Increased miR-3074-5p expression promotes M1 polarization and pyroptosis of macrophages via ERα/NLRP3 pathway and induces adverse pregnancy outcomes in mice.

Decidual macrophages (dMϕs) play critical roles in regulation of immune-microhomeostasis at maternal-fetal interface during pregnancy, but the underlying molecular mechanisms are still unclear. In this study, it was found that litter size and fetal weight were significantly reduced, whereas the rate of embryo resorption was increased in miR-3074-5p knock-in (3074-KI) pregnant mice, compared to that of wild-type (WT) pregnant mice. Plasma levels of pro-inflammatory cytokines in 3074-KI pregnant mice were also significantly elevated compared to WT pregnant mice at GD7.5. The quantity of M1-Mϕs in uterine tissues of 3074-KI pregnant mice was significantly increased compared to WT pregnant mice at GD13.5. Estrogen receptor-α (ERα) was validated to be a target of miR-3074-5p. Either miR-3074-5p overexpression or ERα knockdown promoted transcriptional activity of NF-κB/p65, induced M1-polarization and pyroptosis of THP1-derived Mϕs, accompanied with increased intracellular levels of cleaved Caspase-1, cleaved IL-1ß, NLRP3, cleaved GSDMD and ASC aggregation. Furthermore, ERα could not only bind to NLRP3 or ASC directly, but also inhibit the interaction between NLRP3 and ASC. The endometrial miR-3074-5p expression level at the middle secretory stage of repeated implantation failure (RIF) patients was significantly decreased compared to that of control fertile women. These data indicated that miR-3074-5p could promote M1 polarization and pyroptosis of Mϕs via activation of NLRP3 inflammasome by targeting ERα, and the dysregulation of miR-3074-5p expression in dMϕs might damage the embryo implantation and placentation by interfering with inflammatory microenvironment at the maternal-fetal interface during early pregnancy.

MiR-3074-5p Regulates Trophoblasts Function via EIF2S1/GDF15 Pathway in Recurrent Miscarriage.

Dysfunction of extravillous trophoblasts (EVTs) might cause early pregnancy failure by interfering with embryo implantation and/or placentation. We previously reported that the villus miR-3074-5p expression level was increased, whereas the peripheral level of GDF15, a predict target gene of miR-3074-5p, was decreased in recurrent miscarriages (RM) patients, and miR-3074-5p could enhance apoptosis but reduce invasion of human extravillous trophoblast cells (EVTs). The aim of this study was to further explore roles of miR-3074-5p/GDF15 pathway in regulation of EVTs function. It was validated that GDF15 was not the direct target of miR-3074-5p, whereas EIF2S1, an upstream regulator of GDF15 maturation and secretion, was the direct target of miR-3074-5p. The villus expression levels of GDF15 and EIF2S1 were significantly decreased in RM patients. Knockdown of GDF15 expression presented inhibitory effects on proliferation, migration, and invasion of HTR8/SVneo cells. Up-regulated miR-3074-5p expression led to the significant decreased GDF15 expression in HTR8/SVneo cells, and this effect could be efficiently reversed by the overexpression of EIF2S1. Meanwhile, the suppressive effects of miR-3074-5p on proliferation, migration, and invasion of HTR8/SVneo cells could be intercepted by the treatment of recombinant human GDF15 protein. Collectively, these data suggested that miR-3074-5p could reduce GDF15 production via targeting inhibition of EIF2S1 expression, and the deficiency in GDF15 function might lead to the early pregnancy loss by attenuating proliferation and invasion of EVTs.

Development and validation of Adaptability to Return-to-Work Scale (ARTWS) for cancer patients.

Introduction: The research on cancer patients returning to work in China is still in its infancy, and there is no research and discussion on the adaptability to return-to-work for cancer patients. It is critical to develop the Adaptability to Return-to-Work Scale (ARTWS) for cancer patients and evaluate its psychometric properties. Methods: The items of the initial scale were compiled based on the theoretical model and literature review results. Through two rounds of Delphi expert consultation (N = 15) and a pilot survey (N = 40), the initial scale was further checked and revised. Conduct a large sample survey (N = 376) and the construct validity and reliability of the ARTWS were assessed by confirmatory factor analysis (CFA) and exploratory factor analysis (EFA). Results: The final ARTWS consisted of 24 items. "Focusing on rehabilitation," "Rebuilding Self-efficiency," and "Adjusting plans" as common factors in determining adaptability to return to work for cancer patients, and the cumulative variance contribution rate for these three factors was 66.6%. The S-CVI of the total scale was 0.979. The Cronbach's α coefficient was 0.937 and the 2-week test-retest reliability was 0.814. Discussion: ARTWS has good correlation validity and can be used as a tool to measure the adaptability of cancer patients' return to work. The presentation of the manuscript in Research Square (https://doi.org/10.21203/rs.3.rs-2323264/v1).

SEC5 is involved in M2 polarization of macrophages via the STAT6 pathway, and its dysfunction in decidual macrophages is associated with recurrent spontaneous abortion.

Decidual macrophages (dMϕs) play critical roles in the establishment of microhomeostasis at the maternal-fetal interface during pregnancy. Impaired macrophage polarization during early pregnancy is associated with recurrent spontaneous abortion (RSA). In the present study, the SEC5 expression level was found to be significantly decreased in primary dMϕs of patients with RSA, and downregulation of SEC5 expression inhibited M2 polarization and STAT6 phosphorylation, whereas SEC5 overexpression in the Mϕs promoted M2 polarization and STAT6 phosphorylation in vitro. We subsequently found that SEC5 interacted with STAT6 in THP-1-derived Mϕs. The abundance of phosphorylated STAT6 (pSTAT6) protein was obviously increased, with a predominant distribution in the nucleus, after M2 polarization of Mϕs, and SEC5 protein was colocalized with pSTAT6. Moreover, a significantly reduced pSTAT6 expression level was observed in the dMϕs of patients with RSA. M2 polarization of Mϕs showed a stimulatory effect on the proliferation and invasion of human extravillous trophoblasts (EVTs) in vitro, and downregulation of SEC5 expression in Mϕs effectively reversed this effect. In a mouse model of LPS-induced early pregnancy loss, the uterine SEC5 expression level and the number of M2-Mϕs at the maternal-fetal interface were significantly reduced. More interestingly, heterozygous SEC5-deficient (SEC5-/+) pregnant mice were more sensitive to LPS-induced pregnancy loss. Taken together, these data indicate that SEC5 participates in the regulation of M2 polarization of Mϕs by interacting with STAT6 and that decreased SEC5 expression inhibits the M2 polarization of dMϕs and results in early pregnancy loss by interfering with the physical activities of EVTs and immunotolerance at the maternal-fetal interface.

[Research of preparation quality markers of Yulian Tang with anti-inflammatory activity].

The lipopolysaccharide(LPS)-indused RAW264.7 cells inflammation model was used as a carrier to investigated the effects of the preparation quality markers of Yulian Tang with anti-inflammatory activity in vitro. RAW264.7 cells were treated with LPS(50 ng·mL~(-1)) or/and different concentrations(low dose 0.1 µmol·L~(-1); medium dose 1 µmol·L~(-1); high dose 10 µmol·L~(-1)) of 18 chemical components in Yulian Tang for 24 h. Then the activity of RAW264.7 cell was detected using Cell Counting Kit-8(CCK-8) and the concentrations of inflammatory factors TNF-α and IL-6 in the supernatant of RAW264.7 cell were detected by ELISA assay. As the concentrations of chemical components in Yulian Tang increased, berberine, coptisine, magnoflorine, epiberberine, columbamine and costunolide had stronger inhibitory effects on TNF-α, whereas limonin, dehydroevodiamine, chlorogenic acid, neochlorogenic acid, groenlandicine, evodiamine, rutaecarpine and phellodendrine showed weakened inhibitory effects on TNF-α. The concentrations of palmatine, jatrorrhizine, dehydrocostus lactone and cryptochlorogenic acid had no significant effect on their inhibitory effect on TNF-α. Furthermore, dehydrorutaecarpine, chlorogenic acid, neochlorogenic acid, evodiamine, rutaecarpine, costunolide, phellodendrine and cryptochlorogenic acid showed stronger inhibitory effect on IL-6 as their concentrations increased; berberine, coptisine, magnoflorine, epiberberine, limonin, columbamine, groenlandicine and dehydrocostus lactone had no changes in their inhibitory effects on IL-6 as the concentrations increased. Palmatine and jatrorrhizine had the best inhibitory effect on IL-6. Combining the previous analysis of qualitative and quantitative preparation quality markers of Yulian Tang with the above result of dose-response relationship, we finally identified 15 preparation quality markers of Yulian Tang with anti-inflammatory activity, namely berberine, coptisine, palmatine, magnoflorine, epiberberine, limonin, columbamine, jatrorrhizine, neochlorogenic acid, chlorogenic acid, groenlandicine, evodiamine, rutaecarpine, dehydrocostus lactone and costunolide. In conclusion, our study provides a quick strategy for screening the qualitative preparation quality markers of Yulian Tang with anti-inflammatory activity. Moreover, it also provides an explicit route for the determination of preparation quality markers of Yulian Tang with other activities.

MNSFß Regulates TNFα Production by Interacting with RC3H1 in Human Macrophages, and Dysfunction of MNSFß in Decidual Macrophages Is Associated With Recurrent Pregnancy Loss.

Decidual macrophages (dMϕ) are the second largest population of leukocytes at the maternal-fetal interface and play critical roles in maintaining pregnancy. Our previous studies demonstrated the active involvement of monoclonal nonspecific suppressor factor-ß (MNSFß) in embryonic implantation and pregnancy success. MNSFß is a ubiquitously expressed ubiquitin-like protein that also exhibits immune regulatory potential, but its function in human dMϕ remains unknown. Here, we observed that the proportion of CD11chigh (CD11cHI) dMϕ was significantly increased in dMϕ derived from patients with recurrent pregnancy loss (RPL dMϕ) compared to those derived from normal pregnant women (Control dMϕ). The production of MNSFß and TNFα by RPL dMϕ was also significantly increased compared to that by Control dMϕ. Conditioned medium from RPL dMϕ exerted an inhibitory effect on the invasiveness of human trophoblastic HTR8/SVneo cells, and this effect could be partially reversed by a neutralizing antibody against TNFα. Bioinformatics analysis indicated a potential interaction between MNSFß and RC3H1, a suppressor of TNFα transcription. Immunoprecipitation experiments with human Mϕ differentiated from the human monocyte cell line Thp1 (Thp1-derived Mϕ) proved the binding of MNSFß to RC3H1. Specific knockdown of MNSFß in Thp1-derived Mϕ led to a marked decrease in TNFα production, which could be reversed by inhibiting RC3H1 expression. Interestingly, a significant decrease in the protein level of RC3H1 was observed in RPL dMϕ. Together, our findings indicate that aberrantly increased MNSFß expression in dMϕ may promote TNFα production via its interaction with RC3H1, and these phenomena could result in the disruption of the immune balance at the maternal-fetal interface and thus pregnancy loss.

Silencing SEC5 inhibits trophoblast invasion via integrin/Ca2+ signaling.

The invasion of maternal decidua by extravillous trophoblast (EVT) is essential for the establishment and maintenance of pregnancy, and abnormal trophoblast invasion could lead to placenta-associated pathologies including early pregnancy loss and preeclampsia. SEC5, a component of the exocyst complex, plays important roles in cell survival and migration, but its role in early pregnancy has not been reported. Thus, the present study was performed to explore the functions of SEC5 in trophoblast cells. The results showed that SEC5 expression in human placental villi at first trimester was significantly higher than it was at the third trimester, and it was abundantly localized in the cytotrophoblast (CTB) and the trophoblastic column. SEC5 knockdown was accompanied by reduced migration and invasion in HTR-8/SVneo cells. In addition, the expression and plasma membrane distribution of integrin ß1 was also decreased. Furthermore, shRNA-mediated knockdown of SEC5 inhibited the outgrowth of first trimester placental explants. SEC5 and InsP3R were colocalized in the cytoplasm of HTR-8/SVneo cells, and the cell-permeant calcium chelator BAPTA-AM could significantly inhibit HTR-8/SVneo cell invasion. The Ca2+ imaging results showed that the 10% fetal bovine serum-stimulated cytosolic calcium concentration ([Ca2+]c) was not only reduced by downregulated SEC5 but also was blocked by the InsP3R inhibitor. Furthermore, either the [Ca2+]c was buffered by BAPTA-AM or the knockdown of SEC5 disrupted HTR-8/SVneo cell F-actin stress fibers and caused cytoskeleton derangement. Taken together, our results suggest that SEC5 might be involved in regulating trophoblast cell migration and invasion through the integrin/Ca2+ signal pathway to induce cytoskeletal rearrangement.

Decreased NDRG1 expression is associated with pregnancy loss in mice and attenuates the in vitro decidualization of endometrial stromal cells.

Embryo implantation is an essential step for a successful pregnancy, and any defect in this process can lead to a range of pregnancy pathologies. The objective of this study was to explore the role of N-myc downregulated gene 1 (NDRG1) in embryo implantation. It was found that uterine NDRG1 expression has a dynamic pattern during the estrous cycle in nonpregnant mice and that uterine NDRG1 expression was elevated during the implantation process in pregnant mice. The distinct accumulation of NDRG1 protein signals was observed in the primary decidual zone adjacent to the implanting embryo during early pregnancy. Furthermore, uterine NDRG1 expression could be induced by activated implantation or artificial decidualization in mice. Decreased uterine NDRG1 expression was associated with pregnancy loss in mice and was associated with recurrent miscarriages in humans. The in vitro decidualization of both mouse and human endometrial stromal cells (ESCs) was accompanied by increased NDRG1 expression and downregulated NDRG1 expression in ESCs effectively inhibited decidualization. Collectively, these data suggest that NDRG1 plays an important role in decidualization during the implantation process, and the abnormal expression of NDRG1 may be involved in pregnancy loss.

Association of the peripheral blood levels of circulating microRNAs with both recurrent miscarriage and the outcomes of embryo transfer in an in vitro fertilization process.

BACKGROUND: Implantation failure is not only a major cause of early pregnancy loss, but it is also an obstacle to assisted reproductive technologies. The identification of potential circulating biomarkers for recurrent miscarriage (RM) and/or recurrent implantation failure would contribute to the development of novel diagnosis and prediction techniques. METHODS: MiR (miR-23a-3p, 27a-3p, 29a-3p, 100-5p, 127-3p and 486-5p) expression in the villi, decidual tissues and peripheral blood plasma and serum were validated by qPCR, and the localization of miRs in the villi and decidual tissues of RM and normal pregnancy (NP) women were detected by in situ hybridization. The invasiveness of HTR8/SVneo cells was determined using a Transwell assay. The predictive values of miRs for RM and the outcome of IVF-ET were respectively calculated by the receiver operating characteristic analysis. RESULTS: The signals of six miRs were observed in the villi and decidual tissues of RM and NP women. The villus miR-27a-3p, miR-29a-3p and miR-100-5p were significantly up-regulated, whereas miR-127-3p and miR-486-5p appeared to be down-regulated in RM women compared to NP women. The invasiveness of HTR8/SVneo cells transfected with miR-23a-3p mimics was evidently weakened, whereas that of cells transfected with miR-127-3p mimics was obviously enhanced. The peripheral blood plasma levels of miR-27a-3p, miR-29a-3p, miR-100-5p and miR-127-3p were significantly increased, whereas that of miR-486-5p was remarkably decreased in RM compared to NP women. By contrast, serum miR-23a-3p and miR-127-3p were significantly decreased, whereas that of miR-486-5p was remarkably increased. The combination of six plasma miRs levels discriminated RM with a sensitivity of 100% and a specificity of 83.3%, whereas that of six serum miRs levels showed a sensitivity of 78.3% and a specificity of 93.1%. In the IVF-ET cohort, the significantly decreased peripheral blood plasma levels of miR-23a-3p, miR-27a-3p, miR-100-5p and miR-127-3p, and the serum levels of miR-100-5p and miR-486-5p, in addition to the significantly increased serum level of miR-27a-3p, were found to be associated with the failure of ET. Moreover, the combination of plasma miR-23a-3p, miR-27a-3p, miR-29a-3p, miR-100-5p, miR-127-3p and miR-486-5p levels discriminated the outcome of IVF-ET with a sensitivity of 68.1% and a specificity of 54.1%, whereas the combination of plasma miR-127-3p and miR-486-5p levels showed a sensitivity of 50.0% and a specificity of 75.3%. CONCLUSIONS: Circulating miR-23a-3p, miR-27a-3p, miR-29a-3p, miR-100-5p, miR-127-3p and miR-486-5 might be involved in RM pathogenesis and present potential diagnostic biomarkers for RM. Meanwhile, these miRs, in particular miR-127-3p and miR-486-5p, provide promising prediction indexes for the outcomes of IVF-ET.

miR-3074-5p Promotes the Apoptosis but Inhibits the Invasiveness of Human Extravillous Trophoblast-Derived HTR8/SVneo Cells In Vitro.

OBJECTIVE: The objective of this study was to observe the effects of the overexpression of miR-3074-5p in human trophoblast cells in vitro. DESIGN: Experimental in vitro study in HTR8/SVneo cells. METHODS: HTR8/SVneo cells were transfected with miR-3074-5p mimic. The cell apoptosis and invasion were measured via flow cytometry and transwell assay, respectively. The expression levels of P53, Cyclin Dependent Kinase Inhibitor 1B (P27), BCL-2, BCL2 associated X (BAX), and BCL2 like 14 (BCL-G) in HTR8/SVneo cells were determined by Western blot. The alterations in gene expression profile of HTR8/SVneo cells were evaluated by complementary DNA microarray assay, and the differential expressions of dihydrolipoamide S-succinyltransferase (DLST), growth-associated protein 43 (GAP43), runt-related transcription factor 2 (RUNX2), and C-C type chemokine receptor 3 (CCR3) were validated by Western blot. Biofunctions of these differentially expressed genes were enriched by Gene Ontology analysis. RESULTS: The overexpression of miR-3074-5p in HTR8/SVneo cells promoted cell apoptosis but inhibited cell invasion, being accompanied by the significantly elevated expressions of P27, BCL-2, and BCL-G. Meanwhile, an increased expression of P27 and P57 was also detected in a small sample size of placental villi of recurrent miscarriage (RM) patients. Totally, 411 genes and 397 genes were screened out, respectively, to be downregulated or upregulated at least by 2-folds in miR-3074-5p overexpressed HTR8/SVneo cells. These differentially expressed genes were involved in several important functions related to pregnancy. Subsequently, the reduced expressions of DLST and GAP43 proteins, as well as the increased expressions of CCR3 and RUNX2 proteins, were validated in miR-3074-5p overexpressed HTR8/SVneo cells. CONCLUSION: These data suggested a potential contribution of miR-3074-5p in the pathogenesis of RM by disturbing the normal activities of trophoblast cells.

Validation of the EQ-5D in a general population sample in urban China.

PURPOSE: To evaluate the reliability and validity of the EQ-5D in a general population sample in urban China. METHODS: Thousand and eight hundred respondents in 18 communities of Hangzhou, China were recruited by multi-stage stratified random sampling. Respondents self-administered a questionnaire including the EQ-5D, the SF-36, and demographic questions. Test-retest reliability at 2-week intervals was evaluated using Kappa coefficient, the intraclass correlation coefficient. The standard error of measurement (SEM) was used to indicate the absolute measurement error. Construct validity was established using convergent, discriminant, and known groups analyses. RESULTS: Complete data for all EQ-5D dimensions were available for 1,747 respondents (97%). Kappa values were from 0.35 to 1.0. The ICCs of test-retest reliability were 0.53 for the EQ-5D index score and 0.87 for the EQ VAS score. The SEM values were 0.13 (9.22% range) and 4.20 (4.20% range) for the EQ-5D index and EQ VAS scores, respectively. The Pearson's correlation coefficients between the EQ-5D and the SF-36 were stronger between comparable dimensions than those between less comparable dimensions, demonstrating convergent and discriminant evidence of construct validity. The Chinese EQ-5D distinguished well between known groups: respondents who reported poor general health and chronic diseases had worse HRQoL than those without. Older people, females, people widowed or divorced, and those with a lower socioeconomic status reported poorer HRQoL. Respondents reporting no problems on any EQ-5D dimension had better scores on the SF-36 summary scores than those reporting problems. CONCLUSIONS: The Chinese version of the EQ-5D demonstrated acceptable construct validity and fair to moderate levels of test-retest reliability in an urban general population in China.