Dynamic Change of Prohibitin2 Expression in Rat Sciatic Nerve After Crush

As a novel cell cycle inhibitor, PHB2 controls the G1/S transition in cycling cells in a complex manner. Its aberrant expression is closely related to cell carcinogenesis. While its expression and role in peripheral nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve crush (SNC) model in adult rats to examine the dynamic changes of PHB2. Temporally, PHB2 expression was sharply decreased after sciatic nerve crush and reached a valley at day 5. Spatially, PHB2 was widely expressed in the normal sciatic nerve including axons and Schwann cells. While after injury, PHB2 expression decreased predominantly in Schwann cells. The alteration was due to the decreased expression of PHB2 in Schwann cells after SNC. PHB2 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, PHB2 largely localized with GAP43 in axons in the crushed segment. Collectively, we suggested that PHB2 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Temporal-Spatial Expressions of Spy1 in Rat Sciatic Nerve After Crush

As a novel cell cycle protein, Spy1 enhances cell proliferation, promotes the G1/S transition as well as inhibits apoptosis in response to UV irradiation. Spy1 levels are tightly regulated during mammary development, and overexpression of Spy1 accelerates tumorigenesis in vivo. But little is known about the role of Spy1 in the pathological process of damage and regeneration of the peripheral nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of Spy1. Spy1 expression was elevated gradually after sciatic nerve crush and peaked at day 3. The alteration was due to the increased expression of Spy1 in axons and Schwann cells after SNC. Spy1 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, Spy1 largely localized in axons in the crushed segment, but rarely co-localized with GAP43. These findings suggested that Spy1 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Upregulated Expression of TRIM32 Is Involved in Schwann Cell Differentiation,Migration and Neurite Outgrowth After Sciatic Nerve Crush

Tripartite motif containing 32 (TRIM32), a member of the tripartite motif (TRIM) family, plays an indispensable role in myoblast proliferation. It also regulates neuron and skeletal muscle stem cell differentiation. Although it is of great importance, we know little about the roles of TRIM32 during peripheral nervous system injury. Here, we examined the dynamic changes of TRIM32 in acute sciatic nerve crush (SNC) model. After crush, TRIM32 rapidly increased and reached the climax at 1 week but then gradually declined to the normal level at 4 weeks post-injury. Meanwhile, we observed similar changes of Oct-6. What is more, we found co-localization of TRIM32 with S100 and Oct-6 in 1-week-injured tissues using double immunofluorescent staining. In further vitro experiments, enhancive expression of TRIM32 was detected during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation and nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. More interestingly, specific si-TRIM32-transfected RSC96 cells exhibited obvious reduction in the ability of migration. Taken together, we inferred that upregulated TRIM32 was not only involved in the differentiation and migration of Schwann cells but the neurite elongation after SNC.     

Changes in Ataxin-10 Expression after Sciatic Nerve Crush in Adult Rats

Ataxin-10 is a cytoplasmic protein that belongs to the family of armadillo repeat proteins and the ataxin proteins are ubiquitously expressed in nervous tissue. A loss of Ataxin-10 in primary neuronal cells causes increased apoptosis of cerebellar neurons. Knockdown of ATXN10 with siRNA in HeLa cells results in cytokinesis defects-multinucleation. Because of the essential role of Ataxin-10 in nervous system and cellular cytokinesis, we investigated the spatiotemporal expression of Ataxin-10 in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Ataxin-10 had a significant up-regulation from 3d, peaked at day 5 and then gradually decreased to the normal level at 4 weeks. At its peak expression, Ataxin-10 expressed mainly in Schwann cells and macrophages of the distal sciatic nerve segment from injury, but had few co-localizations in axons. Besides, the peak expression of Ataxin-10 was in parallel with proliferating cell nuclear antigen (PCNA), and Ataxin-10 co-labeled with PCNA. Thus, all of our findings suggested that Ataxin-10 may be involved in the pathophysiology of sciatic nerve after SNC.     

The RNA-binding protein PCBP2 facilitates gastric carcinoma growth by targeting miR-34a

Gastric carcinoma is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. However, the mechanism underling gastric cancer is still not fully understood. Here in the present study, we identify the RNA-binding protein PCBP2 as an oncogenic protein in human gastric carcinoma. Our results show that PCBP2 is up-regulated in human gastric cancer tissues compared to adjacent normal tissues, and that high level of PCBP2 predicts poor overall and disease-free survival. Knockdown of PCBP2 in gastric cancer cells inhibits cell proliferation and colony formation in vitro, whereas opposing results are obtained when PCBP2 is overexpressed. Our in vivo subcutaneous xenograft results also show that PCBP2 can critically regulate gastric cancer cell growth. In addition, we find that PCBP2-depletion induces apoptosis in gastric cancer cells via up-regulating expression of pro-apoptotic proteins and down-regulating anti-apoptotic proteins. Mechanically, we identify that miR-34a as a target of PCBP2, and that miR-34a is critically essential for the function of PCBP2. In summary, PCBP2 promotes gastric carcinoma development by regulating the level of miR-34a.     

THAP11, a novel binding protein of PCBP1, negatively regulates CD44 alternative splicing and cell invasion in a human hepatoma cell line

THAP11 is an essential factor involved in ES cell pluripotency and cell growth. Here, we identified THAP11 as a novel physiological binding partner of PCBP1. In HepG2 cells, THAP11 overexpression inhibited CD44 v6 expression and cell invasion. However, when deleting the binding domain with PCBP1 or endogenous PCBP1 was knocked down, THAP11 failed to inhibit CD44 v6 expression, indicating that THAP11 regulates CD44 v6 expression through interacting with PCBP1. In HCC patients, the expression of THAP11 mRNA significantly correlated with PCBP1 mRNA expression. Our results suggest a novel role of THAP11 in CD44 alternative splicing and hepatoma invasion.     

Up-Regulation of Cdc37 Contributes to Schwann Cell Proliferation and Migration After Sciatic Nerve Crush

Cell division cycle protein 37 (Cdc37), a molecular chaperone takes part in a series of cellular processes including cell signal transduction, cell cycle progression, cell proliferation, cell motility, oncogenesis and malignant progression. It can not only recruit immature protein kinases to HSP90 but also work alone. Cdc37 was reported to be associated with neurogenesis, neurite outgrowth, axon guidance and myelination. However, the roles of Cdc37 on Schwann cells (SC) after peripheral nerve injury (PNI) remain unknown. In this study, we found that the expression of Cdc37 increased and reached the peak at 1 week after sciatic nerve crush (SNC), which was consistent with that of proliferation cell nuclear antigen. Immunofluorescence verified that Cdc37 co-localized with SC in vivo and in vitro. Intriguingly, Cdc37 protein level was potentiated in the model of TNF-α-induced SC proliferation. Moreover, we found that Cdc37 silencing impaired proliferation of SC in vitro. Moreover, Cdc37 suppression attenuated kinase signaling pathways of Raf–ERK and PI3K/AKT which are crucial cell signaling for SC proliferation. Finally, we found that Cdc37 silencing inhibited SC migration in vitro. In conclusion, we demonstrated that the way Cdc37 contributed to SC proliferation is likely via activating kinase signaling pathways of Raf–ERK and PI3K/AKT, and CDC37 was also involved in SC migration after SNC.     

Poly C binding protein, a single-stranded DNA binding protein, regulates mouse mu-opioid receptor gene expression

Previously, a single-stranded (ss) DNA element, polypyrimidine (PPy) element, was found to be important for the proximal promoter activity of mouse micro-opioid receptor (MOR) gene in a neuronal cell model. In this study, we identified the presence of unknown ssDNA binding proteins specifically bound to MOR ssPPy element in the mouse brain, implicating the physiological significance of these proteins. To identify the ssDNA binding proteins, yeast one-hybrid system with PPy element as the bait was used to screen a mouse brain cDNA library. The clone encoding poly C binding protein (PCBP) was obtained. Its full-length cDNA sequence and protein with molecular weight approximately 38 kDa were confirmed. Electrophoretic mobility shift analysis (EMSA) revealed that PCBP bound to ssPPy element, but not doubled-stranded, in a sequence-specific manner. EMSA with anti-PCBP antibody demonstrated the involvement of PCBP in MOR ssPPy/proteins complexes of mouse brain and MOR expressing neuroblastoma NMB cells. Functional analysis showed that PCBP trans-activated MOR promoter as well as a heterologous promoter containing MOR PPy element. Importantly, ectopic expression of PCBP in NMB cells up-regulated the expression level of endogenous MOR gene in vivo in a dose-dependent manner. Collectively, above results suggest that PCBP participates in neuronal MOR gene expression.     

Spatiotemporal Expression of PSD-95 and nNOS After Rat Sciatic Nerve Injury

Neuronal nitric oxide synthase (nNOS) has been implicated to influence peripheral nerve lesion and regeneration. Post-synaptic density-95 (PSD-95) is one of nNOS-anchoring proteins and plays an important role in specifying the sites of reaction of NO in nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of PSD-95 and nNOS. At gene levels, PSD-95 mRNA diminished shortly after crush, and significantly elevated from 2 days to 2 weeks, whereas nNOS decreased progressively post-operation, reached the valley at 1 day, and markedly up-regulated from 1 to 2 weeks after SNC. The expression of both molecules returned to the control level at 4 weeks post-injury. At protein levels, PSD-95 and nNOS underwent the similar changes as their gene expression except for a time lag during up-regulating. At their peak expression, PSD-95 co-labeled with nNOS in Schwann cells (SCs) of sciatic nerve within 0.5 mm from the lesion site, but had few colocalization in axons. In addition, the interaction between PSD-95 and nNOS enhanced significantly at 2 weeks after SNC. These results suggest a correlation of PSD-95 up-regulation with nNOS in reactive SCs of crushed sciatic nerve, which may lead to understanding the function of PSD-95 during peripheral nerve regeneration. Shangfeng Gao and Min Fei contributed equally to this work.     

Poly(C)-binding protein 1 (PCBP1) mediates housekeeping degradation of mitochondrial antiviral signaling (MAVS)

Mitochondrial antiviral signaling (MAVS) is a key adaptor in cellular antiviral innate immunity. We previously identified poly(C)-binding protein 2 (PCBP2) as a feedback inhibitor of MAVS that facilitates its degradation after viral infection, but little is known about the regulatory potential of poly(C)-binding protein 1 (PCBP1), which highly resembles PCBP2. Here we report that PCBP1 mediates housekeeping degradation of MAVS using the same mechanism as PCBP2 employs. Overexpression of PCBP1 impairs MAVS-mediated antiviral responses, while knockdown of PCBP1 exerts the opposite effect. The suppression is due to PCBP1-induced MAVS degradation. We observe that PCBP1 and PCBP2 show synergy in MAVS inhibition, but their expression patterns are distinct: PCBP1 is stably and abundantly expressed, while PCBP2 shows low basal expression with rapid induction after infection. Individual knockdown and subcellular fractionation analyses reveal that unlike the postinfection inhibitor PCBP2, PCBP1 continuously eliminates cellular MAVS. Our findings unravel a critical role of PCBP1 in regulating MAVS for both fine-tuning the antiviral immunity and preventing inflammation.     

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two‐hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation using the two‐hybrid system, and by co‐immunoprecipitation with anti‐PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co‐immunoprecipitation suggested that RACK1‐PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over‐expression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by ³H‐diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.     

Involvement of Upregulated SYF2 in Schwann Cell Differentiation and Migration After Sciatic Nerve Crush

SYF2 is a putative homolog of human p29 in Saccharomyces cerevisiae. It seems to be involved in pre-mRNA splicing and cell cycle progression. Disruption of SYF2 leads to reduced α-tubulin expression and delayed nerve system development in zebrafish. Due to the potential of SYF2 in modulating microtubule dynamics in nervous system, we investigated the spatiotemporal expression of SYF2 in a rat sciatic nerve crush (SNC) model. We found that SNC resulted in a significant upregulation of SYF2 from 3 days to 1 week and subsequently returned to the normal level at 4 weeks. At its peak expression, SYF2 distributed predominantly in Schwann cells. In addition, upregulation of SYF2 was approximately in parallel with Oct-6, and numerous Schwann cells expressing SYF2 were Oct-6 positive. In vitro, we observed enhanced expression of SYF2 during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation. SYF2-specific siRNA-transfected Schwann cells did not show significant morphological change in the process of Schwann cell differentiation. Also, we found shorter and disorganized microtubule structure and a decreased migration in SYF2-specific siRNA-transfected Schwann cells. Together, these findings indicated that the upregulation of SYF2 was associated with Schwann cell differentiation and migration following sciatic nerve crush.     

The histone deacetylase SIRT6 suppresses the expression of the RNA-binding protein PCBP2 in glioma

More than 80% of tumors that occur in the brain are malignant gliomas. The prognosis of glioma patients is still poor, which makes glioma an urgent subject of cancer research. Previous evidence and our present data show that PCBP2 is over-expressed in human glioma tissues and predicts poor outcome. However, the mechanism by which PCBP2 is regulated in glioma remains elusive. We find that SIRT6, one of the NAD⁺-dependent class III deacetylase SIRTUINs, is down-regulated in human glioma tissues and that the level of SIRT6 is negatively correlated with PCBP2 level while H3K9ac enrichment on the promoter of PCBP2 is positively correlated with PCBP2 expression. Furthermore, we identify PCBP2 as a target of SIRT6. We demonstrate that PCBP2 expression is inhibited by SIRT6, which depends upon deacetylating H3K9ac. Finally, our results reveal that SIRT6 inhibits glioma cell proliferation and colony formation in vitro and glioma cell growth in vivo in a PCBP2 dependent manner. In summary, our findings implicate that SIRT6 inhibits PCBP2 expression through deacetylating H3K9ac and SIRT6 acts as a tumor suppressor in human glioma.     

PCBP2 promotes the development of glioma by regulating FHL3/TGF-β/Smad signaling pathway

The purpose of this study was to investigate the role of Poly (C)-binding protein 2 (PCBP2) and the related signaling pathway in glioma progression. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were performed to measure PCBP2 messenger RNA and protein expression in glioma tissues or cells. Cell transfection was completed using Lipofectamine 2000. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Transwell assay and flow cytometry assay were used to explore the effects of PCBP2 expression on biological behaviors of glioma cells. Western blot assay was used for the detection of pathway related proteins. Expression of PCBP2 in glioma tissues and cells were higher than that in paracancerous tissues and normal cells (both p < .01). Moreover, the elevated expression of PCBP2 was significantly correlated with tumor size (p = .001) and WHO stage (p = .010). Knockdown of PCBP2 could suppress proliferation, migration and invasion of glioma cells and promote apoptosis. Besides, the expression of transforming growth factor-β (TGF-β) pathway related proteins TGF-β1, p-Smad2 and p-Smad7 were decreased following the downregulation of PCBP2. PCBP2 also inhibited FHL3 expression by binding to FHL3-3′UTR. The inhibition of FHL3 could reverse the antitumor action caused by PCBP2 silencing. In vivo assay, PCBP2 was also found to inhibit the tumor growth of glioma. PCBP2 activates TGF-β/Smad signaling pathway by inhibiting FHL3 expression, thus promoting the development and progression of glioma.