ABSTRACT
[This corrects the article DOI: 10.3389/fcell.2020.00587.].
ABSTRACT
BACKGROUND: 4-vinylcyclohexene diepoxide (VCD) has long been considered a hazardous occupational chemical that promotes ovarian failure. However, VCD is also used as a research compound to chemically induce animal models of premature ovarian insufficiency (POI), and in related work we unexpectedly found that VCD apparently exhibits both dose- and duration-dependent opposing, hormone-like effects on the maintenance of the primordial follicle pool, follicle development, and ovulation induction. RESULTS: We conducted experiments with cultured murine ovaries and performed transplantation experiments using postnatal day (PD) 2 and PD12 mice and found that low-dose, short-term exposure to VCD (VCDlow) actually protects the primordial/primary follicle pool and improves the functional ovarian reserve (FOR) by disrupting follicular atresia. VCDlow inhibits follicular apoptosis and regulates the Pten-PI3K-Foxo3a pathway. Short-term VCD exposure in vivo (80 mg/kg, 5 days) significantly increases the number of superovulated metaphase II oocytes, preovulatory follicles, and corpus luteum in middle-aged mice with diminished ovarian reserve (DOR). We demonstrate that low-dose but not high-dose VCD promotes aromatase levels in granulosa cells (GCs), thereby enhancing the levels of estradiol secretion. CONCLUSION: Our study illustrates a previously unappreciated, hormone-like action for the occupational "ovotoxin" molecule VCD and strongly suggests that VCDlow should be explored for its potential utility for treating human ovarian follicular development disorders, including subfertility in perimenopausal women.
ABSTRACT
Bag1 transcribes a multifunctional protein that participates in many important biological processes such as cell apoptosis, proliferation, differentiation and embryo development. Despite numerous published studies, the role of Bag1 in the context of embryonic stem (ES) cells, has not been explored. To investigate the function of Bag1 in ES cells, we generated mutant Bag1-/- ES cells using the CRISPR/Cas9 system. We established that the Bag1 double knockout ES cell line maintained their pluripotency, possessed a normal karyotype and the ability to differentiate into all three germ layers.
Subject(s)
CRISPR-Cas Systems , DNA-Binding Proteins/deficiency , Homozygote , Mouse Embryonic Stem Cells/metabolism , Transcription Factors/deficiency , Animals , Cell Line , Mice , Mice, Knockout , Mouse Embryonic Stem Cells/cytologyABSTRACT
OBJECTIVE: To discuss the effect and molecular mechanism of miR-146a on the proliferation of lung cancer cells by targeting and regulating the macrophage migration inhibitory factor (MIF) gene. METHODS: RT-PCR was employed to detect expression of miR-146a; immunohistochemistry was used to detect the expression of MIF. The luciferase reporter gene technique was adopted to verify that MIF was the specific reverse target gene of miR-146a and the liposome Lipofectamine™2000 was employed to transfer the modeled miR-146a mimics, and miR-146a negative control (NC) in NSCLC cells to detect the expression of MIF mRNA and protein. MTT assay was used to detect cell viability, cloning technique to detect cell proliferation ability, AnnexinV-PI to detect cell apoptosis, UV spectrophotometry to detect viability of cysteinyl aspartate specific proteinase 3 (Caspase 3), and western blot to detect expression of nuclear factor-κB (NF-κB) in cells. RESULTS: The expression of miR-146a in NSCLC lung tissues was lower than that in the normal lung tissues besides the lung cancer; while the expression of miR-146a in NSCLC cells was lower than that in normal human embryonic lung tissues. It was chosen as the subsequent cell line for its appropriate expression in A549. The expression of MIF protein in NSCLC lung tissues was higher than that in the normal lung tissues besides the lung cancer. The luciferase reporter gene proved that MIF was the reverse target gene of miR-146a. The miR-146a mimics were transfected into A549 cells through the liposome. Compared with NC group, the expression of MIF protein and mRNA was significantly decreased (P < 0.01), with the decrease in the cell viability (P < 0.01), the decrease in the number of clones (P < 0.01), cell apoptosis (P < 0.01), the increase in the activity of Caspase 3 (P < 0.01), and decrease in the phosphorylation of NF-κB p65 (P < 0.01). CONCLUSIONS: miR-146a has low expression in NSCLC tissues and cell lines, while MIF has the over expression in NSCLC tissues. The increased expression of miR-146a can inhibit the expression of MIF via the gene targeting and thus inhibit the proliferation of A549 cells and induce the apoptosis of cancer cells, which may be realized through NF-κB signaling pathway.
ABSTRACT
AIM: To investigate the effect of hyperthermia on hypoxia-induced epithelial-mesenchymal transition (EMT) in HepG2 hepatocellular carcinoma (HCC) cells, and its mechanism. METHODS: Cells were treated with hyperthermia at 43â °C for 0.5 h, followed by incubation under hypoxic or normoxic conditions for 72 h. Cell morphology was observed. Expressions of E-cadherin and vimentin were determined by immunofluorescence assay or Western blot. The protein and mRNA expressions of Snail were also determined by Western blot and reverse transcription-polymerase chain reaction. Cell migratory capacity was evaluated. RESULTS: Hypoxia induced EMT in HepG2 cells, which was evidenced by morphological, molecular and functional changes, including the formation of a spindle shape and the loss of cell contact. The expression of E-cadherin was decreased but the expression of vimentin was increased; also, the migratory capability was increased by 2.2 ± 0.20-fold as compared with normoxia. However, those effects were inhibited by hyperthermia pretreatment. Furthermore, protein synthesis and mRNA expression of Snail in the cells were enhanced by hypoxia as compared with normoxia, and also significantly inhibited by hyperthermia pretreatment. CONCLUSION: Hyperthermia may inhibit hypoxia-induced EMT in HepG2 HCC cells, and the mechanism may involve inhibition of induced expression of Snail.
