Weilin Jin

The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis

During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in nonneuronal cells filopodia dynamics decrease the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results suggest that F-BAR domains are functionally diverse and highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis.

MicroRNA-218 Inhibits Glioma Invasion, Migration, Proliferation, and Cancer Stem-like Cell Self-Renewal by Targeting the Polycomb Group Gene Bmi1

Malignant gliomas are the most common central nervous system tumors and the molecular mechanism driving their development and recurrence is still largely unknown, limiting the treatment of this disease. Here, we show that restoring the expression of miR-218, a microRNA commonly downregulated in glioma, dramatically reduces the migration, invasion, and proliferation of glioma cells. Quantitative reverse transcription PCR and Western blotting analysis revealed that expression of the stem cell-promoting oncogene Bmi1 was decreased after overexpression of miR-218 in glioma cells. Mechanistic investigations defined Bmi1 as a functional downstream target of miR-218 through which miR-218 ablated cell migration and proliferation. We documented that miR-218 also blocked the self-renewal of glioma stem-like cells, consistent with the suggested role of Bmi1 in stem cell growth. Finally, we showed that miR-218 regulated a broad range of genes involved in glioma cell development, including Wnt pathways that suppress glioma cell stem-like qualities. Taken together, our findings reveal miR-218 as a tumor suppressor that prevents migration, invasion, proliferation, and stem-like qualities in glioma cells.

EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice

Programmed cell death is essential for the development of multicellular organisms, yet pathways of plant programmed cell death and its regulation remain elusive. Here we report that ETERNAL TAPETUM 1, a basic helix-loop-helix transcription factor conserved in land plants, positively regulates programmed cell death in tapetal cells in rice anthers. eat1 exhibits delayed tapetal cell death and aborted pollen formation. ETERNAL TAPETUM 1 directly regulates the expression of OsAP25 and OsAP37, which encode aspartic proteases that induce programmed cell death in both yeast and plants. Expression and genetic analyses revealed that ETERNAL TAPETUM 1 acts downstream of TAPETUM DEGENERATION RETARDATION, another positive regulator of tapetal programmed cell death, and that ETERNAL TAPETUM 1 can also interact with the TAPETUM DEGENERATION RETARDATION protein. This study demonstrates that ETERNAL TAPETUM 1 promotes aspartic proteases triggering plant programmed cell death, and reveals a dynamic regulatory cascade in male reproductive development in rice.

Trans-activator of Transcription-mediated Delivery of NEP1-40 Protein into Brain Has a Neuroprotective Effect against Focal Cerebral Ischemic Injury via Inhibition of Neuronal Apoptosis

Background:The Nogo-66 antagonistic peptide (NEP1-40) is a potential candidate for therapeutic intervention of neuronal injury. However, delivery of the proteins across the blood–brain barrier is severely limited by its size and biochemical properties. The current study was designed to evaluate the transducible effects of the trans-activator of transcription (TAT) transduction system for NEP1-40 to cross the blood–brain barrier and to clarify whether intraperitoneal administration of TAT-NEP1-40 could protect cerebral neurons from ischemic injury. Methods:Adult male Sprague-Dawley rats were submitted to a 120-min focal ischemia and received an intraperitoneal injection of No-TAT-NEP1-40, TAT-NEP1-40, TAT-&bgr;-galactosidase, or vehicle. The existence of the proteins in the brain was analyzed with immunofluorescence and Western blot techniques at 6 h after injection. Brain ischemic injury was evaluated by neurologic deficit scores, infarction volumes, terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labeling staining, and assay of caspase-3 activity. Results:Western blot analysis and immunofluorescence staining confirmed the presence of TAT-NEP1-40 protein in the brains 6 h after injection. Intraperitoneal injection of TAT-NEP1-40 could attenuate the numbers of terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end labeling–positive cells and activated caspase-3 positive cells, and increase the viability of the cells in the ischemic bounder zone, compared with that treated with No-TAT-NEP1-40, TAT-&bgr;-Gal, or vehicle. Furthermore, treatment with TAT-NEP1-40 significantly improved the neurologic outcomes and reduced the size of the infarction in rats. Conclusions:The results demonstrate that the TAT-NEP1-40 could be efficiently delivered into the rat brains and improve ischemia-induced neurologic outcomes through attenuating cell apoptosis in ischemic brains.

Circulating MiR-16-5p and MiR-19b-3p as Two Novel Potential Biomarkers to Indicate Progression of Gastric Cancer

Gastric cancer (GC) is the second most common cancer in China and the second leading cause of cancer-related death in the world. Identifying circulating biomarkers is helpful to improve theranostics of gastric cancer. Herein, we are for the first time to report miR-16-5p and miR-19b-3p were identified to be the novel potential plasma biomarkers to detect gastric cancer. Differentially expressed miRNAs were initially screened out by genome-wide miRNA profiling microarrays between 16 plasma samples of gastric cancer and 18 matched normal controls, and then were quantified and validated by quantitative reverse transcription-PCR method between 155 gastric cancer cases and 111 normal controls. Additionally, 30 plasma samples from precancerous lesions and 18 paired samples from gastric cancer patients with gastrectomy were further detected. Results showed that based on two normalization methods, miR-16-5p and miR-19b-3p in plasma were found to be capable of distinguishing normal population from GC cases with different TNM stages and differentiation grades, particularly including the early cancer cases (P<0.05). And the two miRNAs were down-regulated in GC cases (FC<0.5). Especially, the down-regulation degree was correlated with the progression of the GC cases from the early stage to the advanced stage (0.2< rs<0.3, P<0.01). And the same weak down-regulation of the two biomarkers as the early GC occurred initially in the precancerous diseases (P<0.05). The corresponding performance of the two miRNAs to detect GC in ROC analysis gradually performed better with the disease progression from the earlier stages or lower grades to the advanced stages (TNM Ⅳ stage: AUC=0.832 for miR-16-5p; TNM Ⅲ stage: AUC=0.822 for miR-19b-3p) or high grade (Poorly differentiated: AUC=0.801, 0.791 respectively for miR-16-5p and miR-19b-3p). Additionally, miR-19b-3p remained down-regulated in patient plasma within 9 days after gastrectomy. In conclusion, miR-19b-3p and miR-16-5p maybe prospective biomarkers to detect gastric cancer and indicate its progression, and thus may own great potential in applications such as early screening and progression evaluation of gastric cancer in the near future.

An in vitro study on the involvement of LINGO-1 and Rho GTPases in Nogo-A regulated differentiation of oligodendrocyte precursor cells

Nogo-A has been considered as one of the most important myelin-associated axonal regeneration inhibitors in the central nervous system. Recent studies have demonstrated various additional physiological roles of Nogo family members. To understand the possible effect of Nogo-A on the differentiation of oligodendrocytes, antibodies against distinct extracellular domains of Nogo-A were applied in cell cultures. Oligodendrocyte precursor cells from P2 rat cortex were grown in the presence of monoclonal antibody against the N-terminal inhibitory domain of Nogo-A or the C-terminal 66 amino acid loop of Nogo-A for 3 days, and the antibody treatment resulted in stunted process extension and inhibited differentiation of oligodendrocytes. Concomitant with morphology changes, Rho GTPases activity was greatly increased upon the antibody treatment and the expression level of LINGO-1, which was recently shown to be a negative regulator for the oligodendrocyte maturation, was upregulated in the process of antibody treatment. These results indicate that endogenous Nogo-A expressed in oligodendrocyte may act though Rho GTPase and LINGO-1 to influence the morphological differentiation of oligodendrocytes and will help us to understand the physiology role of Nogo-A in oligodendrocyte biology.

The emerging role of tumor-suppressive microRNA-218 in targeting glioblastoma stemness

Glioblastoma multiforme (GBM) is by far the most common and most aggressive malignant primary tumor in humans and has poor outcomes despite many advances in treatment using combinations of surgery, radiotherapy and chemotherapy. Recent studies demonstrate that GBM contains a subpopulation of cancer cells with stem cell characteristics, including self-renewal and multipotentiality, and that these cancer stem cells contribute to disease progression. MicroRNAs (miRNAs) are small non-coding regulatory RNA molecules that regulate a variety of cellular processes, including stem cell maintenance. An accumulating body of evidence shows that miR-218 may act as a tumor suppressor by inhibiting glioblastoma invasion, migration, proliferation and stemness through its different targets, indicating the great potential and relevance of miR-218 as a novel class of therapeutic target in glioblastoma.

Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

Nogo-A is originally identified as an inhibitor of axon regeneration from the CNS myelin. Nogo-A is mainly expressed by oligodendrocytes, and also by some neuronal subpopulations, particularly in the developing nervous system. Although extensive studies have uncovered regulatory roles of Nogo-A in neurite outgrowth inhibition, precursor migration, neuronal homeostasis, plasticity and neurodegeneration, its cell-autonomous functions in neurons are largely uncharacterized. Here, we show that HIV-1 trans-activating-mediated amino-Nogo-A protein transduction into cultured primary cortical neurons achieves an almost complete neuroprotection against oxidative stress induced by exogenous hydrogen peroxide (H2O2). Endogenously expressed neuronal Nogo-A is significantly downregulated upon H2O2 treatment. Furthermore, knockdown of Nogo-A results in more susceptibility to acute oxidative insults and markedly increases neuronal death. Interacting with peroxiredoxin 2 (Prdx2), amino-Nogo-A reduces reactive oxygen species (ROS) generation and extracellular signal-regulated kinase phosphorylation to exert neuroprotective effects. Structure–function mapping experiments reveal that, out of NiG-Δ20, a novel region comprising residues 290–562 of amino-Nogo-A is indispensable for preventing oxidative neuronal death. Moreover, mutagenesis analysis confirms that cysteine residues 424, 464 and 559 are involved in the inhibition of ROS generation and neuroprotective role of amino-Nogo-A. Our data suggest that neuronal Nogo-A might play a cell-autonomous role in improving neuronal survival against oxidative insult through interacting with Prdx2 and scavenging of ROS.

The Inverse F-BAR Domain Protein srGAP2 Acts through srGAP3 to Modulate Neuronal Differentiation and Neurite Outgrowth of Mouse Neuroblastoma Cells

The inverse F-BAR (IF-BAR) domain proteins srGAP1, srGAP2 and srGAP3 are implicated in neuronal development and may be linked to mental retardation, schizophrenia and seizure. A partially overlapping expression pattern and highly similar protein structures indicate a functional redundancy of srGAPs in neuronal development. Our previous study suggests that srGAP3 negatively regulates neuronal differentiation in a Rac1-dependent manner in mouse Neuro2a cells. Here we show that exogenously expressed srGAP1 and srGAP2 are sufficient to inhibit valporic acid (VPA)-induced neurite initiation and growth in the mouse Neuro2a cells. While ectopic- or over-expression of RhoGAP-defective mutants, srGAP1R542A and srGAP2R527A exert a visible inhibitory effect on neuronal differentiation. Unexpectedly, knockdown of endogenous srGAP2 fails to facilitate the neuronal differentiation induced by VPA, but promotes neurite outgrowth of differentiated cells. All three IF-BAR domains from srGAP1-3 can induce filopodia formation in Neuro2a, but the isolated IF-BAR domain from srGAP2, not from srGAP1 and srGAP3, can promote VPA-induced neurite initiation and neuronal differentiation. We identify biochemical and functional interactions of the three srGAPs family members. We propose that srGAP3-Rac1 signaling may be required for the effect of srGAP1 and srGAP2 on attenuating neuronal differentiation. Furthermore, inhibition of Slit-Robo interaction can phenocopy a loss-of-function of srGAP3, indicating that srGAP3 may be dedicated to the Slit-Robo pathway. Our results demonstrate the interplay between srGAP1, srGAP2 and srGAP3 regulates neuronal differentiation and neurite outgrowth. These findings may provide us new insights into the possible roles of srGAPs in neuronal development and a potential mechanism for neurodevelopmental diseases.

POH1 deubiquitylates and stabilizes E2F1 to promote tumour formation.

Hyperactivation of the transcriptional factor E2F1 occurs frequently in human cancers and contributes to malignant progression. E2F1 activity is regulated by proteolysis mediated by the ubiquitin–proteasome system. However, the deubiquitylase that controls E2F1 ubiquitylation and stability remains undefined. Here we demonstrate that the deubiquitylase POH1 stabilizes E2F1 protein through binding to and deubiquitylating E2F1. Conditional knockout of Poh1 alleles results in reduced E2F1 expression in primary mouse liver cells. The POH1-mediated regulation of E2F1 expression strengthens E2F1-downstream prosurvival signals, including upregulation of Survivin and FOXM1 protein levels, and efficiently facilitates tumour growth of liver cancer cells in nude mice. Importantly, human hepatocellular carcinomas (HCCs) recapitulate POH1 regulation of E2F1 expression, as nuclear abundance of POH1 is increased in HCCs and correlates with E2F1 overexpression and tumour growth. Thus, our study suggests that the hyperactivated POH1–E2F1 regulation may contribute to the development of liver cancer.