High Homocysteine-Thiolactone Leads to Reduced MENIN Protein Expression and an Impaired DNA Damage Response: Implications for Neural Tube Defects.

DNA damage is associated with hyperhomocysteinemia (HHcy) and neural tube defects (NTDs). Additionally, HHcy is a risk factor for NTDs. Therefore, this study examined whether DNA damage is involved in HHcy-induced NTDs and investigated the underlying pathological mechanisms involved. Embryonic day 9 (E9) mouse neuroectoderm cells (NE4C) and homocysteine-thiolactone (HTL, active metabolite of Hcy)-induced NTD chicken embryos were studied by Western blotting, immunofluorescence. RNA interference or gene overexpression techniques were employed to investigate the impact of Menin expression changes on the DNA damage. Chromatin immunoprecipitation-quantitative polymerase chain reaction was used to investigate the epigenetic regulation of histone modifications. An increase in γH2AX (a DNA damage indicator) was detected in HTL-induced NTD chicken embryos and HTL-treated NE4C, accompanied by dysregulation of phospho-Atr-Chk1-nucleotide excision repair (NER) pathway. Further investigation, based on previous research, revealed that disruption of NER was subject to the epigenetic regulation of low-expressed Menin-H3K4me3. Overexpression of Menin or supplementation with folic acid in HTL-treated NE4C reversed the adverse effects caused by high HTL. Additionally, by overexpressing the Mars gene, we tentatively propose a mechanism whereby HTL regulates Menin expression through H3K79hcy, which subsequently influences H3K4me3 modifications, reflecting an interaction between histone modifications. Finally, in 10 human fetal NTDs with HHcy, we detected a decrease in the expression of Menin-H3K4me3 and disorder in the NER pathway, which to some extent validated our proposed mechanism. The present study demonstrated that the decreased expression of Menin in high HTL downregulated H3K4me3 modifications, further weakening the Atr-Chk1-NER pathway, resulting in the occurrence of NTDs.

The lncRNA MEG3/miRNA-21/P38MAPK axis inhibits coxsackievirus 3 replication in acute viral myocarditis.

Evidence is emerging on the roles of long noncoding RNAs (lncRNAs) as regulatory factors in a variety of viral infection processes, but the mechanisms underlying their functions in coxsackievirus group B type3 (CVB3)-induced acute viral myocarditis have not been explicitly delineated. We previously demonstrated that CVB3 infection decreases miRNA-21 expression; however, lncRNAs that regulate the miRNA-21-dependent CVB3 disease process have yet to be identified. To evaluate lncRNAs upstream of miRNA-21, differentially expressed lncRNAs in CVB3-infected mouse hearts were identified by microarray analysis and lncRNA/miRNA-21 interactions were predicted bioinformatically. MEG3 was identified as a candidate miRNA-21-interacting lncRNA upregulated in CVB3-infected mouse hearts. MEG3 expression was verified to be upregulated in HeLa cells 48 h post CVB3 infection and to act as a competitive endogenous RNA of miRNA-21. MEG3 knockdown resulted in the upregulation of miRNA-21, which inhibited CVB3 replication by attenuating P38-MAPK signaling in vitro and in vivo. Knockdown of MEG3 expression before CVB3 infection inhibited viral replication in mouse hearts and alleviated cardiac injury, which improved survival. Furthermore, the knockdown of CREB5, which was predicted bioinformatically to function upstream of MEG3, was demonstrated to decrease MEG3 expression and CVB3 viral replication. This study identifies the function of the lncRNA MEG3/miRNA-21/P38 MAPK axis in the process of CVB3 replication, for which CREB5 could serve as an upstream modulator.

Engineered coxsackievirus B3 containing multiple organ-specific miRNA targets showed attenuated viral tropism and protective immunity.

Coxsackievirus B3 (CVB3) can cause viral myocarditis, pancreatitis, and aseptic meningitis. This study aimed to construct an engineered CVB3 harboring three different tissue-specific miRNA targets (CVB3-miR3*T) to decrease the virulence of CVB3 in muscles, pancreas, and brain. CVB3-miR3*T and CVB3-miR-CON (containing three sequences not found in the human genome) were engineered and replicated in HELA cells. A viral plaque assay was used to determine the titers in HELA cells and TE671 cells (high miRNA-206 expression), MIN-6 cells (high miRNA-29a-3p expression), and mouse astrocytes (high miRNA-124-3p expression). We found that engineered CVB3 showed attenuated replication and reduced cytotoxicity, the variability of each type of cell was also increased in the CVB3-miR3*T group. Male BALB/c mice were infected to determine the LD50 and examine heart, pancreas, and brain titers and injury. Viral replication of the engineered viruses was restricted in infected mouse heart, pancreas, and brain, and viral plaques were about 100 fold lower compared with the control group. Mice immunized using CVB3-miR3*T, UV-inactivated CVB3-WT, and CVB3-miR-CON were infected with 100 × LD50 of CVB3-WT to determine neutralization. CVB3-miRT*3-preimmunized mice exhibited complete protection and remained alive after lethal virus infection, while only 5/15 were alive in the UV-inactivated mice, and all 15 mice were dead in the PBS-immunized group. The results demonstrate that miR-206-, miRNA-29a-3p-, and miRNA-124-3p-mediated CVB3 detargeting from the pancreas, heart, and brain might be a highly effective strategy for viral vaccine development.

Identification of histone malonylation in the human fetal brain and implications for diabetes-induced neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are severe congenital malformations. Diabetes during pregnancy is a risk factor for NTDs, but its mechanism remains elusive. Emerging evidence suggests that protein malonylation is involved in diabetes. Here, we report the correlation between histone lysine malonylation in diabetes-induced NTDs. METHODS: Nano-HPLC/MS/MS was used to screen the histone malonylation profile in human embryonic brain tissue. Then, the histone malonylation level was compared between the brains of normal control mice and mice with diabetes-induced NTDs. Finally, the histone malonylation level was compared under high glucose exposure in an E9 neuroepithelial cell line (NE4C). RESULTS: A total of 30 histone malonylation sites were identified in human embryonic brain tissue, including 18 novel sites. Furthermore, we found an increased histone malonylation level in brain tissues from mice with diabetes-induced NTDs. Finally, both the histone malonylation modified sites and the modified levels were proved to be increased in the NE4C treated with high glucose. CONCLUSION: Our results present a comprehensive map of histone malonylation in the human fetal brain. Furthermore, we provide experimental evidence supporting a relationship between histone malonylation and NTDs caused by high glucose-induced diabetes. These findings offer new insights into the pathological role of histone modifications in human NTDs.

The protective role of microRNA-21 against coxsackievirus B3 infection through targeting the MAP2K3/P38 MAPK signaling pathway.

BACKGROUND: The P38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in CVB3-induced diseases. We previously demonstrated microRNA-21 has potential inhibitory effect on the MAP2K3 which locates upstream of P38 MAPK and was upregulated in mouse hearts upon CVB3 infection. However, the effect and underlying mechanism of miRNA-21 on CVB3 infection remain unclear. METHODS: We detected continuous changes of cellular miRNA-21 and P38 MAPK proteins expression profiling post CVB3 infection in vitro within 12 h. P38 MAPK signaling was inhibited by the specific inhibitor, small interfering RNA and miRNA-21 mimic in vitro, CVB3 replication, cell apoptosis rate and proliferation were detected. Viral load in the mice heart, cardiomyocyte apoptosis rate and histological of the heart were also detected in the mice model of viral myocarditis pretreated with miRNA-21-lentivirus. RESULTS: We observed significant upregulation of miRNA-21 expression followed by suppression of the MAP2K3/P38 MAPK signaling in CVB3-infected Hela cells. The inactivation of the MAP2K3/P38 MAPK signaling by P38 MAPK specific inhibitor, small interfering RNA against MAP2K3, or miRNA-21 overexpression significantly inhibited viral progeny release from CVB3-infected cells. Mechanistically, when compared with control miRNA, miRNA-21 showed no effect on capsid protein VP1 expression and viral load within host cells, while significantly reversing CVB3-induced caspase-3 activation and cell apoptosis rate, further promoting proliferation of infected cells, which indicates the inhibitory effect of miRNA-21 on CVB3 progeny release. In the in vivo study, when compared with control miRNA, miRNA-21 pretreatment remarkably inactivated the MAP2K3/P38 MAPK signaling in mice and protected them against CVB3 infection as evidenced by significantly alleviated cell apoptosis rate, reduced viral titers, necrosis in the heart as well as by remarkably prolonged survival time. CONCLUSIONS: miRNA-21 were reverse correlated with P38 MAPK activation post CVB3 infection, miRNA-21 overexpression significantly inhibited viral progeny release and decreased myocytes apoptosis rate in vitro and in vivo, suggesting that miRNA-21 may serve as a potential therapeutic agent against CVB3 infection through targeting the MAP2K3/P38 MAPK signaling.

Plasma interleukin-37 is increased and inhibits the production of inflammatory cytokines in peripheral blood mononuclear cells in systemic juvenile idiopathic arthritis patients.

BACKGROUND: Interleukin (IL)-37 has emerged as a novel anti-inflammatory cytokine that play an immunosuppressive role in regulating inflammatory response. This study aimed to measure IL-37 levels in the plasma and peripheral blood mononuclear cells (PBMCs) of patients with systemic juvenile idiopathic arthritis (sJIA), and to establish the correlation between IL-37 levels and disease activity, laboratory parameters and inflammatory cytokines. METHODS: The mRNA levels of IL-37 in PBMCs and plasma IL-37 concentrations in 46 sJIA patients and 30 age- and sex-matched healthy controls were measured by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The correlations between plasma IL-37 levels and disease activity, laboratory parameters and inflammatory cytokines in sJIA were analyzed by Spearman correlation test. PBMCs from the sJIA patients were stimulated with recombinant human IL-37 (rhIL-37) protein, expressions of IL-1ß, IL-6, TNF-α and IL-17 were detected by RT-PCR and ELISA. RESULTS: Plasma levels of IL-37 and relative IL-37 mRNA expression were significantly elevated in sJIA patients, especially in active sJIA patients, when compared with the healthy controls (P < 0.001). Furthermore, patients with active disease showed higher IL-37 mRNAs and plasma protein levels than those with inactive disease as well as healthy controls. Plasma IL-37 levels were correlated with disease activity and inflammatory cytokines (IL-6, TNF-α, IL-17 and GM-CSF) in sJIA patients. The productions of inflammatory cytokines such as IL-6, TNF-α, IL-17 in PBMCs from sJIA patients were obviously decreased after recombinant IL-37 stimulation, whereas the production of IL-1ß was not changed. CONCLUSIONS: Our results demonstrate that levels of IL-37 were higher in sJIA patients, which were correlated with disease activity and sJIA related inflammatory cytokines. In addition, rhIL-37 down-regulates the expressions of inflammatory cytokines form PBMCs in sJIA patients, suggesting that IL-37 may have the potential role as a natural inhibitor for the pathogenesis and therapy of sJIA.

Coxsackievirus B3 induces autophagy in HeLa cells via the AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways.

In a previous study, the number of autophagosomes increased after coxsackievirus B3 (CVB3) infection. However, the exact mechanism by which CVB3 regulates the number of autophagosomes is unclear. Earlier studies have found that infection with CVB3 activates extracellular signal-regulated kinase (ERK). ERK is essential for CVB3 replication and can increase the number of autophagosomes. In the current study, extracellular signal-regulated kinase 1/2 was activated in HeLa cells after CVB3 infection. The ERK kinase inhibitor, U0126, was then used to inhibit the activity of ERK. Treatment with U0126 led to a significant reduction in the number of autophagosomes indicating that the CVB3-induced autophagosome accumulation may have occurred via the ERK pathway. The relationship between CVB3 infection and ERK pathway activation was also investigated. The results showed that the RasGAP protein could be further cleaved, leading to the activation of the Ras/Raf/MEK (mitogen/extracellular signal-regulated kinase)/ERK pathway and that CVB3 infection could result in an increase in the concentration of calcium in the cytoplasm, resulting in mitochondrial damage, a decrease in the concentration of ATP and activation of the AMPK (AMP-activated protein kinase)/MEK/ERK pathway. In summary, CVB3 might directly or indirectly induce autophagy via AMPK/MEK/ERK and Ras/Raf/MEK/ERK signaling pathways in the host cells, representing a pivotal mechanism for CVB3 pathogenesis.

Coxsackievirus B3 engineered to contain microRNA targets for muscle-specific microRNAs displays attenuated cardiotropic virulence in mice.

UNLABELLED: Coxsackievirus B3 (CVB3) is trophic for cardiac tissue and is a major causative agent for viral myocarditis, where local viral replication in the heart may lead to heart failure or even death. Recent studies show that inserting microRNA target sequences into the genomes of certain viruses can eradicate these viruses within local host tissues that specifically express the cognate microRNA. Here, we demonstrated both in vitro and in vivo that incorporating target sequences for miRNA-133 and -206 into the 5' untranslated region of the CVB3 genome ameliorated CVB3 virulence in skeletal muscle and myocardial cells that specifically expressed the cognate cellular microRNAs. Compared to wild-type CVB3, viral replication of the engineered CVB3 was attenuated in human TE671 (rhabdomyosarcoma) and L6 (skeletal muscle) cell lines in vitro that expressed high levels of miRNA-206. In the in vivo murine CVB3-infection model, viral replication of the engineered CVB3 was attenuated specifically in the heart that expressed high levels of both miRNAs, but not in certain tissues, which allowed the host to retain the ability to induce a strong and protective humoral immune response against CVB3. The results of this study suggest that a microRNA-targeting strategy to control CVB3 tissue tropism and pathogenesis may be useful for viral attenuation and vaccine development. IMPORTANCE: Coxsackievirus B3 (CVB3) is a major causative agent for viral myocarditis, and viral replication in the heart may lead to heart failure or even death. Limiting CVB3 replication within the heart may be a promising strategy to decrease CVB3 pathogenicity. miRNAs are ∼21-nucleotide-long, tissue-specific endogenous small RNA molecules that posttranscriptionally regulate gene expression by imperfectly binding to the 3' untranslated region (UTR), the 5' UTR, or the coding region within a gene. In our study, muscle-specific miRNA targets (miRT) were incorporated into the CVB3 genome. Replication of the engineered viruses was restricted in the important heart tissue of infected mice, which reduced cardiac pathology and increased mouse survival. Meanwhile, replication ability was retained in other tissues, thus inducing a strong humoral immune response and providing long-term protection against CVB3 rechallenge. This study suggests that a microRNA-targeting strategy can potentially control CVB3 tissue tropism and pathogenesis and may be useful for viral attenuation and vaccine development.

Coxsackievirus B3 induces crosstalk between autophagy and apoptosis to benefit its release after replicating in autophagosomes through a mechanism involving caspase cleavage of autophagy-related proteins.

Coxsackievirus B3 (CVB3) is known to induce both autophagy and apoptosis, but whether a relationship exists between these processes upon infection, and whether and how they influence the viral life cycle are currently unknown. We observed here that while autophagosome formation increased in CVB3-infected HeLa cells at the early stage of infection, it decreased at the late stage of infection along with increased apoptosis. Examining whether a functional relationship existed between autophagy and apoptosis during CVB3 infection, we found that increasing levels of autophagy inhibited apoptosis and that some apoptotic proteins in the endogenous and exogenous apoptosis pathways played a role in the transition from autophagy to apoptosis by cleaving the autophagy-related proteins Beclin-1 and Atg5. However, the transcription and translation of full-length Atg5 and Beclin-1 also increased, which likely counteracted the cleavage effect in order to prevent cells from dying too early and to ensure that CVB3 replication was complete in the autophagosomes. Using pharmacological inducers and inhibitors of autophagy as well as inhibitors of apoptosis, we found that while CVB3 replication relied on the autophagosomes, its release from the cell depended on apoptosis. Therefore, autophagy and apoptosis are two important processes that interact with each other during CVB3 infection, promoting the CVB3 life cycle.

Inhibition of IL-2 inducible T-cell kinase alleviates T-cell activation and murine myocardial inflammation associated with CVB3 infection.

BACKGROUND: Coxsackievirus B3 (CVB3) infection causes myocarditis, pancreatitis, and aseptic meningitis. Targeting antigen-specific T cell reactions might be a promising way to alleviate the inflammatory response induced by CVB3 infection. IL-2-inducible T-cell kinase (ITK), a member of Tec kinase family expressed mainly in T cells, plays an important role in the activation of T cells. The role of ITK in viral myocarditis induced by CVB3 has not been documented. METHODOLOGY: In this study, we inhibited the ITK expression in Jurkat cells, primary human peripheral blood mononuclear cells (PBMC), and mouse splenocytes by ITK-specific siRNA. The inhibition efficiently suppressed cell proliferation (P<0.05) and T-cell related cytokine secretion (P<0.05). In order to inhibit ITK in vivo, the pGCSIL plasmid containing short hairpin RNAs targeting ITK was constructed and transduced into mice infected with CVB3. ITK-inhibited mice showed reduced cell proliferation (3, 5, and 7 days post-challenge, P<0.05) as well as CD4+ and CD8+ T cells (5 days post-challenge, P<0.05). The altered production of inflammatory cytokines alleviated pathologic heart damage and improved mice survival rate (P<0.05). CONCLUSION: ITK played an important role in the T cell development and represented a new target for the modulation of T-cell-mediated inflammatory response by CVB3 infection.

MicroRNAs regulate the pathogenesis of CVB3-induced viral myocarditis.

AIMS: To evaluate the role of microRNAs (miRNAs) in the pathogenesis of Coxsackievirus B3 (CVB3)-induced viral myocarditis. METHODS: We detected miRNA expression profiling by microarray utilizing a mouse model on day 4 after CVB3 infection. Then we validated differentially expressed miRNAs using real-time polymerase chain reaction (PCR). We predicted target genes using miRNA target prediction databases and assessed them using mRNA microarray and qualitative reverse transcription PCR measurements. By analyzing the target function of differentially expressed miRNAs, we initially explored the regulating role of miRNAs in viral myocarditis. RESULTS: We found five differentially expressed miRNAs that are involved in regulating several important innate immune and antiviral pathways such as the Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine- cytokine receptor interaction, MAPK signaling pathway, JAK-STAT signaling pathway, and natural killer cell-mediated cytotoxicity. CONCLUSION: miRNAs regulate the pathogenesis of viral myocarditis. This study may provide a new perspective and a deeper understanding of the pathogenesis of viral myocarditis that may help with the development of novel therapies against CVB3 infection.

[Inhibition of virus infection in coxsackievirus-induced myocarditis model by RNA interference].

To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.

Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both in vitro and in vivo.

BACKGROUND: Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection. METHODS: To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B) expressed by a recombinant vector (pGCL-2B) or a recombinant lentivirus (Lenti-2B) were tansfected in HeLa cells or transduced in mice infected with CVB3. RESULTS: ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo. CONCLUSIONS: Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.

[Recurrent miscarriage and the quality of semen and sperm: a case-control study].

OBJECTIVE: To analyze the relation between recurrent miscarriage and routine semen parameters. METHODS: We compared the semen parameters of normal healthy men with those of the spouses of recurrent miscarriage women through 1: 1 age-matched case-control study. RESULTS: Compared with the healthy controls, the men of the case group showed a significantly lower mean semen volume ([1.95 +/- 1.11] ml vs [2.74 +/- 1.43] ml), sperm concentration ([48.68 +/- 20.07] x 10(6)/ml vs [59.26 +/- 25.35] x 10(6)/ml), percentage of grade b sperm ([12.07 +/- 3.34] % vs [16.18 +/- 6.74] %), fruit-sugar content ([1.73 +/- 0.64] g/L vs [2.21 +/- 0.75] g/L), acrosomal enzyme activity ([84.34 +/- 26.69] U/mg prot vs [94.20 +/- 26.35] U/mg prot), alpha-glucuronidase (alpha-GLU) content ([36.28 +/- 15.98] U/ml vs [44.45 +/- 12. 54] U/ml), and acid phosphatase (ACP) content ([68.55 +/- 35.45] U/ml vs [84.78 +/- 51. 10] U/ml) (P < 0.05), but remarkably higher percentages of head teratospermia ([47.36 +/- 4.59] % vs [46.50 +/- 6.32] %) and tail teratospermia ([7.56 +/- 2.27] % vs [7.28 +/- 3.10] %), and elastase content ([885.64 +/- 1 272.30] ng/ml vs [661.08 +/- 764.64] ng/ml) (P < 0.05). Based on the results of discriminant analysis, the semen volume, percentages of grade b sperm and combined teratospermia, and contents of fruit-sugar, alpha-GLU and ACP could be used to evaluate the semen and sperm quality of the spouses of recurrent miscarriage women. CONCLUSION: Routine semen and sperm tests might help evaluate the seminal factors of recurrent miscarriage, but they lack specificity and need comprehensive analysis. Poorer semen quality is associated with higher incidence of recurrent miscarriage.

[Effect of Itk down regulation on cytokines production in Jurkat cell].

OBJECTIVE: To study the effect of Itk down regulation on Jurkat cell proliferation and inflammatory cytokines production, and provide useful data for Itk as an attractive target for potential drugs. METHODS: Three shRNAs against different region of Itk were constructed and cotransfected with pEGFP-C1-hItk. The shRNA, which can knock down Itk, was selected and packed into lentivirus. After Jurkat cells were transfected with shRNA lentivirus, the change of Itk protein expression, cell proliferation and cytokines production was observed. RESULTS: Itk mRNA was reduced about 55% in Jurkat cells transfected with Itk-shRNA1, compared with that in control cells shRNAnon (P < 0.05). Knocking down Itk expression had a profound inhibitory effect on Jurkat cell proliferation. In addition, there was a substantial decrease in level of cytokines, such as IL-2, IL-5, IL-10 and IFN-gamma, produced by cell transfected with Itk-shRNA1. CONCLUSION: Knocking down Itk expression can inhibit Jurkat cell proliferation and inflammatory cytokines production.

[Research of ITK regulation on mouse spleen lymphocytes proliferation and differentiation].

OBJECTIVE: By using the RNAi method to inhibit Itk protein expression specificity, to observe lymphocytes proliferation and cytokines production, verify its function as a drug target. METHODS: Designed siRNA aims at Itk sequence according to its sequence and solid structure, then electrotransfected into mouse spleen lymphocytes, We validated the decrease of Itk protein by Western-Blot, and detected the change of the cell proliferation by MTS and the change of inflammatory cytokines by ELISA. RESULTS: Itk protein can be suppressed by Itk-siRNA, there were significantly reduced compared to its control group on cell proliferation as well as cytokine secretion such as IL-2, IL-4, IL-5, IFN-gamma. They all have statistical difference (P < 0.05). CONCLUSION: Itk has an important immunomodulatory effect in mouse spleen lymphocytes proliferation and secretion of inflammatory cytokines.This can supply an experimental basis to regard Itk as drug target for inflammation therapy.

[Intracellular clearance of Coxsakievirus B3 infection by short interfering RNA and its mechanism study].

OBJECTIVE: To evaluate the possibility of short interfering RNA (siRNA) inhibiting Coxsackievirus B3 (CVB3) infection in vitro, and discover the mechanism initially. METHODS: We obtained proper effective dosage of siRNA by observing cytopathic effect (CPE). Estimate its antiviral activities and its pathway of siRNA by Western Blot assay and RT-PCR. RESULTS: Results showed that siRNA-3753 can be effectively transfected into HeLa cells, we can achieve a high transfection efficiency up to 98.77% and its effect can last for 48 h stably in cells. 0.6 micromol/L siRNA-3753 got a high inhibiting effect of virus and didn't show any toxicity to cells. So we consider this concentration as the experimental concentration. siRNA-3753 can debase virus reproduction. The antiviral effect is sequence-specific and is not attributable to either interferon or the interferon response effectors protein kinase R (PKR). CONCLUSION: The data confirmed that siRNA can effectively inhibit CVB3 infection in vitro, its antivirus effect was gained from specific debase of virus genome.

[Study on inhibition of coxsackievirus B3 infection in HeLa cell by short interfering RNA targeting 2B protein].

To study the inhibitory effect and function characteristics of small interfering RNA (siRNA) on cosxackievirus B3(CVB3) infection by RNA interference technique, siRNA-2B against the viral 2B region was synthesized and transfected into HeLa cell, which was then infected with CVB3. The efficiency of siRNA transfection was examined by FCM, the cell toxicity of siRNA-2B by MTT, and the antiviral ability of siRNA-2B by cytopathic effect (CPE), plaque reduction assay and RT-PCR. The results showed that siRNA-2B could be transfected efficiently into HeLa cell and lasted at least 48h. High concentration of siRNA-2B didn't show any sign of toxicity to cells. siRNA-2B exhibited a significant protective effect on cell viability by specific inhibition of viral replication. It showed a close relationship between the concentrations of siRNA-2B and the antiviral effects. siRNA-2B led to dramatical reduction of viral titers in supernatant of cell culture and weakened the reinfection ability of the virus. These data proposed that siRNA-2B, targeting 2B protein, can effectively inhibit CVB3 infection in HeLa cell and exhibits its transfection efficiency, viral inhibition specificity and adose-dependant manner, suggesting its potential role in prevention and treatement of CVB3 infection.

[Short interfering RNA-mediated inhibition of coxsakievirus B3 infection in vitro].

OBJECTIVE: To evaluate feasibility of inhibiting coxsackievirus B3 (CVB3) infection at cellular, protein and gene levels by using small interfering RNA (siRNA). METHODS: Antiviral activities of siRNAs were evaluated by observing cytopathic effect (CPE), using plaque reduction Western blotting assays and RT-PCR. RESULTS: Eight siRNAs were synthesized, among them, SiRNA-2, SiRNA-3, SiRNA-6 and SiRNA-7 which were targeted against sequences located in 2B, VP4, 2A and 3C section of CVB3 genome, were designed to have different effect of inhibiting CVB3 infection in vitro. SiRNA-2 showed the best protective effect, 95 percent inhibition of CVB3 cytopathic effect and plaque forming effect was observed at 0.0001 MOI, viral protein synthesis and replication were inhibited. SiRNA-2 showed 30 percent inhibition of virus at 0.1 MOI, 70 percent inhibition at 0.01 MOI, 88 percent inhibition at 0.001 MOI, and 99 percent inhibition at 0.0001 MOI 48 hours after CVB3 infection. CONCLUSION: SiRNA could effectively inhibit CVB3 infection in vitro, siRNA-2, which is targeted against sequence in 2B section of CVB3 genome, seemed to be the best one among those synthesized in this study.

[Antisense oligonucleotide inhibition of coxsackievirus B3 gene expression in HeLa cells and dose-response experiments].

OBJECTIVE: In this study, the authors investigated inhibition of coxsackievirus B (CVB) gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon and structural protein coding sequences and also observed the dose-response of the sequence specific inhibition of CVB plaque formation by antisense oligonucleotides. METHODS: Antiviral activities of these oligonucleotides were evaluated by using plaque reduction assay, yield reduction assay, cytopathic effect (CPE) and Western blot analysis. The cells were treated with random oligonucleotides as a specificity control. RESULTS: At a screening concentration of 5 micromole, 6 of the phosphorothioate oligonucleotide demonstrated some reduction of virus replication relative to untreated cells. 70%-90% inhibition of virus at 0.1 MOI (multiplicity of infection), 50% inhibition of virus infection at 10 MOI. The levels of the VP1 were reduced in CVB-infected cells treated with Scb561 and Scb733. VP1 was significantly reduced after treatment with 0.625 micromole Scb561 and almost undetectable in cells treated with 2.5 micromole Scb561. Dose response experiments implied that sequence specific oligonucleotide doses were related to effect on inhibition of CVB3 infection. When oligonucleotide doses were increased from 1.25 to 5 micromole, 75% to 90% inhibition were observed with Scb561 and 65% to 80% inhibition with Scb733, whereas random control failed to inhibit CVB replication (8% inhibition for each). CONCLUSION The present studies showed that antisense oligonucleotides against internal ribosome entry site (IRES) and translation initiation codon were capable of specifically inhibiting the synthesis of viral protein and subsequent productive CVB replication.The selective inhibition using antisense oligonucleotide might lead to development of an effective antiviral agent for future clinical evaluation.