A, and HIF1α levels were increased in hypoxia conditions. The 2-h hypoxia induced ferroptosis resistance. KDM4A and HIF1α were highly-expressed in CC tissues and positively correlated with each other. KDM4A knockdown attenuated cell resistance to Erastin, increased H3K9me3 level in the HIF1α promoter region, and downregulated HIF1α transcription and translation. H3K9me3 level was increased in the HIF1α promoter after hypoxia. HIF1α overexpression abrogated the function of KDM4A knockdown on ferroptosis in hypoxia conditions. Iron uptake/release and TfR1/DMT1 levels were increased after hypoxia. Hypoxia activated HRE sequence in TfR1 and DMT1 promoters. Conclusion: Hypoxia upregulated KDM4A, enhanced HIF1α transcription, and activated HRE sequence in TfR1 and DMT1 promoters via H3K9me3, thus inducing ferroptosis resistance in CC cells.

Endometrial stromal cells treated by tumor necrosis factor-α stimulate macrophages polarized toward M2 via interleukin-6 and monocyte chemoattractant protein-1.

AIM: This study aimed to investigate the effects of endometrial stromal cells (ESC)-derived interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 on macrophage polarization in endometriosis. METHODS: Macrophage polarization was measured in eutopic endometrium of control participants ('normal endometrium'), eutopic endometrium of patients with endometriosis ('eutopic endometrium') and ectopic endometrium of endometriosis patients ('ectopic endometrium') by immunohistochemical staining. Expression of IL-6 and MCP-1 were measured in the eutopic and ectopic endometrium through enzyme-linked immunosorbent assays. Expression of CD163 was measured in human acute monocytic leukemia (THP-1) cell-derived macrophages that were treated with conditional medium induced by tumor necrosis factor (TNF)-α, TNF-α + anti-IL-6 or TNF-α + anti-MCP-1 via flow cytometry. RESULTS: The ratio of CD163+/CD68+ macrophages in the normal endometrium was higher than that in the eutopic endometrium, while differences between the eutopic and ectopic endometrium were not statistically significant. IL-6 and MCP-1 exhibited enhanced expression in the ectopic endometrium group and decreased expression in the eutopic endometrium group. TNF-α could promote the expression of ESC-derived IL-6 and MCP-1. Intervention with TNF-α-induced conditioned medium resulted in the upregulation of CD163 in THP-1 cells, while conditional medium induced with IL-6 and MCP-1 neutralizing antibodies decreased the proportion of CD163+ macrophages significantly. CONCLUSION: In endometriosis patients, the macrophages of the eutopic endometrium polarize toward M1 compared with the normal endometrium, and those of the ectopic endometrium were mainly M2-polarized. Under the action of TNF-α, ESC-derived IL-6 and MCP-1 could stimulate peritoneal macrophages toward M2-polarization, which could modulate endometriosis.

Serum and Ectopic Endometrium from Women with Endometriosis Modulate Macrophage M1/M2 Polarization via the Smad2/Smad3 Pathway.

OBJECTIVE: This study investigated the alterations in macrophage polarization in patients with endometriosis as well as the underlying molecular mechanisms. METHODS: Peritoneal washings, serum samples, and endometrial tissues were collected from endometriosis patients and control subjects. Endometrial stromal cells (ESCs) were isolated from endometrial tissue, and conditioned medium was prepared by treating ESCs with or without various concentrations of interleukin- (IL-) 6, estrogen, or progestin. The frequencies of CD86+ and CD163+ cells and expression levels of these markers as well as the cytokines IL-12 and IL-10 were measured in THP-1- (human monocytic leukemia cell) derived macrophages. RESULTS: There was a decrease in the percentage of CD86+ macrophages in the peritoneal wash solution of patients with endometriosis. Ectopic endometrial homogenates could promote M1 to M2 macrophage polarization in response to lipopolysaccharide (LPS), as evidenced by the increased percentage of CD163+ macrophages and increased IL-10 expression as well as a decreased percentage of CD86+ cells and lower IL-12 expression. In contrast, addition of serum from women with endometriosis to THP-1 cells resulted in the polarization of macrophages towards both M1 and M2 phenotypes. Upregulation of Smad2/Smad3 in macrophages upon exposure to eutopic and ectopic endometrial homogenates as well as serum of women with endometriosis was observed, and blockage of Smad2/Smad3 with their inhibitor SB431542 could reverse the macrophage polarization from M1 to M2. Conditioned medium induced by IL-6, but neither estrogen nor progestin, could facilitate M2 polarization. Neutralization of IL-6 diminished macrophage M2 polarization in endometriosis. CONCLUSION: This study provides detailed evidence supporting alterations in M1 to M2 macrophage polarization that may contribute to the initiation as well as progression of endometriosis.

[Effect of IL-1ß or TNF-α-treated human decidual cells on balance between Th1 and Th2 or Th17 and Treg immunity].

OBJECTIVE: To investigate the effect of IL-1ß or TNF-α-treated human decidual cells on the balance between Th1 and Th2 or Th17 and Treg.
 Methods: Human decidual cells were isolated and treated with estrogen (E2) and progesterone (P) for 7 days and then with IL-1ß or TNF-α for 24 h. The culture medium was collected to prepare conditioned medium. The T cells isolated from human decidual tissue were cultured in presence of conditioned medium for 72 h. ELISA was used to detect concentration of IFN-γ, IL-2, IL-5, IL-17 IL-10, and TGF-1ß in supernatants and quantitative real-time PCR was used to detect the mRNA expression of IFN-γ, IL-17, IL-10, and TGF-1ß.
 Results: The levels of IFN-γ, IL-2 and IL-17 were significantly increased when T cells were cultured in the conditioned medium compared with the control group (E2+P), while the levels of IL-5, IL-10, and TGF-1ß were not significantly changed. The mRNA expressions of IFN-γ and IL-17 mRNA were significantly increased when T cells were cultured in the conditioned medium compared with the control group (E2+P), while the mRNA expression of IL-10 and TGF-1ß was not significantly different.
 Conclusion: Human decidual cells treated by IL-1ß or TNF-α can shift the balance of Th1 and Th2 or Th17 and Treg toward Th1 and Th17 immunity.

Letrozole ovulation induction: an effective option in endometrial preparation for frozen-thawed embryo transfer.

PURPOSE: To evaluate the clinical efficacy of letrozole on ovulation induction and hormone replacement therapy (HRT) during endometrial preparation for frozen-thawed embryo transfer (FET). METHODS: We analyzed totally 1,230 cycles of patients that underwent FET from October 2010 to September 2012. Seven hundred and thirteen cycles of patients with ovulation disorders that underwent FET were randomly assigned to two groups by case control study. 359 cycles received letrozole ovulation induction and 354 cycles received HRT during endometrial preparation for FET, respectively. In the corresponding period, 517 cycles of patients with normal ovulation in the natural cycle group for FET endometrial preparation served as controls. Reproduction-related clinical outcomes of patients in the three groups were compared. RESULTS: The embryo implantation rate of patients in letrozole group (30.4 %) was significantly higher than the HRT group (22.8 %, P < 0.05). The clinical pregnancy rate of patients in the letrozole group (53.2 %) was significantly higher than the HRT group (44.4 %, P < 0.05), while no significant difference was observed between the letrozole and natural cycle groups (51.3 %, P > 0.05). Estradiol levels on the day of human chorionic gonadotropin administration in the letrozole group were significantly lower than those in the natural cycle group (280.32 ± 125.39 pg/ml and 351.06 ± 123.03 pg/ml, respectively; P < 0.05). The live birth rate of patients in letrozole group (44.6 %) was significantly higher than the HRT group (32.5 %, P < 0.05), while abortion rate (12.0 %) was significantly lower than the HRT group (21.0 %, P < 0.05). There were no significant differences in number of mature follicles, endometrial thickness, duration of follicle growth between the letrozole and the natural cycle groups, and there were no significant differences in twin birth rate and ectopic pregnancy rate among the three groups (all P values >0.05). CONCLUSIONS: Ovulation induction with letrozole during endometrial preparation for FET has a higher rate of pregnancy success and a lower abortion rate than HRT. Letrozole treatment exhibits clinical progression and outcomes similar to those patients undergoing a natural cycle or normal ovulation cycle. Therefore, letrozole treatment may be an effective option in endometrial preparation for FET in patients with ovulation disorders or irregular menstruation.

[Expression of RUNX3 in cervical carcinoma and its clinical significance].

OBJECTIVE To explore the role of runt-related transcription factor 3(RUNX3) in the tumorgenesis and progression of cervical carcinoma. METHODS The immunohistochemical staining technique was used to detect the expression of RUNX3 protein in 25 cases of normal cervix, 34 intraepithelia neoplasia (CIN), and 48 cervical carcinomas. SYBR Green I chimeric fluorescence Real-time PCR was applied to detect the expression of RUNX3 mRNA in 10 cases of normal cervix, 24 CIN, and 30 cervical carcinomas. RESULTS The expressions of RUNX3 protein and mRNA in normal cervix, CINI,CINII-III, and cervical carcinoma tissues tended to be down-regulated. There was significant difference among these groups (P<0.05). The expressions of RUNX3 protein and mRNA in the cervical carcinoma tissues were correlated with the histological differentiation, clinical stage, and lymphatic metastasis (P<0.05), but had no relationship with the age, high-risk human papillomavirus infection, and histological classification (P> 0.05). CONCLUSION RUNX3 may function as a tumor suppressor gene in the occurrence and progression of cervical carcinoma.

[Inhibitory effects of human papilloma virus 18 E6 gene in HeLa cell transfected with shRNA].

OBJECTIVE: To investigate the inhibition of HPV18E6 gene in HeLa cell transfected with plasmid expressing human papilloma virus 18 E6 (HPV18E6) short hairpin RNA (shRNA). METHODS: We synthesized two HPV18E6 shRNA frames and sub-cloned them into pSUPER which can express shRNA in mammalian cells to construct pE6-1shRNA and pE6-2shRNA which were mutant in E6 shRNA frame. The pE6-1shRNA, pE6-2shRNA and pcDNA3.1 were co-transfected into HeLa cells by cationic liposome respectively and the positive transfectants were selected by G418. The HPV18E6 mRNA and protein expression level was detected by semi-quantitative RT-PCR and streptavidin-peroxidase conjugated method (SP) to assay the inhibitory effects of pE6shRNA. RESULTS: We successfully constructed several new HeLa cell lines transfected with pE6-1shRNA and pE6-2shRNA. In the HeLa cells without transfection and the HeLa cells transfected with pE6-1shRNA plasmid, the HPV18E6 mRNA levels were 1.14 +/- 0.45, 0.76 +/- 0.28 respectively, and the difference of HPV18E6 mRNA levels was significant (P < 0.05). The inhibition efficiency of HPV18E6 gene mRNA was 33.3% and the HPV18E6 protein levels were declined after transfection with pE6-1shRNA. In the HeLa cells transfected with pE6-2shRNA and pSUPER plasmids, HPV18E6 mRNA and protein expression levels were not different from those in wild HeLa cells. CONCLUSIONS: The pE6-1shRNA plasmid can inhibit HPV18E6 expression in HeLa cells, which is persistent, specific and heritable.