Epidermal growth factor: Expression in goat endometrial epithelia during early pregnancy and regulation by interferon tau and FOXO1.

In ruminants, establishment and maintenance of pregnancy depends upon a well-coordinated interaction between the conceptus and the maternal endometrium. Epidermal growth factor (EGF) is important for embryo implantation and pregnancy establishment. However, the regulatory mechanisms of EGF expression remain unclear. FOXO1, a member of the Forkhead box O (FOXO) subfamily of transcription factors, is currently accepted as a novel endometrial receptivity marker for humans and mice owing to its timely and specific expression at the window of implantation. In this study, we examined the spatiotemporal expression profile of EGF in goat uterus during early pregnancy (Day 0 to Day 50 of pregnancy) and verified that EGF expression was regulated by FOXO1 and interferon tau (IFNT). Our results showed that EGF was highly expressed in the luminal epithelium (LE) and the glandular epithelium (GE) during conceptus adhesion (Day 16 to Day 25 of pregnancy). After implantation, EGF protein signals were continuously detected in the endometrial epithelia and appeared in the conceptus trophectoderm. Furthermore, EGF expression could be up-regulated by IFNT in goat uterus and primary endometrial epithelium cells (EECs). The luciferase assay results showed that FOXO1 could promote EGF transcription by binding to its promoter. And FOXO1 positively regulates EGF expression in goat EECs. These findings contribute to better understanding the role and regulation mechanisms of EGF during ruminant early pregnancy.

An IFNT/FOXO1/PTGS2 axis regulates prostaglandin F2α synthesis in goat uterus during early pregnancy.

In ruminants, IFN-tau (IFNT) regulates the production of prostaglandins (PG) in the endometrium, which is crucial for conceptus adhesion. However, the related molecular regulatory mechanisms remain unclear. Forkhead box O1 (FOXO1), a member of the FOXO subfamily of transcription factors, is known to be important for mouse implantation and decidualization. In this study, we determined the spatiotemporal expression profile of FOXO1 in goat endometrium during early pregnancy. FOXO1 was highly expressed in the glandular epithelium since the onset of conceptus adhesion (d 16 of pregnancy). Then, we validated that FOXO1 could bind to the promoter of prostaglandin-endoperoxide synthase 2 (PTGS2) and increase its transcription. And the expression profile of PTGS2 was similar to that of FOXO1 in the peri-implantation uterus. Moreover, IFNT could upregulate the levels of FOXO1 and PTGS2 in goat uterus and primary endometrial epithelium cells (EEC). In EEC, the intracellular content of PGF2α was positively correlated with the levels of IFNT and FOXO1. Altogether, we found an IFNT/FOXO1/PTGS2 axis that controls the synthesis of PGF2α but not prostaglandin E2 in goat uterine glands. These findings contribute to better understanding the function of FOXO1 in the reproductive physiology of goats and provide more insights into the implantation of small ruminants.

METTL3 promotes proliferation of goat endometrial epithelial cells by regulating CTGF in an m6A-dependent manner†.

N6-methyladenosine (m6A), an epigenetic modification on RNAs, plays an important role in many physiological and pathological processes. However, the involvement of m6A in goat uterus during early pregnancy remains largely unknown. In this study, we found that the total m6A level was increasing in goat uterus as early pregnancy progressed. Methyltransferase-like 3 (METTL3) is a core catalytic subunit of the m6A methyltransferase. We thus determined the expression and regulation of METTL3 in goat uterus. METTL3 was highly expressed in the luminal and glandular epithelia from day 16 (D16) to D25 of pregnancy, and it could be up-regulated by estrogen and progesterone in goat uterus and primary endometrial epithelial cells (EECs). In EECs, knockdown or overexpression of METTL3 resulted in a significant decrease or increase of cell proliferation, respectively. METTL3 knockdown reduced the m6A level of not only total RNA but also connective tissue growth factor (CTGF) mRNA. Luciferase assay suggested that METTL3 might target the potential m6A sites in the 3'untranslated region (3'UTR) of CTGF mRNA. Moreover, METTL3 positively regulated CTGF expression, and CTGF knockdown significantly counteracted the promoting effect of METTL3 overexpression on EEC proliferation. Collectively, METTL3 is dynamically expressed in goat uterus and can affect EEC proliferation by regulating CTGF in an m6A-dependent manner. Our results will lay a foundation for further studying the crucial mechanism of METTL3-mediated m6A modification in goat uterus during early pregnancy.

Expression and hormone regulation of UCP2 in goat uterus.

Pregnancy success is closely related to the molecular mechanisms that control energy metabolism balance. However, the mechanisms have not been fully understood. Uncoupling protein 2 (UCP2) plays a physiological role by regulating energy metabolism in numerous tissues. In this study, we determined the expression and hormone regulation of UCP2 in goat uterus. UCP2 is expressed in the luminal and glandular epithelia of goat uterus during early pregnancy, as revealed by in situ hybridization and immunohistochemistry conducted on pregnant goats. The signals were detected from day 0 (D0) to D30 of pregnancy, though weak on D16 (the adhesion period). The low levels of UCP2 on D16 were confirmed by RT-qPCR and western blot. In goat uterus and endometrial epithelial cells (EECs), UCP2 was up-regulated by progesterone and estrogen. In addition, after goat EECs were treated with genipin (an inhibitor of UCP2), not only UCP2 expression but also cell proliferation was inhibited. Collectively, UCP2 is dynamically expressed in goat uterus and can affect EEC proliferation, suggesting that it may participate in regulating the energy metabolism balance of goat uterus during early pregnancy.

Peri-implantation expression and regulation of ITGB8 in goat uterus.

Ruminants have a superficial implantation pattern. The extended conceptus attaches to the receptive endometrium to form the cotyledonary placenta. During the attachment, a large number of events occur at the maternal-fetal interface. However, the related molecular mechanisms have not been fully understood. Integrin beta8 (ITGB8) is a subunit of integrin beta involved in embryo implantation. In this study, we determined peri-implantation expression and regulation of ITGB8 in goat uterus. The mRNA and protein levels of ITGB8 were both high in goat endometrial luminal epithelium (LE) and superficial glandular epithelium (sGE) during the adhesion period (Days 16-19 of pregnancy). Such expression profile was opposite to that of microRNA-187 (miR-187). Then, we validated that miR-187 targeted the 3' untranslated region (UTR) of ITGB8 in primary goat endometrial epithelial cells (EECs). In EECs, inhibition of miR-187 resulted in not only up-regulated ITGB8 level but also reduced cell proliferation and focal adhesion kinase (FAK) activity. Moreover, ITGB8 and miR-187 were regulated by interferon tau (IFNT). Altogether, in goat, the miR-187/ITGB8 axis may be involved in conceptus attachment and is downstream of IFNT. Our results will help us better understand the mechanisms of ruminant implantation and may provide a useful tool to improve the reproduction ratio for ruminants.

Mus81 knockdown sensitizes colon cancer cells to chemotherapeutic drugs by activating CHK1 pathway.

PURPOSE: The inhibition of Mus81, a critical DNA repair gene, is recently related to the chemosensitivity of several human cancer cells such as hepatocellular carcinoma (HCC) cells. However, the role of Mus81 knockdown in chemotherapy response of colon cancer cells remains largely unknown. METHODS AND MATERIALS: The effects of Mus81 knockdown by lentivirus-mediated short hairpin RNA in sensitivity of HCT116 and LS180 colon cancer cell lines to four therapeutic drugs, including cisplatin (CDDP), were evaluated by MTT assay as well as a mouse model. Apoptosis and cell cycle distribution of HCT116 cell line was detected by flow cytometric analysis. Western blot was also employed to determine the expression of CHK1 pathway and apoptosis-related proteins in HCT116 cells and the xenograft mouse tumors. RESULTS: Mus81 knockdown could significantly improve the chemosensitivity of colon cancer cells in vitro and in vivo, especially to CDDP. Mus81 knockdown also induced S phase arrest and elevated apoptosis in CDDP treated HCT116 cells through activating CHK1/CDC25A/CDK2 and CHK1/p53/Bax pathways, while these effects could be counteracted by CHK1 inhibition. CONCLUSION: Mus81 knockdown improves the chemosensitivity of colon cancer cells by inducing S phase arrest and promoting apoptosis through activating CHK1 pathway.

STC2 as a novel mediator for Mus81-dependent proliferation and survival in hepatocellular carcinoma.

Methyl methansulfonate and UV sensitive gene clone 81 (Mus81) is a critical DNA repair gene that has been implicated in development of several cancers including hepatocellular carcinoma (HCC). However, whether Mus81 can affect proliferation and survival of HCC remains unknown. In the present study, we demonstrated that the knockdown of Mus81 was associated with suppressed proliferation and elevated apoptosis of HCC cells in vitro and in vivo. Multilayered screenings, including DNA microarray, high content screen, and real-time PCR validation, identified STC2 as a proliferation-facilitating gene significantly down-regulated in HCC cells upon Mus81 knockdown. STC2 expression was also closely correlated to Mus81 expression in HCC tissues. More importantly, the restoration of STC2 expression recovered the compromised cell proliferation and survival in Mus81 depleted HCC cells. Furthermore, Mus81 knockdown was associated with the activation of APAF1, APC, and PTEN pathways and concurrent inhibition of MAPK pathway through decreasing STC2 expression. In conclusion, Mus81 knockdown suppresses proliferation and survival of HCC cells likely by downregulating STC2 expression, implicating Mus81 as a therapeutic target for HCC.