LINC00894 inhibited neuron cellular apoptosis and regulated activating transcription factor 3 expression.

LINC00894 may be associated with synaptic function, but its biology function in neural cells is still unknown. In this study, LINC00894 knockdown decreased the EdU incorporated into newly synthesized DNA and cell viability in MTT or CCK-8 assay in HEK-293T and BE(2)-M17 (M17) neuroblastoma cells. And LINC00894 knockdown increased cellular apoptosis in Annexin V-FITC staining, the expression of activated Caspase3 and the level of reactive oxygen species (ROS) both in HEK-293T and M17 cells. Moreover, LINC00894 also protected cells from hydrogen peroxide induced apoptosis in in vitro models. Utilizing RNA sequencing (RNA-seq) integrated with quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoblot, we identified that LINC00894 affected activating transcription factor 3 (ATF3) expression in HEK-293T, M17, and SH-SY5Y neuroblastoma cells. Finally, we found that ectopic expression of ATF3 restored cell proliferation and inhibited cell apoptosis in LINC00894 downregulated M17 cells. While knockdown of ATF3 also significantly increased the cell viability inhibition and apoptosis promotion induced by LINC00894 knockdown in M17 cells. Our results from in vitro models revealed that LINC00894 could promote neuronal cell proliferation and inhibit cellular apoptosis by affecting ATF3 expression.

A novel variant in NBAS identified from an infant with fever-triggered recurrent acute liver failure disrupts the function of the gene.

Mutations in the neuroblastoma amplified sequence (NBAS) gene correlate with infantile acute liver failure (ALF). Herein, we identified a novel NBAS mutation in a female infant diagnosed with recurrent ALF. Whole-exome and Sanger sequencing revealed that the proband carried a compound heterozygous mutation (c.938_939delGC and c.1342 T > C in NBAS). NBAS c.938_939delGC was presumed to encode a truncated protein without normal function, whereas NBAS c.1342 T > C encoded NBAS harboring the conserved Cys448 residue mutated to Arg448 (p.C448R). The proportion of CD4 + T cells decreased in the patient's peripheral CD45 + cells, whereas that of CD8 + T cells increased. Moreover, upon transfecting the same amount of DNA expression vector (ectopic expression) encoding wild-type NBAS and p.C448R NBAS, the group transfected with the p.C448R NBAS-expressing vector expressed less NBAS mRNA and protein. Furthermore, ectopic expression of the same amount of p.C448R NBAS protein as the wild-type resulted in more intracellular reactive oxygen species and the induction of apoptosis and expression of marker proteins correlating with endoplasmic reticulum stress in more cultured cells. This study indicated that p.C448R NBAS has a function different from that of wild-type NBAS and that the p.C448R NBAS mutation potentially affects T-cell function and correlates with ALF.

A Patient with MELAS Syndrome Carried an M.3243A>G Mutation in Mitochondrial DNA and Multiple Nuclear Genetic Variants: A Case Report.

We discuss the involvement of nuclear genetic variants correlating to observed phenotype in this case study. In January 2020, the 19-year-old boy from Nantong, Jiangsu Province, China with epilepsy symptom was identified to have myelin loss in the motor and sensory nerves in the electromyogram examination. Brain magnetic resonance imaging (MRI) demonstrated high-intensity areas of small multifocal gray matter regions in the bilateral temporal, parietal, and occipital lobes. In the serum of the patient, the levels of lactate dehydrogenase (LDH) and lactic acid were higher than the normal range values in multiple tests. By subsequent whole exome sequencing (WES) including analysis of the mitochondrial genome, the patient was revealed to carry an m.3243A>G mutation in mitochondria MTTL1 gene which was confirmed by direct Sanger sequencing analysis. Thus, disease of the patient was diagnosed as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. According to WES analysis, the patient also carried multiple homozygous variants, which correlating to myelinloss and epilepsy in nuclear genes. The peripheral neuropathy of the patient carrying single mitochondrial m.3243A>G mutation could be caused by multiple nuclear DNA defect.

Protein O-GlcNAcylation alleviates small intestinal injury induced by ischemia-reperfusion and oxygen-glucose deprivation.

Protein O-GlcNAcylation is a dynamic post-translational protein modification that regulates fundamental cellular functions in both normal physiology and diseases. The levels of protein O-GlcNAcylation are determined by flux of the hexosamine biosynthetic pathway (HBP), which is a branch of glycolysis, and are directly controlled by a pair of enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). An increase in protein O-GlcNAcylation has been shown to have protective effects on ischemia-related insults in the heart and brain. To determine whether O-GlcNAcylation plays a beneficial role in ischemia-reperfusion (IR)-induced intestinal injury, we used pharmacological manipulation of O-GlcNAc to induce loss- and gain-of-function conditions and evaluated the viability and apoptosis of intestinal epithelioid cells in an in vitro oxygen-glucose deprivation (OGD) model and tissue injury grade in a small intestinal ischemia-reperfusion (SIIR) mouse model. We found that 1) Upregulation of O-GlcNAcylation induced by glucosamine (GlcN, increase in HBP flux) or thiamet G (an OGA inhibitor) enhanced intestinal cell survival in the OGD model. In contrast, downregulation of O-GlcNAcylation induced by DON (due to a reduction in HBP flux) or OMSI-1 (an OGT inhibitor) made the cells more susceptible to hypoxia injury. 2) Reducing the increase in O-GlcNAcylation levels with a combination of either GlcN with DON or thiamet G with OMSI-1 partly canceled its protective effect on OGD-induced cell injury. 3) In the in vivo SIIR mouse model, GlcN augmented intestinal protein O-GlcNAcylation and significantly alleviated intestinal injury by inhibiting cell apoptosis. These results indicate that acute increases in protein O-GlcNAcylation confer protection against intestinal ischemia insults, suggesting that O-GlcNAcylation, as an endogenous stress sensor, could be a universal protective mechanism and could be a potential therapeutic target for intestinal ischemic disease.

GPR126 regulates colorectal cancer cell proliferation by mediating HDAC2 and GLI2 expression.

The G-protein-coupled receptor 126 (GPR126) may play an important role in tumor development, although its role remains poorly understood. We found that GPR126 had higher expression in most colorectal cancer cell lines than in normal colon epithelial cell lines, and higher expression levels in colorectal cancer tissues than in normal adjacent colon tissues. GPR126 knockdown induced by shRNA inhibited cell viability and colony formation in HT-29, HCT116, and LoVo cells, decreased BrdU incorporation into newly synthesized proliferating HT-29 cells, led to an arrest of cell cycle progression at the G1 phase in HCT-116 and HT-29 cells, and suppressed tumorigenesis of HT-29, HCT116, and LoVo cells in nude mouse xenograft models. GPR126 knockdown engendered decreased transcription and translation of histone deacetylase 2 (HDAC2), previously implicated in the activation of GLI1 and GLI2 in the Hedgehog signaling pathway. Ectopic expression of HDAC2 in GPR126-silenced cells restored cell viability and proliferation, GLI2 luciferase reporter activity, partially recovered GLI2 expression, and reduced the cell cycle arrest. HDAC2 regulated GLI2 expression and, along with GLI2, it bound to the PTCH1 promoter, as evidenced by a chip assay with HT-29 cells. Purmorphamine, a hedgehog agonist, largely restored the cell viability and expression of GLI2 proteins in GPR126-silenced HT-29 cells, whereas GANT61, a hedgehog inhibitor, further enhanced the GPR126 knockdown-induced inhibitory effects. Our findings demonstrate that GPR126 regulates colorectal cancer cell proliferation by mediating the expression of HDAC2 and GLI2, therefore it may represent a suitable therapeutic target for colorectal cancer treatment.

Senescence is a Spi1-induced anti-proliferative mechanism in primary hematopoietic cells.

Transcriptional deregulation caused by epigenetic or genetic alterations is a major cause of leukemic transformation. The Spi1/PU.1 transcription factor is a key regulator of many steps of hematopoiesis, and limits self-renewal of hematopoietic stem cells. The deregulation of its expression or activity contributes to leukemia, in which Spi1 can be either an oncogene or a tumor suppressor. Herein we explored whether cellular senescence, an anti-tumoral pathway that restrains cell proliferation, is a mechanism by which Spi1 limits hematopoietic cell expansion, and thus prevents the development of leukemia. We show that Spi1 overexpression triggers cellular senescence both in primary fibroblasts and hematopoietic cells. Erythroid and myeloid lineages are both prone to Spi1-induced senescence. In hematopoietic cells, Spi1-induced senescence requires its DNA-binding activity and a functional p38MAPK14 pathway but is independent of a DNA-damage response. In contrast, in fibroblasts, Spi1-induced senescence is triggered by a DNA-damage response. Importantly, using our well-established Spi1 transgenic leukemia mouse model, we demonstrate that Spi1 overexpression also induces senescence in erythroid progenitors of the bone marrow in vivo before the onset of the pre-leukemic phase of erythroleukemia. Remarkably, the senescence response is lost during the progression of the disease and erythroid blasts do not display a higher expression of Dec1 and CDKN1A, two of the induced senescence markers in young animals. These results bring indirect evidence that leukemia develops from cells which have bypassed Spi1-induced senescence. Overall, our results reveal senescence as a Spi1-induced anti-proliferative mechanism that may be a safeguard against the development of acute myeloid leukemia.

GPR126 protein regulates developmental and pathological angiogenesis through modulation of VEGFR2 receptor signaling.

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for development, wound healing, and tumor progression. The VEGF pathway plays irreplaceable roles during angiogenesis, but how other signals cross-talk with and modulate VEGF cascades is not clearly elucidated. Here, we identified that Gpr126, an endothelial cell-enriched gene, plays an important role in angiogenesis by regulating endothelial cell proliferation, migration, and tube formation. Knockdown of Gpr126 in the mouse retina resulted in the inhibition of hypoxia-induced angiogenesis. Interference of Gpr126 expression in zebrafish embryos led to defects in intersegmental vessel formation. Finally, we identified that GPR126 regulated the expression of VEGFR2 by targeting STAT5 and GATA2 through the cAMP-PKA-cAMP-response element-binding protein signaling pathway during angiogenesis. Our findings illustrate that GPR126 modulates both physiological and pathological angiogenesis through VEGF signaling, providing a potential target for the treatment of angiogenesis-related diseases.

Lgr4 gene regulates corpus luteum maturation through modulation of the WNT-mediated EGFR-ERK signaling pathway.

Luteal-phase insufficiency is one of the major causes of female infertility, but the molecular mechanisms are still largely unknown. Here we found that disruption of Lgr4/Gpr48, the newly identified receptor for R-spondins, greatly reduced female fertility in mice. The expression of Lgr4 was induced specifically in granulosa-lutein cells during luteinization. In Lgr4-deficient female mice, the estrous cycle was prolonged and serum progesterone levels were dramatically downregulated. In Lgr4(-/-) corpora lutea, the expression of key enzymes for steroidogenesis as well as common luteal marker genes was significantly decreased. Additionally, the activity of epidermal growth factor receptor (EGFR)-ERK signaling was attenuated in Lgr4(-/-) granulosa-lutein cells. We found that the maturation of Lgr4(-/-) cells was impaired in cultured primary granulosa cells, but the defect was partially rescued by reactivation of EGFR signaling by heparin-binding EGF-like growth factor treatment. We found that the expression of wingless-type MMTV integration site family (WNT)/catenin (cadherin associated protein), beta 1 (CTNNB1) downstream targets, including matrix metalloproteinase 9, which is a critical matrix metalloproteinase for activation of EGF-like factors, was significantly downregulated in Lgr4(-/-) ovaries. Matrix metalloproteinase 9 inhibitor treatment attenuated human chorionic gonadotropin- but not heparin-binding EGF-like growth factor-induced ERK activation and luteinization in primary granulosa cells. Together, we report that Lgr4 modulates WNT-mediated EGFR-ERK signaling to facilitate corpus luteum maturation and ovarian steroidogenesis to maintain female reproduction.

Heparanase enhances nerve-growth-factor-induced PC12 cell neuritogenesis via the p38 MAPK pathway.

Heparanase is involved in the cleavage of the HS (heparan sulfate) chain of HSPGs (HS proteoglycans) and hence participates in remodelling of the ECM (extracellular matrix) and BM (basement membrane). In the present study we have shown that NGF (nerve growth factor) promoted nuclear enrichment of EGR1 (early growth response 1), a transcription factor for heparanase, and markedly induced heparanase expression in rat adrenal pheochromocytoma (PC12) cells. K252a, an antagonist of the NGF receptor TrkA (tyrosine kinase receptor A), decreased heparanase protein expression induced by NGF in PC12 cells. Suramin, a heparanase inhibitor, decreased heparanase in PC12 cells and blocked NGF-induced PC12 neuritogenesis. Stable overexpression of heparanase activated p38 MAPK (mitogen-activated protein kinase) by phosphorylation and enhanced the neurite outgrowth induced by NGF, whereas knock down of heparanase impaired this process. However, overexpression of latent pro-heparanase with a Y156A mutation still led to enhanced NGF-induced neurite outgrowth and increased p38 MAPK phosphorylation. Inhibition of p38 MAPK by SB203580 suppressed the promotion of NGF-induced neuritogenesis by the wild-type and mutant heparanase. The impaired differentiation by knock down of heparanase could be restored by transfection of wild-type or mutant heparanase in PC12 cells. The results of the present study suggest that heparanase, at least in the non-enzymatic form, may promote NGF-induced neuritogenesis via the p38 MAPK pathway.

PAK1IP1, a ribosomal stress-induced nucleolar protein, regulates cell proliferation via the p53-MDM2 loop.

Cell growth and proliferation are tightly controlled via the regulation of the p53-MDM2 feedback loop in response to various cellular stresses. In this study, we identified a nucleolar protein called PAK1IP1 as another regulator of this loop. PAK1IP1 was induced when cells were treated with chemicals that disturb ribosome biogenesis. Overexpression of PAK1IP1 inhibited cell proliferation by inducing p53-dependent G1 cell-cycle arrest. PAK1IP1 bound to MDM2 and inhibited its ability to ubiquitinate and to degrade p53, consequently leading to the accumulation of p53 levels. Interestingly, knockdown of PAK1IP1 in cells also inhibited cell proliferation and induced p53-dependent G1 arrest. Deficiency of PAK1IP1 increased free ribosomal protein L5 and L11 which were required for PAK1IP1 depletion-induced p53 activation. Taken together, our results reveal that PAK1IP1 is a new nucleolar protein that is crucial for rRNA processing and plays a regulatory role in cell proliferation via the p53-MDM2 loop.