CDT1 is the major functional regulatory subunit of the pre‐replication complex in zygotes

Pre‐replication complex (pre‐RC) is critical for DNA replication initiation. CDT1 and MCM2 are the subunits of pre‐RC, and proper regulation of CDT1 and MCM2 are necessary for DNA replication and cell proliferation. The present study aimed to explore the role of CDT1 and MCM2 in oocyte meiotic maturation and early embryonic development. The depletion and overexpression of Cdt1 and Mcm2 in oocyte and zygote were achieved by microinjecting specific siRNA and mRNA to explored their functions in oocyte meiotic maturation and embryonic development. Then, we examined the effect of CDT1 and MCM2 on other signal pathways by immunostaining the expression of related maker genes. We showed that neither depletion nor overexpression of Cdt1 affected oocyte meiotic progressions. The CDT1 was degraded in S phase and remained at a low level in G2 phase of zygote. Exogenous expression of Cdt1 in G2 phase led to embryo attest at zygote stage. Mechanistically, CDT1 overexpression induced DNA re‐replication and thus DNA damage check‐point activation. Protein abundance of MCM2 was stable throughout the cell cycle, and embryos with overexpressed MCM2 could develop to blastocysts normally. Overexpression or depletion of Mcm2 also had no effect on oocyte meiotic maturation. Our results indicate that pre‐RC subunits CDT1 and MCM2 are not involved in oocyte meiotic maturation. In zygote, CDT1 but not MCM2 is the major regulator of DNA replication in a cell cycle dependent manner. Furthermore, its'' degradation is essential for zygotes to prevent from DNA re‐replication in G2 stage.

Pre‐replication complex (pre‐RC) is formed in G1 phase during which CDT1 is localized in nucleus. When zygote enters S phase, CDT1 is degraded and stays at a low level in G2 phase. However, the expression of exogenous Cdt1 mRNA in G2 phase of fertilized egg, mimicking events in which CDT1 degradation is disrupted, pre‐RC reassembles and leads to DNA re‐replication, and thus DNA damage check point activation, which results in embryo arrest at G2/M phase.     

Increased SPON1 promotes pancreatic ductal adenocarcinoma progression by enhancing IL‐6 trans‐signalling

ObjectivesThis study investigated the specific molecular mechanism and the roles of extracellular matrix protein Spondin 1 (SPON1) in the development of pancreatic ductal adenocarcinoma (PDAC).Materials and MethodsThe expression pattern and clinical relevance of SPON1 was determined in GEO, Ren Ji and TCGA datasets, further validated by immunohistochemical staining and Kaplan‐Meier analysis. Loss and gain of function experiments were employed to investigate the cellular function of SPON1 in vitro. Gene set enrichment analysis, luciferase assay, immunofluorescence and Western blot and immunoprecipitation were applied to reveal the underlying molecular mechanisms. Subcutaneous xenograft model was used to test the role of SPON1 in tumour growth and maintenance in vivo.ResultsSPON1 is significantly upregulated in PDAC tumour tissues and correlated with progression of PDAC. Loss and gain of function experiments showed that SPON1 promotes the growth and colony formation ability of pancreatic cancer cells. Combining bioinformatics assays and experimental signalling evidences, we found that SPON1 can enhance the IL‐6/JAK/STAT3 signalling. Mechanistically, SPON1 exerts its oncogenic roles in pancreatic cancer by maintaining IL‐6R trans‐signalling through stabilizing the interaction of soluble IL‐6R (sIL‐6R) and glycoprotein‐130 (gp130) in PDAC cells. Furthermore, SPON1 depletion greatly reduced the tumour burden, exerted positive effect with gemcitabine, prolonging PDAC mice overall survival.ConclusionsOur data indicate that SPON1 expression is dramatically increased in PDAC and that SPON1 promotes tumorigenicity by activating the sIL‐6R/gp130/STAT3 axis. Collectively, our current work suggests SPON1 may be a potential therapy target for PDAC patient.

Extracellular matrix protein spondin 1 is significantly upregulated in PDAC tumour cell, which exerts its oncogenic roles in pancreatic cancer by maintaining IL6R trans‐signalling through stabilizing the interaction of sIL6R and GP130 in PDAC cell, resulting in STAT3 signalling activating and tumour cell growth.     

Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization

The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P < 0.05) increased polyspermy during porcine IVF and reduced (P < 0.05) UCH enzymatic activity measured in motile boar spermatozoa using a specific fluorometric UCH substrate, ubiquitin-AMC. Antibodies against two closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6-8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility.     

HOIL‐1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation

HOIL‐1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase‐inactive HOIL‐1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL‐1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL‐1 monoubiquitylates glycogen and α1:4‐linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester‐linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100‐fold by the interaction of Met1‐linked or Lys63‐linked ubiquitin oligomers with the RBR domain of HOIL‐1. HOIL‐1 also transferred pre‐formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL‐1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL‐1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.     

Adverse PFAS effects on mouse oocyte in vitro maturation are associated with carbon‐chain length and inclusion of a sulfonate group

ObjectivesPer‐ and polyfluoroalkyl substances (PFAS) are man‐made chemicals that are widely used in various products. PFAS are characterized by their fluorinated carbon chains that make them hard to degrade and bioaccumulate in human and animals. Toxicological studies have shown PFAS toxic effects: cytotoxicity, immunotoxicity, neurotoxicity, and reproductive toxicity. However, it is still unclear how the structures of PFAS, such as carbon‐chain length and functional groups, determine their reproductive toxicity.Methods and ResultsBy using a mouse‐oocyte‐in‐vitro‐maturation (IVM) system, we found the toxicity of two major categories of PFAS, perfluoroalkyl carboxylic acid (PFCA) and perfluoroalkyl sulfonic acid (PFSA), is elevated with increasing carbon‐chain length and the inclusion of the sulfonate group. Specifically, at 600 μM, perfluorohexanesulfonic acid (PFHxS) and perfluorooctanesulfonic acid (PFOS) reduced the rates of both germinal‐vesicle breakdown (GVBD) and polar‐body extrusion (PBE) as well as enlarged polar bodies. However, the shorter PFSA, perfluorobutanesulfonic acid (PFBS), and all PFCA did not show similar adverse cytotoxicity. Further, we found that 600 μM PFHxS and PFOS exposure induced excess reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP). Cytoskeleton analysis revealed that PFHxS and PFOS exposure induced chromosome misalignment, abnormal F‐actin organization, elongated spindle formation, and symmetric division in the treated oocytes. These meiotic defects compromised oocyte developmental competence after parthenogenetic activation.ConclusionsOur study provides new information on the structure‐toxicity relationship of PFAS.

Reproductive toxicity of PFAS is determined by their chemical structures. An inverted U‐shaped relationship is shown between toxic effects of PFAS and carbon‐chain length; carbon‐chain length around 10 is the most toxic. PFSA has higher and unique toxicity effects on mouse oocyte maturation compared to PFCA because PFSA has an extra sulfonate group.     

Lack of SIRP‐alpha reduces lung cancer growth in mice by promoting anti‐tumour ability of macrophages and neutrophils

ObjectivesSignal regulatory protein‐alpha (SIRPα) is a transmembrane glycoprotein specifically expressed on myeloid cells. Blockade of SIRPα/CD47 interaction is effective in combinational therapy of some cancers. This study aimed to explore into the role and underlying molecular mechanisms of SIRPα in lung cancer growth.Materials and MethodsA mouse model with lung cancer in wild‐type (WT) and SIRPα‐knockout mouse (KO) mice was established by subcutaneous injection of Lewis murine lung cancer cells (LLC). Circulating monocytes and neutrophils were depleted in mice by intraperitoneal administration of clodronate liposomes and anti‐Ly6G antibody, respectively. Phenotypes and phagocytosis of macrophages and neutrophils were analysed by flow cytometry. Transwell assay was used to analyse LLC cells migration and invasion.ResultsLack of SIRPα inhibited LLC cells growth in KO mice, associated with reduced infiltrating PD‐1⁺CD8⁺ T cells and production of IL‐6 from infiltrating macrophages and neutrophils in tumour tissues. Depletion of circulating monocytes and neutrophils reduced LLC cells growth in WT mice, which was abolished in KO mice. Studies in vitro showed that lack of SIRPα increased M1/M2 ratio, and reduced LLC cell migration and invasion via attenuated IL‐6 secretion. Lack of SIRPα expression in neutrophils effectively increased the cytotoxic activity to LLC cells in vitro.ConclusionsLack of SIRPα suppressed lung cancer cell growth in mice, dependent on circulating macrophages and neutrophils, in association with improved phagocytosis and reduced IL‐6 expression.

Targeting SIRPα alone was qualified to inhibit LLC growth. SIRPα inhibits macrophages and neutrophils phagocytosis. Tumour‐derived mediators induce M2 cell polarisation, IL‐6 expression in macrophages and neutrophils through SIRPα/SHP‐1/p38 MAPK/STAT3 signalling. IL‐6 induces epithelial–mesenchymal transition of LLC cells and PD‐1 expression in CD8⁺ T cells.     

RAB14 GTPase is essential for actin‐based asymmetric division during mouse oocyte maturation

ObjectivesRAB14 is a member of small GTPase RAB family which localizes at the endoplasmic reticulum (ER), Golgi apparatus and endosomal compartments. RAB14 acts as molecular switches that shift between a GDP‐bound inactive state and a GTP‐bound active state and regulates circulation of vesicles between the Golgi and endosomal compartments. In present study, we investigated the roles of RAB14 during oocyte meiotic maturation.Materials and methodsMicroinjection with siRNA and exogenous mRNA for knock down and rescue, and immunofluorescence staining, Western blot and real‐time RT‐PCR were utilized for the study.ResultsOur results showed that RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 did not affect polar body extrusion but caused large polar bodies, indicating the failure of asymmetric division. We found that absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK‐cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc‐Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection.ConclusionsTaken together, our results suggest that RAB14 affects ROCK‐cofilin pathway for actin‐based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation.     

Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth

The ER‐bound kinase/endoribonuclease (RNase), inositol‐requiring enzyme‐1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box‐binding protein‐1 (XBP1) or the RNA substrates of IRE1‐dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide‐derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross‐talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P₃) 5‐phosphatase‐2 (INPPL1) is a direct target of miR‐2137, which controls PI(3,4,5)P₃ levels in macrophages. The modulation of cellular PI(3,4,5)P₃/PIP₂ ratio and anabolic mTOR signaling by the IRE1‐induced miR‐2137 demonstrates how the ER can provide a critical input into cell growth decisions.     

N6 ‐methyladenosine‐modified circRNA RERE modulates osteoarthritis by regulating β‐catenin ubiquitination and degradation

ObjectivesN⁶‐methyladenosine (m6A) is one of the most abundant internal RNA modifications. We investigated the role of m6A‐modified circRERE in osteoarthritis (OA) and its mechanism.Materials and MethodsCircRERE and IRF2BPL were screened by microarrays. The role of m6A‐modification in circRERE was examined by methylated RNA precipitation and morpholino oligo (MOs) treatment. The axis of circRERE/miR‐195‐5p/IRF2BPL/β‐catenin was determined using flow cytometry, western blotting and immunofluorescence in human chondrocytes (HCs) and corroborated using a mouse model of destabilization of medial meniscus (DMM) with intra‐articular (IA) injection of adeno‐associated viruses (AAV).ResultsCircRERE was decreased in OA cartilage and chondrocytes compared with control. CircRERE downregulation was likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2‐HRSP12‐RNase P/MRP in OA chondrocytes. MOs transfection targeting HRSP12 binding motifs in circRERE partially reversed decreased circRERE expression and increased apoptosis in HCs treated with IL‐1β for 6 h. CircRERE exerted chondroprotective effects by targeting miR‐195‐5p/IRF2BPL, thus regulating the ubiquitination and degradation of β‐catenin. CircRere (mouse homologue) overexpression by IA‐injection of AAV‐circRere into mice attenuated the severity of DMM‐induced OA, whereas AAV‐miR‐195a‐5p or AAV‐sh‐Irf2bpl reduced the protective effects. The detrimental effects of AAV‐sh‐Irf2bpl on DMM‐induced OA were substantially counteracted by ICG‐001, an inhibitor of β‐catenin.ConclusionsOur study is a proof‐of‐concept demonstration for targeting m6A‐modified circRERE and its target miR‐195‐5p/IRF2BPL/β‐catenin as potential therapeutic strategies for OA treatment.

Liu et al. demonstrate that circRERE expression is downregulated in human osteoarthritis (OA) cartilage and chondrocytes, and circRERE downregulation is likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2‐HRSP12‐RNase P/MRP. Mechanistically, circRERE downregulation leads to aberrant β‐catenin ubiquitin and degradation by targeting miR‐195‐5p/IRF2BPL during OA pathogenesis.     

Oral intake of Lactobacillus plantarum L‐14 extract alleviates TLR2‐ and AMPK‐mediated obesity‐associated disorders in high‐fat‐diet‐induced obese C57BL/6J mice

ObjectivesWhether periodic oral intake of postbiotics positively affects weight regulation and prevents obesity‐associated diseases in vivo is unclear. This study evaluated the action mechanism of Lactobacillus plantarum L‐14 (KTCT13497BP) extract and the effects of its periodic oral intake in a high‐fat‐diet (HFD) mouse model.Materials and methodsMouse pre‐adipocyte 3T3‐L1 cells and human bone marrow mesenchymal stem cells (hBM‐MSC) were treated with L‐14 extract every 2 days during adipogenic differentiation, and the mechanism underlying anti‐adipogenic effects was analysed at cellular and molecular levels. L‐14 extract was orally administrated to HFD‐feeding C57BL/6J mice every 2 days for 7 weeks. White adipose tissue was collected and weighed, and liver and blood serum were analysed. The anti‐adipogenic mechanism of exopolysaccharide (EPS) isolated from L‐14 extract was also analysed using Toll‐like receptor 2 (TLR2) inhibitor C29.ResultsL‐14 extract inhibited 3T3‐L1 and hBM‐MSC differentiation into mature adipocytes by upregulating AMPK signalling pathway in the early stage of adipogenic differentiation. The weight of the HFD + L‐14 group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g). L‐14 extract also significantly decreased the serum triacylglycerol/high‐density lipoprotein cholesterol ratio (an insulin resistance marker) and steatohepatitis. In addition, EPS activated the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis.ConclusionsEPS from L‐14 extract inhibits adipogenesis via TLR2 and AMPK signalling pathways, and oral intake of L‐14 extract improves obesity and obesity‐associated diseases in vivo. Therefore, EPS can be used to prevent and treat obesity and metabolic disorders.     

Combination of chemically modified SDF‐1α mRNA and small skin improves wound healing in diabetic rats with full‐thickness skin defects

ObjectivesDiabetes mellitus is associated with refractory wound healing, yet current therapies are insufficient to accelerate the process of healing. Recent studies have indicated chemically modified mRNA (modRNA) as a promising therapeutic intervention. The present study aimed to explore the efficacy of small skin engineered to express modified mRNAs encoding the stromal cell‐derived factor‐1α (SDF‐1α) facilitating wound healing in a full‐thickness skin defect rat model. This study, devised therapeutic strategies for diabetic wounds by pre‐treating small skin with SDF‐1α modRNA.Materials and MethodsThe in vitro transfection efficiency was evaluated using fluorescence microscopy and the content of SDF‐1α in the medium was determined using ELISA after the transfection of SDF‐1α into the small skin. To evaluate the effect of SDF‐1α modRNA and transplantation of the small skin cells on wound healing, an in vivo full‐thickness skin defect rat model was assessed.ResultsThe results revealed that a modRNA carrying SDF‐1α provided potent wound healing in the small skin lesions reducing reduced scar thickness and greater angiogenesis (CD31) in the subcutaneous layer. The SDF‐1α cytokines were significantly secreted by the small skin after transfection in vitro.ConclusionsThis study demonstrated the benefits of employing small skin combined with SDF‐1α modRNA in enhancing wound healing in diabetic rats having full‐thickness skin defects.

An illustration of how the small skin was transfected with the SDF‐1α modRNA and the design of the in vivo experiments.     

L‐Se‐methylselenocysteine sensitizes lung carcinoma to chemotherapy

ObjectivesOrganic Selenium (Se) compounds such as L‐Se‐methylselenocysteine (L‐SeMC/SeMC) have been employed as a class of anti‐oxidant to protect normal tissues and organs from chemotherapy‐induced systemic toxicity. However, their comprehensive effects on cancer cell proliferation and tumour progression remain elusive.Materials and MethodsCCK‐8 assays were conducted to determine the viabilities of cancer cells after exposure to SeMC, chemotherapeutics or combined treatment. Intracellular reactive oxygen species (ROS) levels and lipid peroxidation levels were assessed via fluorescence staining. The efficacy of free drugs or drug‐loaded hydrogel against tumour growth was evaluated in a xenograft mouse model.ResultsAmong tested cancer cells and normal cells, the A549 lung adenocarcinoma cells showed higher sensitivity to SeMC exposure. In addition, combined treatments with several types of chemotherapeutics induced synergistic lethality. SeMC promoted lipid peroxidation in A549 cells and thereby increased ROS generation. Significantly, the in vivo efficacy of combination therapy was largely potentiated by hydrogel‐mediate drug delivery.ConclusionsOur study reveals the selectivity of SeMC in the inhibition of cancer cell proliferation and develops an efficient strategy for local combination therapy.