Fanconi anemia complementation group D2 promotes sensitivity of endometrial cancer cells to chemotherapeutic agents by inhibiting the ferroptosis pathway.

BACKGROUND: Resistance can develop during treatment of advanced endometrial cancer (EC), leading to unsatisfactory results. Fanconi anemia complementation group D2 (Fancd2) has been shown to be closely related to drug resistance in cancer cells. Therefore, this study was designed to explore the correlation of Fancd2 with EC resistance and the mechanism of Fancd2. METHODS: Real-time quantitative PCR (RT-qPCR) was used to detect the expression of Fancd2 in EC tissues and cells. EC cells (Ishikawa) and paclitaxel-resistant EC cells (Ishikawa/TAX) were transfected to knock down Fancd2. In addition, the ferroptosis inhibitor Ferrostatin-1 was adopted to treat Ishikawa/TAX cells. The sensitivity of cancer cells to chemotherapeutic agents was observed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and inhibitory concentration (IC)50 was calculated. Reactive oxygen species (ROS) levels were measured by flow cytometry, the activity of malondialdehyde (MDA) and the levels of glutathione (GSH) and Fe2+ in cells were detected by corresponding kits, and protein expression of solute farrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) was obtained through western blot. RESULTS: Compared with the normal tissues and endometrial epithelial cells, Fancd2 expression was significantly increased in EC tissues and Ishikawa cells, respectively. After knock-down of Fancd2, Ishikawa cells showed significantly increased sensitivity to chemotherapeutic agents. Besides, compared with Ishikawa cells, the levels of ROS, the activity of MDA, and the levels of GSH and Fe2+ were significantly decreased in Ishikawa/TAX cells, while the expression levels of SLC7A11 and GPX4 were significantly increased. Knock-down of Fancd2 significantly increased the ferroptosis levels in Ishikawa/TAX cells, but this effect could be reversed by Ferrostatin-1. CONCLUSION: Fancd2 increases drug resistance in EC cells by inhibiting the cellular ferroptosis pathway.

Comparison of the efficacy and safety of total laparoscopic hysterectomy without and with uterine manipulator combined with pelvic lymphadenectomy for early cervical cancer.

OBJECTIVE: Some studies have reported that the prognosis of total laparoscopic hysterectomy (TLH) for early-stage cervical cancer (CC) is worse than that of open surgery. And this was associated with the use of uterine manipulator or not. Therefore, this study retrospectively analyzes the efficacy and safety of TLH without uterine manipulator combined with pelvic lymphadenectomy for early-stage CC. METHODS: Fifty-eight patients with CC (stage IB1-IIA1) who received radical hysterectomy from September 2019 to January 2020 were divided into no uterine manipulator (n = 26) and uterine manipulator group (n = 32). Then, clinical characteristics were collected and intraoperative/postoperative related indicators were compared. RESULTS: Patients in the no uterine manipulator group had significantly higher operation time and blood loss than in the uterine manipulator group. Notably, there was no significant difference in hemoglobin change, blood transfusion rate, number of pelvic nodules, anal exhaust time, complications and recurrence rate between the two groups. Additionally, patients in the uterine manipulator group were prone to urinary retention (15.6%) and lymphocyst (12.5%), while the no uterine manipulator group exhibited high probability of bladder dysfunction (23.1%) and urinary retention (15.4%). Furthermore, the 1-year disease-free survival rate and the 1-year overall survival rate were not significantly different between the two groups. CONCLUSION: There was no significant difference in the efficacy and safety of TLH with or without uterine manipulator combined with pelvic lymphadenectomy in the treatment of patients with early-stage CC. However, the latter requires consideration of the negative effects of high operation time and blood loss.

Long non-coding RNAs in hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs) refer to a class of RNAs greater than 200 nucleotides in length, most of which are considered unable to encode proteins, thus deemed to be junk genes formerly. But with emerging studies about lncRNAs coming out in recent years, it is much more clearly depicted that they can regulate gene expression at different levels, with various mechanisms, thus participating in diverse biological or pathological processes, including complicated tumor-associated pathways. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, the third leading cause of cancer-related mortality worldwide, which has been found to tightly associate with aberrant expression of a variety of lncRNAs regulating tumor proliferation, invasion, drug resistance, and so on, making it a potential novel tumor marker and therapeutic target. In this review, we highlight a few lncRNAs that are closely related to the occurrence and progression of HCC and try to cover their multifarious roles from different layers.

Removal of nonspecific binding proteins is required in co-immunoprecipitation with nuclear proteins.

Whether protein samples should be pretreated to remove nonspecific binding proteins in co-immunoprecipitation (CO-IP) is controversial. In this work, nonspecific binding of proteins to agarose beads was found to be greater than that to magnetic beads. The nonspecific binding was increased with the decrease of ion concentrations but reduced by Nonidet P40. Western blot indicated that p65 and ß-actin were present as nonspecifically bound protein to the beads. p53 and ß-actin were present in the CO-IP precipitates of nuclear proteins but pretreatment cleared the nonspecifically pulled down p53 and ß-actin. These data suggest that magnetic beads are better for CO-IP, but preclearing is necessary to minimize false positive regardless of which bead is used, particularly for nuclear proteins.

PDIA3 regulates trophoblast apoptosis and proliferation in preeclampsia via the MDM2/p53 pathway.

Protein disulfide isomerase 3 (PDIA3) is a chaperone protein that modulates the folding of newly synthesized glycoproteins, has isomerase and redox activity, and has been implicated in the pathogenesis of many diseases. However, the role of PDIA3 in pregnancy-associated diseases remains largely unknown. Our present study reveals a key role for PDIA3 in the biology of placental trophoblasts from women with preeclampsia (PE). Immunohistochemistry and Western blot analysis revealed that PDIA3 expression was decreased in villous trophoblasts from women with PE compared to normotensive pregnancies. Further, using a Cell Counting Kit-8 assay, flow cytometry, and 5-ethynyl-2'-deoxyuridine (EdU) staining, we found that siRNA-mediated PDIA3 knockdown significantly promoted apoptosis and inhibited proliferation in the HTR8/SVneo cell line, while overexpression of PDIA3 reversed these effects. Furthermore, RNA sequencing and Western blot analysis demonstrated that knockdown of PDIA3 inhibited MDM2 protein expression in HTR8 cells, concurrent with marked elevation of p53 and p21 expression. Conversely, overexpression of PDIA3 had the opposite effects. Immunohistochemistry and Western blot further revealed that MDM2 protein expression was downregulated and p21 was increased in trophoblasts of women with PE compared to women with normotensive pregnancies. Our findings indicate that PDIA3 expression is decreased in the trophoblasts of women with PE, and decreased PDIA3 induces trophoblast apoptosis and represses trophoblast proliferation through regulating the MDM2/p53/p21 pathway.

Downregulation of CCNA2 disturbs trophoblast migration, proliferation, and apoptosis during the pathogenesis of recurrent miscarriage.

PROBLEM: Recurrent miscarriage (RM) is defined as two or more pregnancy losses until 24 weeks of gestation, which distresses up to 1-5% of couples worldwide. Cyclin A2 (CCNA2) regulates the cell cycle by promoting transition through G1/S and G2/M. Little is known about CCNA2's functions in trophoblast, although it is highly expressed in the placenta. We therefore sought to explore the role of CCNA2 in RM and early pregnancy. METHOD OF STUDY: First-trimester human placental tissues were collected from patients with RM and normal pregnant women to clarify the expression level of CCNA2. First-trimester human villi explants culture was performed as an ex vivo model to study the functions of CCNA2. The HTR8/SVneo cells were studied for in vitro experiments. The migration, proliferation, and apoptosis levels of trophoblast were examined in the CCNA2-knockdown and CCNA2-overexpressing HTR8 cells. Results Our study revealed that CCNA2 was downregulated in trophoblast of RM first-trimester villi. Results showed that CCNA2 promotes migration of HTR8 cells via the RhoA-ROCK signaling and that CCNA2 increased HTR8 proliferation and inhibited their apoptosis via p53 pathway. In addition, the relationship between ROCK signaling and p53 pathway was found. Further, mechanistic research attributed the regulatory function of CCNA2 on p53 pathway to the involvement of DNA damage. Conclusion Our study revealed the downregulation of CCNA2 in first-trimester chorionic villi of patients with RM and showed that this protein regulates migration, proliferation, and apoptosis in HTR8 cells, suggesting that CCNA2 may be the potential therapeutic target for RM.

EIF5A1 promotes trophoblast migration and invasion via ARAF-mediated activation of the integrin/ERK signaling pathway.

Trophoblast dysfunction is one mechanism implicated in the etiology of recurrent miscarriage (RM). Regulation of trophoblast function, however, is complex and the mechanisms contributing to dysregulation remain to be elucidated. Herein, we found EIF5A1 expression levels to be significantly decreased in cytotrophoblasts in RM villous tissues compared with healthy controls. Using the HTR-8/SVneo cell line as a model system, we found that overexpression of EIF5A1 promotes trophoblast proliferation, migration and invasion in vitro. Knockdown of EIF5A1 or inhibiting its hypusination with N1-guanyl-1,7-diaminoheptane (GC7) suppresses these activities. Similarly, mutating EIF5A1 to EIF5A1K50A to prevent hypusination abolishes its effects on proliferation, migration and invasion. Furthermore, upregulation of EIF5A1 increases the outgrowth of trophoblasts in a villous explant culture model, whereas knockdown has the opposite effect. Suppression of EIF5A1 hypusination also inhibits the outgrowth of trophoblasts in explants. Mechanistically, ARAF mediates the regulation of trophoblast migration and invasion by EIF5A1. Hypusinated EIF5A1 regulates the integrin/ERK signaling pathway via controlling the translation of ARAF. ARAF level is also downregulated in trophoblasts of RM villous tissues and expression of ARAF is positively correlated with EIF5A1. Together, our results suggest that EIF5A1 may be a regulator of trophoblast function at the maternal-fetal interface and low levels of EIF5A1 and ARAF may be associated with RM.