Dual blockages of a broad and potent neutralizing IgM antibody targeting GH loop of EV-As.

The reported enterovirus A 71 (EVA71) vaccines and immunoglobin G (IgG) antibodies have no cross-antiviral efficacy against other enterovirus A (EV-A) which caused hand, foot and mouth disease (HFMD). Here we constructed an IgM antibody (20-IgM) based on our previous discovery to address the resistance encountered by IgG-based immunotherapy. Although binding to the same conserved neutralizing epitope within the GH loop of EV-As VP1, the antiviral breath and potency of 20-IgM are still higher than its parental 20-IgG1. The 20-IgM blocks the interaction between the EV-As and its receptors, scavenger receptor class B, member 2 (SCARB2) and Kringle-containing transmembrane protein 1(KREMEN1) of the host cell. The 20-IgM also neutralizes the EV-As at the post-attachment stages, including postattachment neutralization, uncoating and RNA release inhibition after internalization. Mechanistically, the dual blockage effect of 20-IgM is dependent on both a conserved site targeting and high affinity binding. Meanwhile, 20-IgM provides cross-antiviral efficacy in EV-As orally infected neonatal ICR mice. Collectively, 20-IgM and its property exhibit excellent antiviral activity with a dual-blockage inhibitory effect at both the pre- and post-attachment stages. The finding enhances our understanding of IgM-mediated immunity and highlights the potential of IgM subtype antibodies against enterovirus infections.

A broad and potent IgM antibody against tetra-EV-As induced by EVA71 and CVA16 co-immunization.

OBJECTIVE: To determine the potent and broad neutralizing monoclonal antibody (mAb) against enterovirus A (EV-A) in vitro and in vivo induced by enterovirus A71(EVA71) and coxsackievirus 16 (CVA16) co-immunization. METHODS: The mAb was Generated by co-immunization with EVA71 and CVA16 through hybridomas technology. The characteristics and neutralizing ability of mAb were analysed in vitro and in mice. RESULTS: We screened three mAb, the IgM antibody M20 and IgG antibody B1 and C31. All three antibodies showed cross-reactivity against tetra-EV-As. However, M20 showed potent and broad neutralizing ability against tetra-EV-As than B1 and C31. Meanwhile, M20 provided cross-antiviral efficacy in tetra-EV-As orally infected mice. Moreover, M20 binds to a conserved neutralizing epitope within the GH loop of tetra-EV-As VP1. CONCLUSIONS: M20 and its property exhibited potent and broad antiviral activity against tetra-EV-As, and that is expected to be a potential preventive and therapeutic candidate against EV-As.

Establishment and transcriptomic features of an immortalized hepatic cell line of the Chinese tree shrew.

BACKGROUND: The Chinese tree shrew (Tupaia belangeri chinesis) is a rising experimental animal and has been used for studying a variety of human diseases, such as metabolic and viral infectious diseases. METHODS: In this study, we established an immortalized tree shrew hepatic cell line, ITH6.1, by introducing the simian virus 40 large T antigen gene into primary tree shrew hepatocytes (PTHs). RESULTS: The ITH6.1 cell line had a stable cell morphology and proliferation activity. This cell line could be infected by enterovirus 71 (EV71), but not hepatitis C virus (HCV), although the known HCV entry factors, including CD81, SR-BI, CLDN1 and OCLN, were all expressed in the PTHs and ITH6.1 of different passages. Comparison of the transcriptomic features of the PTHs and different passages of the ITH6.1 cells revealed the dynamic gene expression profiles during the transformation. We found that the DNA replication- and cell cycle-related genes were upregulated, whereas the metabolic pathway-related genes were downregulated in early passages of immortalized hepatocytes compared to the PTHs. Furthermore, expression of hepatocytes function-related genes were repressed in ITH6.1 compared to that of PTHs. CONCLUSION: We believe these cellular expression alterations might cause the resistance of the ITH6.1 cell to HCV infection. This tree shrew liver cell line may be a good resource for the field. KEY POINTS: • A tree shrew hepatic cell line (ITH6.1) was established. • ITH6.1 cells could be infected by EV71, but not HCV. • ITH6.1 had an altered expression profiling compared to the primary hepatocytes.

Transcriptome analysis following enterovirus 71 and coxsackievirus A16 infection in respiratory epithelial cells.

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major pathogens responsible for hand, foot and mouth disease (HFMD), but the mechanism by which these viruses cause disease remains unclear. In this study, we used transcriptome sequencing technology to investigate changes in the transcriptome profiles after infection with EV-A71 and CV-A16 in human bronchial epithelial (16HBE) cells. Using systematic bioinformatics analysis, we then searched for useful clues regarding the pathogenesis of HFMD. As a result, a total of 111 common differentially expressed genes were present in both EV-A71- and CV-A16-infected cells. A trend analysis of these 111 genes showed that 91 of them displayed the same trend in EV-A71 and CV-A16 infection, including 49 upregulated genes and 42 downregulated genes. These 91 genes were further used to conduct GO, pathway, and coexpression network analysis. It was discovered that enriched GO terms (such as histone acetylation and positive regulation of phosphorylation) and pathways (such as glycosylphosphatidylinositol (GPI)-anchor biosynthesis and DNA replication) might be closely associated with the pathogenic mechanism of these two viruses, and key genes (such as TBCK and GPC) might be involved in the progression of HFMD. Finally, we randomly selected 10 differentially expressed genes for qRT-PCR to validate the transcriptome sequencing data. The experimental qRT-PCR results were roughly in agreement with the results of transcriptome sequencing. Collectively, our results provide clues to the mechanism of pathogenesis of HFMD induced by EV-A71 and CV-A16.

High-efficiency nonhomologous insertion of a foreign gene into the herpes simplex virus genome.

Efficient, accurate and convenient foreign-gene insertion strategies are crucial for the high-throughput and rapid construction of large DNA viral vectors, but relatively inefficient and labour-intensive methods have limited the application of recombinant viruses. In this study, we applied the nonhomologous insertion (NHI) strategy, which is based on the nonhomologous end joining (NHEJ) repair pathway. Compared to the currently used homologous recombination (HR) strategy, we obtained a higher efficiency of foreign-gene insertion into the herpes simplex virus (HSV) genome that reached 45 % after optimization. By using NHI, we rapidly constructed recombinant reporter viruses using a small amount of clinical viruses, and the recombinant virus was stable for at least ten consecutive passages. The fidelity of NHI ranged from 70-100% and was related to the sequence background of the insertion site according to the sequencing results. Finally, we depict the dynamic process by which the foreign-gene donor plasmid and viral genome are rapidly cleaved by Cas9, as revealed by quantitative pulse analysis. Furthermore, the NHI strategy exerted selection pressure on the wild-type and reverse-integrated viral genomes to efficiently integrate the foreign gene in a predetermined direction. Our results indicate that the use of a rationally designed NHI strategy can allow rapid and efficient foreign gene knock-in into the HSV genome and provide useful guidance for gene insertion into large DNA viral genomes using NHI.

The art of partnerships for vaccines.

The Developing Countries Vaccine Manufacturers Network (DCVMN) convened vaccine manufacturing experts and leaders from local and global public health organizations for its 19th Annual General Meeting. Lectures and panel discussions centered on international cooperation for better access to vaccines, and partnerships in areas ranging from vaccine research and process development, to clinical studies, regulatory, supply chain and emergency preparedness and response. Global vaccine market trends and changes that will impact vaccine financing and procurement methods were discussed as well as capital sources, including funding, for the development of new or improved vaccines. DCVMN members presented their progress in developing novel Hexavalent, Meningitis, Pneumococcal Conjugate Vaccine, Shigella, Mumps, Rotavirus, Yellow Fever, Polio, Hepatitis E and Dengue vaccines, and a novel monoclonal antibody cocktail for post-bite prophylaxis against rabies infections. Access to and availability of vaccines is enhanced through sharing of best practices for vaccine quality control, reducing redundant testing and promoting development of harmonized common standards. Eligible stakeholders were encouraged to join the WHO-National Control Laboratory Network for Biologicals which serves as a platform for collaboration and technical exchange in this area. Increasing regulatory convergence at the regional and global levels through mechanisms such as joint dossier review and the WHO Collaborative Registration Procedure can help to accelerate vaccine access globally. Additionally, four proposals for streamlining procedures and alignment of dossiers were discussed. Successful partnerships between a broad range of stakeholders, including international organizations, manufacturers, academic research institutes and regulators have provided support for, and in some cases accelerated, vaccine innovation, clinical trials and registration, WHO prequalification, vaccine introduction and access. Strong partnerships, based on experience and trust, help leverage opportunities and are critically important to advancing the shared goal of providing quality vaccines for all people.

Comparative analysis of putative novel microRNA expression profiles induced by enterovirus 71 and coxsackievirus A16 infections in human umbilical vein endothelial cells using high-throughput sequencing.

Hand, foot and mouth disease (HFMD) is mainly caused by human enterovirus 71 (EV71) and coxsackievirus A16 (CA16), which circulate alternatively or together in epidemic areas. Although the two viruses exhibit genetic homology, their clinical manifestations have some discrepancies. However, the factors underlying these differences remain unclear. Herein, we mainly focused on the alterations and roles of putative novel miRNAs in human umbilical vein endothelial cells (HUVECs) following EV71 and CA16 infections using high-throughput sequencing. The results identified 247 putative novel, differentially expressed miRNAs, of which only 11 miRNAs presented an opposite trend between the EV71- and CA16-infected samples and were used for target prediction. Gene ontology (GO) and pathway enrichment analysis of the predicted targets displayed the top 15 significant biological processes, molecular functions, cell components and pathways. Subsequently, regulatory miRNA-predicted targets and miRNA-GO and miRNA-pathway networks were constructed to further reveal the complex regulatory mechanisms of the miRNAs during infection. Therefore, our data provide useful insights that will help elucidate the different host-pathogen interactions following EV71 and CA16 infections and may offer novel therapeutic targets for these infections.

High-Throughput Sequencing of Putative Novel microRNAs in Rhesus Monkey Peripheral Blood Mononuclear Cells following EV71 and CA16 Infection.

OBJECTIVES: Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) remain the major pathogens in hand, foot, and mouth disease (HFMD) cases, but the mechanisms of the different pathogeneses that follow EV71 and CA16 infection remain largely unknown. METHODS: Herein, we utilized microRNA (miRNA) deep sequencing to investigate the roles of novel differentially expressed miRNAs in peripheral blood mononuclear cells (PBMCs) infected with EV71 and CA16. RESULTS: The results identified 13 novel differentially expressed miRNAs in each group. Additionally, the target genes were predicted by the miRanda and RNAhybrid programs, and a total of 2,501 targets were found in the two databases. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that these targets were mainly involved in cell development and were associated with nervous system development, system development, multicellular organism development, the Wnt signaling pathway, the PDGF signaling pathway, and the EGF receptor signaling pathway. Finally, a coexpression regulatory network was built with the key targets to further extrapolate the functional interactions of the targets and their coexpressed genes. CONCLUSION: Our results not only revealed potential biomarkers or targets for the diagnosis and treatment of HFMD, but also provided new insights to explore the mechanisms of EV71 and CA16 pathogenesis.

miR-1303 regulates BBB permeability and promotes CNS lesions following CA16 infections by directly targeting MMP9.

Coxsackievirus A16 (CA16) is a member of the Picornaviridae family and causes mild and self-limiting hand, foot, and mouth disease (HFMD) in infants and young children. CA16 infection can also progress to central nervous system (CNS) complications; however, the underlying mechanism by which CA16 penetrates the blood-brain barrier (BBB) and then causes CNS damage remains unclear. This study aimed to explore the mechanism of CA16 neurotropic tropism by establishing an in vitro BBB model with CA16 infection and an in vivo CA16 rhesus monkey infant infection model. The results showed that CA16 infection induced increased permeability of the BBB accompanied by upregulation of matrix metalloproteinase 9 (MMP9) expression. Subsequently, high-throughput miRNA sequencing technology and bioinformatics analysis revealed that miR-1303 may regulate BBB permeability by targeting MMP9. Next, we used dual-luciferase, qRT-PCR, and western blot assays to provide evidence of MMP9 targeting by miR-1303. Further experiments revealed that CA16 infection promoted the degradation of junctional complexes (Claudin4, Claudin5, VE-Cadherin, and ZO-1), likely by downregulating miR-1303 and upregulating MMP9. Finally, EGFP-CA16 infection could enter the CNS by facilitating the degradation of junctional complexes, eventually causing neuroinflammation and injury to the CNS, which was confirmed using the in vivo rhesus monkey model. Our results indicate that CA16 might penetrate the BBB and then enter the CNS by downregulating miR-1303, which disrupts junctional complexes by directly regulating MMP9 and ultimately causing pathological CNS changes. These results provide new therapeutic targets in HFMD patients following CA16 infection.

Profiling of novel microRNAs elicited by EV71 and CA16 infection in human bronchial epithelial cells using high-throughput sequencing.

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are two major etiologic agents associated with hand, foot, and mouth disease (HFMD) worldwide. Despite that they both belong to the Enterovirus genus of the Picornaviridae family, there are many differences in the infection process of these viruses. However, the underlying mechanisms have not been elucidated. Multiple studies indicated that microRNAs (miRNAs) can play critical roles in the host-pathogen interaction. Our previous study reported that EV71 and CA16 infection leads to differential expression of miRNAs in human bronchial epithelial (16HBE) cells. Herein, we aimed to further explore the expression profile and possible roles of other differentially expressed miRNAs in 16HBE cells following EV71 and CA16 infections using high-throughput sequencing. We describe 44 novel differentially expressed miRNAs in all samples. Among these miRNAs, 7 novel differentially expressed miRNAs show an opposite expression trend during the progression of EV71 and CA16 infections. Subsequently, bioinformatics analyses, including Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, were used to identify the biological processes, molecular functions, cellular components, and pathways involved. The top 10 significant GO and Pathway annotations indicated that 849 target genes are involved in cell development, such as nervous system development, multicellular organism development, and developmental biology. Finally, the genes identified in both the GO and Pathway analysis were used to construct a co-expression network to further identify the potential function of these co-expressed genes. Thus, our data may be beneficial in guiding further studies on the molecular mechanism of developmental regulation in HFMD pathogenesis caused by EV71 and CA16. In addition, it provided new candidate biomarkers or therapeutic targets for HFMD.

Development of a tree shrew-specific interferon-gamma assay.

Tree shrews (Tupaia belangeri) are small squirrel-like mammals closely related to primates. Due to their susceptibility to several human viruses, tree shrews have been proposed as potential animal models for the study of human viral infections. However, there are no standardized assays currently available for the detection of tree shrew-specific interferon (IFN)-γ, a major cytokine secreted during the antiviral immune response. Herein, we developed a novel enzyme-linked immunosorbent assay (ELISA) for the quantification of IFN-γ in tree shrew serum samples. Tree shrew-specific IFN-γ was expressed in Escherichia coli via fusion with glutathione S-transferase (GST-TS-IFN-γ) to obtain recombinant IFN-γ. To generate anti-IFN-γ monoclonal antibodies, mice were immunized with the GST-TS-IFN-γ recombinant fusion protein, and hybridoma cell lines were established. Similarly, anti-IFN-γ polyclonal antibodies were obtained from immunized rabbits, purified, and conjugated to horseradish peroxidase (HRP). Based on the results obtained from the antibody matching test, we optimized the monoclonal antibody (1:2000) and the HRP-conjugated polyclonal antibody (1:8000) as coating and detection antibodies, respectively. Titration curves were generated with recombinant IFN-γ to develop a sensitive sandwich ELISA; the lowest detection limit of the assay was 20 ng/mL. We also tested mitogen-stimulated tree shrew blood samples in this ELISA, and found significantly higher levels of IFN-γ in the stimulated versus the unstimulated samples. Most importantly, our ELISA system detected native IFN-γ in serum samples from 50 healthy tree shrews. We have thus developed a novel ELISA, and have demonstrated the first ELISA-based measurement of IFN-γ in tree shrew serum samples.

[Expression and purification of rabies virus glycoprotein and analysis of its specific binding capacity to memory B cells].

We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells. We successfully constructed three expression vectors pGEX-5X-1-RVG, pET28a-RVG and pET30a-G. Three glycoproteins GST-RVG, His-RVG and His-G were obtained by optimized expression and purification conditions. The antigen specificity of purified GST-RVG, His-RVG and His-G were identified by Western blotting and ELISA. The affinity of these three purified glycoproteins to anti-rabies virus antibody were detected by competitive ELISA. Anti-rabies virus specific memory B cells in positive PBMCs gained from people who had ever been injected with the vaccine can be detected by flow cytometry. Thus, we got a recombinant rabies virus glycoprotein that had high-affinity and could sort antigen specific memory B cells.

Extracellular Interactions between Hepatitis C Virus and Secreted Apolipoprotein E.

Interactions between hepatitis C virus (HCV) and lipoproteins in humans play an important role in the efficient establishment of chronic infection. Apolipoprotein E (ApoE) on the HCV envelope mediates virus attachment to host cells as well as immune evasion. This interaction is thought to occur in hepatocytes, as ApoE plays dual functions in HCV assembly and maturation as well as cell attachment. In the present study, we found that secreted ApoE (sApoE) can also bind to viral particles via its C-terminal domain after HCV is released from the cell. Furthermore, the binding affinity of interactions between the sApoE N terminus and cell surface receptors affected HCV infectivity in a dose-dependent manner. The extracellular binding of sApoE to HCV is dependent on HCV envelope proteins, and recombinant HCV envelope proteins are also able to bind to sApoE. These results suggest that extracellular interactions between HCV and sApoE may potentially complicate vaccine development and studies of viral pathogenesis.IMPORTANCE End-stage liver disease caused by chronic HCV infection remains a clinical challenge, and there is an urgent need for a prophylactic method of controlling HCV infection. Because host immunity against HCV is poorly understood, additional investigations of host-virus interactions in the context of HCV are important. HCV is primarily transmitted through blood, which is rich in lipoproteins. Therefore, it is of interest to further determine how HCV interacts with lipoproteins in human blood. In this study, we found that secreted ApoE (sApoE), an exchangeable component found in lipoproteins, participates in extracellular interactions with HCV virions. More significantly, different variants of sApoE differentially affect HCV infection efficiency in a dose-dependent manner. These findings provide greater insight into HCV infection and host immunity and could help propel the development of new strategies for preventing HCV infection.

Assessment of HCV genotypes in Yunnan Province of Southwest China.

Recently, we reported that the frequency of hepatitis C virus (HCV) genotypes and subtypes has rapidly changed among intravenous drug users (IDUs) in Yunnan Province over the last 5 years; this is especially true for subtype 6a which has increased in frequency from 5 to 15%. Here, we assessed 120 HCV-positive plasma samples from the general population (GP). HCV NS5B fragments were amplified and sequenced by PCR. We identified four HCV genotypes (1, 2, 3 and 6) and seven HCV subtypes (1b, 2a, 3a, 3b, 6a, 6n, and 6k) in this population. Genotype 3 was predominant, with a distribution frequency of 0.484, followed by genotype 1 (0.283), genotype 6 (0.133) and genotype 2 (0.100). HCV subtypes 3b (frequency 0.292) and 1b (frequency 0.283) were the most common subtypes. A comparison of the current data with previous results reported for IDUs showed that the distribution frequencies of genotypes 1, 2 and 6 were significantly different between patients in the GP and IDUs (P < 0.05). Among the HCV subtypes, the distribution frequencies of 1b, 2a, 6a, and 6n were significantly different between patients in the GP and IDU groups (P < 0.05). Moreover, Phylogenetic analyses showed that HCV subtype 6a strains isolated from IDUs and the GP were intermixed and not separately clustered. HCV subtype 6a was predominant not only among IDUs but also among those in the GP in the Guangdong Province and Vietnam. However, HCV subtype 6a was predominant only among IDUs and not among those in the GP in the Yunnan and Guangxi Provinces. Our results indicate that the HCV subtype 6a could rapidly spread across China.

Hepatitis C virus genotype diversity among intravenous drug users in Yunnan Province, Southwestern China.

BACKGROUND: Recently, high proportions (15.6%-98.7%) of intravenous drug users (IDUs) in China were found to be positive for hepatitis C virus (HCV). Yunnan Province is located in southwestern China and borders one of the world's most important opium-producing regions, thus it is an important drug trafficking route to other regions of China. METHODOLOGY/PRINCIPAL FINDINGS: Here, we assessed 100 HCV-positive plasma samples from IDUs who were enrolled through the Kunming Center for Disease Control and Prevention in 2012. HCV C/E1 fragments were PCR-amplified and sequenced. We identified eight HCV subtypes (1a, 1b, 3a, 3b, 6a, 6n, 6u and 6v), of which genotype 6 was most predominant (frequency, 47%) followed by genotypes 3 (41%) and 1 (12%). HCV subtypes 6n (30%) and 3b (29%) were most common and were identified in 59% of the IDUs. We compared HCV genotypes among IDUs in Yunnan Province with those from other regions and found that the distribution patterns of HCV genotypes in Yunnan Province were similar to those in southern China, but different from those in eastern China. However, the distribution patterns of HCV subtypes varied among Yunnan Province and southern China, despite the shared similar genotypes. A comparison of the current data with those previously reported showed that the frequency of HCV genotype 6 increased from 25% to 47% within 5 years, especially subtypes 6a (5% to 15%) and 6n (11.2% to 30%). In contrast, the frequencies of subtypes 3b and 1b decreased by almost 50% within 5 years. CONCLUSION/SIGNIFICANCE: Our results provided further information to support the assertion that drug trafficking routes influence HCV transmission patterns among IDUs in Yunnan Province. The frequency of HCV genotypes and subtypes changed rapidly among IDUs in Yunnan Province and subtypes 6a and 6n may have originated in Vietnam and Myanmar, respectively.

Investigation of myoepithelial cell differentiation into Schwann-like cells in salivary adenoid cystic carcinoma associated with perineural invasion.

Perineural invasion (PNI) is common in salivary adenoid cystic carcinoma (SACC). The aim of the present study was to explore the association of the Schwann-like cell differentiation with PNI in SACC. Twenty-eight cases of SACC and 10 cases of acinic cell carcinoma (ACA) were examined for the expression of the Schwann cell markers Leu-7 by immunohistochemical staining. The correlation between Leu-7 expression and PNI was analyzed using κ analysis. Immunofluorescence double-staining and pre-embedding immunogold-silver cytochemistry were used to detect the co-expression and the location of Leu-7 and the myoepithelial cell marker α-smooth muscle actin (α-SMA). PNI was identified in 16 SACCs (57.1%) and 1 ACA (10%) and the overexpression of Leu-7 was detected in 22 SACCs (78.6%) and in none of the ACAs (0%). The differences between PNI and Leu-7 expression in SACC and ACA were significant (P<0.05). A correlation was identified between the expression of Leu-7 and PNI in SACC (κ=0.533, P=0.01). In SACC, Leu-7 and α-SMA were co-expressed in the cytoplasm in the same myoepithelial cells. We suggest that Schwann­like cell differentiation correlates with PNI in SACC and that the differentiation of myoepithelial into Schwann­like cells may be one of the mechanisms through which PNI occurs in SACC.

A comparative study of the characteristics of two Coxsackie A virus type 16 strains (genotype B).

Coxsackie A virus is one of the major pathogens associated with hand, foot and mouth disease (HFMD). The etiological characteristics of Coxsackie A virus type 16 (CA16) are thought to correlate with the pathological process of its infection. Two CA16 strains that were isolated from a severe HFMD patient presented with different plaque forms. This observation, along with biological analysis, indicated that the differences in the strains' biological characteristics, such as proliferation kinetics and immunogenicity, correlate with differences in their pathogenicity toward neonatal mice. Furthermore, these differences are thought to be associated with the sequence of the 5' non-coding region of the viral genome and the VP1 structural region sequence. The results suggest that the biological and genetic characteristics of the CA16 viral strains are relevant to their pathogenicity.

[Effects of RhoA siRNA on the proliferation, adhesion, migration and invasion of tongue squamous cell carcinoma Tca8113 cells in vitro].

OBJECTIVE: to investigate the effect of RhoA on the metastasis of tongue squamous cell carcinoma Tca8113 cells in vitro. METHODS: a group of RhoA specific small interfering RNAs (siRNA) was constructed and confined by sequencing analysis. The siRNA of RhoA gene was transfected into human tongue squamous cell carcinoma Tca8113 cells line by Lipofectamine(TM) 2000. The protein transient expression of RhoA in the transfectants was examined 48 h after transfection. The cell line with stable expression of siRNA of RhoA was obtained by blasticidin screening and colony culture. Cell growth rate was determined with a cell counting. Millicell chambermodel and wound healing assay were used to examine the abilities of migration and invasion, respectively in vitro. RESULTS: RhoA was overexpressed in tongue squamous cell carcinoma Tca8113 cell line. Silencing of endogenous RhoA gene expression in Tca8113 cells resulted in inhibition of the proliferation, adhesion, chemotaxis and invasion of Tca8113 cells in vitro. CONCLUSIONS: RhoA siRNA inhibits the proliferation, adhesion, chemotaxis and invasion of oral squamous cell carcinoma Tca8113 cell lines. siRNA of RhoA is a potential factor controlling the proliferation and metastasis of Tca8113 cells. RhoA may play an important role in metastasis of oral squamous cell carcinoma.

Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth.

The Warburg effect describes a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose even in the presence of oxygen. To better understand how tyrosine kinase signaling, which is commonly increased in tumors, regulates the Warburg effect, we performed phosphoproteomic studies. We found that oncogenic forms of fibroblast growth factor receptor type 1 inhibit the pyruvate kinase M2 (PKM2) isoform by direct phosphorylation of PKM2 tyrosine residue 105 (Y(105)). This inhibits the formation of active, tetrameric PKM2 by disrupting binding of the PKM2 cofactor fructose-1,6-bisphosphate. Furthermore, we found that phosphorylation of PKM2 Y(105) is common in human cancers. The presence of a PKM2 mutant in which phenylalanine is substituted for Y(105) (Y105F) in cancer cells leads to decreased cell proliferation under hypoxic conditions, increased oxidative phosphorylation with reduced lactate production, and reduced tumor growth in xenografts in nude mice. Our findings suggest that tyrosine phosphorylation regulates PKM2 to provide a metabolic advantage to tumor cells, thereby promoting tumor growth.

Fibroblast growth factor receptor 3 associates with and tyrosine phosphorylates p90 RSK2, leading to RSK2 activation that mediates hematopoietic transformation.

Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase, TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529, which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation, through a putative disruption of the autoinhibitory alphaL-helix on the C terminus of RSK2, unlike Y529 phosphorylation, which facilitates ERK binding. Moreover, we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707, as well as the subsequent RSK2 activation. Furthermore, in a murine bone marrow transplant assay, genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells, suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases.