Fractional amplitude of low-frequency fluctuation and voxel-mirrored homotopic connectivity in patients with persistent postural-perceptual dizziness: resting-state functional magnetic resonance imaging study.

PURPOSE: Persistent postural-perception dizziness (PPPD) is a chronic subjective form of dizziness characterized by the exacerbation of dizziness with active or passive movement, complex visual stimuli, and upright posture. Therefore, we aimed to analyze the resting-state functional magnetic resonance imaging (fMRI) in patients with PPPD using fractional amplitude of low-frequency fluctuation (fALFF) and voxel-mirrored homotopic connectivity (VMHC) and evaluate the correlation between abnormal regions in the brain and clinical features to investigate the pathogenesis of PPPD. METHODS: Thirty patients with PPPD (19 females and 11 males) and 30 healthy controls (HC) (18 females and 12 males) were closely matched for age and sex. The fALFF and VMHC methods were used to investigate differences in fMRI (BOLD sequences) between the PPPD and HC groups and to explore the associations between areas of functional abnormality and clinical characteristics (Dizziness, Anxiety, Depression, and Duration). RESULT: Compared to the HC group, patients with PPPD displayed different functional change patterns, with increased fALFF in the right precuneus and decreased VMHC in the bilateral precuneus. Additionally, patients with PPPD had a positive correlation between precuneus fALFF values and dizziness handicap inventory (DHI) scores, and a negative correlation between VMHC values and the disease duration. CONCLUSIONS: Precuneus dysfunction was observed in patients with PPPD. The fALFF values correlated with the degree of dizziness in PPPD, and changes in VMHC values were associated with the duration of dizziness, suggesting that fMRI changes in the precuneus of patients could be used as a potential imaging marker for PPPD.

Increased functional connectivity between default mode network and visual network potentially correlates with duration of residual dizziness in patients with benign paroxysmal positional vertigo.

Objective: To assess changes in static and dynamic functional network connectivity (sFNC and dFNC) and explore their correlations with clinical features in benign paroxysmal positional vertigo (BPPV) patients with residual dizziness (RD) after successful canalith repositioning maneuvers (CRM) using resting-state fMRI. Methods: We studied resting-state fMRI data from 39 BPPV patients with RD compared to 38 BPPV patients without RD after successful CRM. Independent component analysis and methods of sliding window and k-means clustering were adopted to investigate the changes in dFNC and sFNC between the two groups. Additionally, temporal features and meta-states were compared between the two groups. Furthermore, the associations between fMRI results and clinical characteristics were analyzed using Pearson's partial correlation analysis. Results: Compared with BPPV patients without RD, patients with RD had longer duration of BPPV and higher scores of dizziness handicap inventory (DHI) before successful CRM. BPPV patients with RD displayed no obvious abnormal sFNC compared to patients without RD. In the dFNC analysis, patients with RD showed increased FNC between default mode network (DMN) and visual network (VN) in state 4, the FNC between DMN and VN was positively correlated with the duration of RD. Furthermore, we found increased mean dwell time (MDT) and fractional windows (FW) in state 1 but decreased MDT and FW in state 3 in BPPV patients with RD. The FW of state 1 was positively correlated with DHI score before CRM, the MDT and FW of state 3 were negatively correlated with the duration of BPPV before CRM in patients with RD. Additionally, compared with patients without RD, patients with RD showed decreased number of states and state span. Conclusion: The occurrence of RD might be associated with increased FNC between DMN and VN, and the increased FNC between DMN and VN might potentially correlate with the duration of RD symptoms. In addition, we found BPPV patients with RD showed altered global meta-states and temporal features. These findings are helpful for us to better understand the underlying neural mechanisms of RD and potentially contribute to intervention development for BPPV patients with RD.

Investigation on biomechanical responses in bilateral semicircular canals and nystagmus in vestibulo-ocular reflex experiments under different forward-leaning angles.

Different head positions affect the responses of the vestibular semicircular canals (SCCs) to angular movement. Specific head positions can relieve vestibular disorders caused by excessive stimulating SCCs. In this study, we quantitatively explored responses of human SCCs using numerical simulations of fluid-structure interaction and vestibulo-ocular reflex (VOR) experiments under different forward-leaning angles of the head, including 0°, 10°, 20°, 30°, 40°, 50°, and 60°. It was found that the horizontal nystagmus slow-phase velocity and corresponding biomechanical responses of the cupula in horizontal SCC increased with the forward-leaning angles of the head, reached a maximum when the head was tilted 30° forward, and then gradually decreased. However, no obvious vertical or torsional nystagmus was observed in the VOR experiments. In the numerical model of bilateral SCCs, the biomechanical responses of the cupula in the left anterior SCC and the right anterior SCC showed the same trends; they decreased with the forward-leaning angles, reached a minimum at a 40° forward tilt of the head, and then gradually increased. Similarly, the biomechanical responses of the cupula in the left posterior SCC and in the right posterior SCC followed a same trend, decreasing with the forward-leaning angles, reaching a minimum at a 30° forward tilt of the head, and then gradually increasing. Additionally, the biomechanical responses of the cupula in both the anterior and posterior SCCs consistently remained lower than those observed in the horizontal SCCs across all measured head positions. The occurrence of these numerical results was attributed to the consistent maintenance of mutual symmetry in the bilateral SCCs with respect to the mid-sagittal plane containing the axis of rotation. This symmetry affected the distribution of endolymph pressure, resulting in biomechanical responses of the cupula in each pair of symmetrical SCCs exhibiting same tendencies under different forward-leaning angles of the head. These results provided a reliable numerical basis for future research to relieve vestibular diseases induced by spatial orientation of SCCs.

MiR129-5p-loaded exosomes suppress seizure-associated neurodegeneration in status epilepticus model mice by inhibiting HMGB1/TLR4-mediated neuroinflammation.

BACKGROUND: Neuroinflammation contributes to both epileptogenesis and the associated neurodegeneration, so regulation of inflammatory signaling is a potential strategy for suppressing epilepsy development and pathological progression. Exosomes are enriched in microRNAs (miRNAs), considered as vital communication tools between cells, which have been proven as potential therapeutic method for neurological disease. Here, we investigated the role of miR129-5p-loaded mesenchymal stem cell (MSC)-derived exosomes in status epilepticus (SE) mice model. METHODS: Mice were divided into four groups: untreated control (CON group), kainic acid (KA)-induced SE groups (KA group), control exosome injection (KA + Exo-con group), miR129-5p-loaded exosome injection (KA + Exo-miR129-5p group). Hippocampal expression levels of miR129-5p, HMGB1, and TLR4 were compared among groups. Nissl and Fluoro-jade B staining were conducted to evaluate neuronal damage. In addition, immunofluorescence staining for IBA-1 and GFAP was performed to assess glial cell activation, and inflammatory factor content was determined by ELISA. Hippocampal neurogenesis was assessed by BrdU staining. RESULTS: The expression of HMGB1 was increased after KA-induced SE and peaking at 48 h, while hippocampal miR129-5p expression decreased in SE mice. Exo-miR129-5p injection reversed KA-induced upregulation of hippocampal HMGB1 and TLR4, alleviated neuronal damage in the hippocampal CA3, reduced IBA-1 + and GFAP + staining intensity, suppressed SE-associated increases in inflammatory factors, and decreased BrdU + cell number in dentate gyrus. CONCLUSIONS: Exosomes loaded with miR129-5p can protect neurons against SE-mediated degeneration by inhibiting the pro-inflammatory HMGB1/TLR4 signaling axis.

Altered parietal operculum cortex 2 functional connectivity in benign paroxysmal positional vertigo patients with residual dizziness: A resting-state fMRI study.

AIMS: To investigate changes in functional connectivity (FC) focusing on parietal operculum cortex 2 (OP2) in benign paroxysmal positional vertigo (BPPV) patients with residual dizziness (RD) after successful canalith repositioning procedure (CRP). METHODS: High-resolution three-dimensional T1 and resting-state functional magnetic resonance imaging (fMRI) were performed on 55 healthy controls (HCs), 55 BPPV patients with RD, and 55 patients without RD after successful CRP. Seed-based (bilateral OP2) FC was calculated to investigate the changes in FC among the three groups. Additionally, we further explored the associations between abnormal FC and clinical symptoms. RESULTS: One-way analysis of covariance showed significant FC differences among the three groups. Post-hoc analysis showed that patients with RD exhibited decreased FC between left OP2 and regions of left angular gyrus (AG), thalamus, precuneus, middle frontal gyrus (MFG), and right cerebellum posterior lobe (CPL) in comparison with HCs. In addition, compared with patients without RD, patients with RD showed decreased FC between left OP2 and regions of left MFG, AG, middle temporal gyrus, and right CPL. Moreover, in patients with RD, the FC between left thalamus and OP2 was negatively correlated with duration of RD, and the FC between left AG and OP2 was negatively correlated with duration of BPPV. CONCLUSION: BPPV patients with RD showed reduced FC between brain regions involved in vestibular processing and spatial cognition; These results suggested that BPPV patients with RD might have diminished central processing of vestibular information and impaired spatial cognition.

Altered functional activity of the precuneus and superior temporal gyrus in patients with residual dizziness caused by benign paroxysmal positional vertigo.

Objective: Benign paroxysmal positional vertigo (BPPV) is a common clinical vertigo disease, and the most effective treatment for this disease is canal repositioning procedures (CRP). Most patients return to normal after a single treatment. However, some patients still experience residual dizziness (RD) after treatment, and this disease's pathogenesis is currently unclear. The purpose of this study is to explore whether there are abnormal brain functional activities in patients with RD by using resting-state functional magnetic resonance imaging (rs-fMRI) and to provide imaging evidence for the study of the pathogenesis of RD. Materials and methods: The BPPV patients in the Second Affiliated Hospital of Xuzhou Medical University had been included from December 2021 to November 2022. All patients had been received the collection of demographic and clinical characteristics (age, gender, involved semicircular canal, affected side, CRP times, BPPV course, duration of RD symptoms, and whether they had hypertension, diabetes, coronary heart disease.), scale assessment, including Dizziness Handicap Inventory (DHI), Hamilton Anxiety Inventory (HAMA), Hamilton Depression Inventory (HAMD), rs-fMRI data collection, CRP treatment, and then a one-month follow-up. According to the follow-up results, 18 patients with RD were included. At the same time, we selected 19 healthy individuals from our hospital's physical examination center who matched their age, gender as health controls (HC). First, the amplitude of low-frequency fluctuations (ALFF) analysis method was used to compare the local functional activities of the two groups of subjects. Then, the brain regions with different ALFF results were extracted as seed points. Functional connectivity (FC) analysis method based on seed points was used to explore the whole brain FC of patients with RD. Finally, a correlation analysis between clinical features and rs-fMRI data was performed. Results: Compared to the HC, patients with RD showed lower ALFF value in the right precuneus and higher ALFF value in the right superior temporal gyrus (STG). When using the right STG as a seed point, it was found that the FC between the right STG, the right supramarginal gyrus (SMG), and the left precuneus was decreased in RD patients. However, no significant abnormalities in the FC were observed when using the right precuneus as a seed point. Conclusion: In patients with RD, the local functional activity of the right precuneus is weakened, and the local functional activity of the right STG is enhanced. Furthermore, the FC between the right STG, the right SMG, and the left precuneus is weakened. These changes may explain the symptoms of dizziness, floating sensation, walking instability, neck tightness, and other symptoms in patients with RD to a certain extent.

Aberrant dynamic functional network connectivity in vestibular migraine patients without peripheral vestibular lesion.

PURPOSE: This study aimed to investigate changes in dynamic functional network connectivity (FNC) in patients with vestibular migraine (VM) and explore their relationship with clinical manifestations. METHODS: Resting-state functional magnetic resonance imaging (rs-fMRI) data were scanned from 35 VM patients without peripheral vestibular lesion and 40 age-, sex- and education-matched healthy controls (HC). Independent component analysis (ICA), sliding window (SW) and k-means clustering analysis were performed to explore the difference in FNC and temporal characteristics between two groups. Additionally, Pearson's partial correlation analysis was adopted to investigate the relationship between clinical manifestations and rs-fMRI results in patients with VM. RESULTS: Compared with HC, patients with VM showed increased FNC in pairs of extrastriate visual network (eVN)-ventral attention network (VAN), eVN-default mode network (DMN) and eVN-left frontoparietal network (lFPN), and exhibited decreased FNC in pairs of VAN-auditory network (AuN). The altered FNC was correlated with clinical manifestations of patients with VM. Additionally, we found increased mean dwell time and fractional windows in state 2 in VM patients compared with HC. Mean dwell time was positively correlated with headache impact test-6 (HIT-6) scores, fractional windows was positively associated with dizziness handicap inventory (DHI) scores. CONCLUSION: Our results indicated that patients with VM showed altered FNC primarily between sensory networks and networks related to cognitive, emotional and attention implementation, with more time spent in a state characterized by positive FNC between sensor cortex system and dorsal attention network (DAN). These findings could help reinforce the understanding on the neural mechanisms of VM.

Altered brain function in patients with vestibular migraine: a study on resting state functional connectivity.

PURPOSE: To characterize the altered brain function in patients with vestibular migraine (VM) using resting-state functional magnetic resonance imaging (fMRI). METHODS: In this prospective study, fMRI images as well as clinical characteristics and behavioral scales were collected from 40 VM patients and 40 healthy controls (HC). All patients received neurological, neuro-otological, and conventional MRI examinations to exclude peripheral vestibular lesions, focal lesions, and other neurological diseases. Seed-based (bilateral parietal operculum cortex 2, OP2) functional connectivity (FC) and independent component analysis (ICA)-based functional network connectivity (FNC) were performed to investigate the brain functional changes in patients with VM. Additionally, the correlations between the altered FC/FNC and behavioral results were analyzed. RESULTS: Compared with HC, patients with VM showed increased FC between the left OP2 and right precuneus and exhibited decreased FC between the left OP2 and left anterior cingulate cortex. We also observed increased FC between the right OP2 and regions of the right middle frontal gyrus and bilateral precuneus, as well as decreased FC between the bilateral OP2. Furthermore, patients with VM showed decreased FNC between visual network (VN) and networks of auditory and default mode, and exhibited increased FNC between VN and executive control network. A correlation analysis found that FC between the left OP2 and right precuneus was positively correlated with scores of dizziness handicap inventory (DHI) in patients with VM. CONCLUSION: The present study demonstrated altered brain function in patients with VM.

Altered thalamo-cortical functional connectivity in patients with vestibular migraine: a resting-state fMRI study.

PURPOSE: To explore the functional connectivity (FC) between the bilateral thalamus and the other brain regions in patients with vestibular migraine (VM). METHODS: Resting-state fMRI and 3D-T1 data were collected from 37 patients with VM during the interictal period and 44 age-, gender-, and years of education-matched healthy controls (HC). The FC of the bilateral thalamus was analyzed using a standard seed-based whole-brain correlation method. Furthermore, the correlations between thalamus FC and clinical characteristics of patients were investigated using Pearson's partial correlation. RESULTS: Compared with HC, VM patients showed decreased FC between the left thalamus and the left anterior cingulate cortex (ACC), bilateral insular and right supplementary motor cortex. We also observed decreased FC between the right thalamus and the left insular and ACC in VM patients. Furthermore, patients with VM also exhibited increased FC between the left thalamus and the right precuneus and middle frontal gyrus, between the right thalamus and superior parietal lobule. FC between the right thalamus and the left insular was negatively correlated with disease duration (p = 0.019, r = - 0.399), FC between the left thalamus and the left ACC was negatively correlated with HIT-6 score (p = 0.004, r = - 0.484). CONCLUSION: VM patients showed altered FC between thalamus and brain regions involved in pain, vestibular and visual processing, which are associated with specific clinical features. Specifically, VM patients showed reduced thalamo-pain and thallamo-vestibular pathways, while exhibited enhanced thalamo-visual pathway, which provided first insight into the underlying functional brain connectivity in VM patients.

Porcine epidemic diarrhea virus E protein suppresses RIG-I signaling-mediated interferon-ß production.

Porcine epidemic diarrhea virus (PEDV) encodes many multifunctional proteins that inhibit host innate immune response during virus infection. As one of important structural proteins, PEDV E protein has been found to block the production of type I interferon (IFN) in virus life cycle, but little is known about this process that E protein subverts host innate immune. Thus, in this present study, we initiated the construction of eukaryotic expression vectors to express PEDV E protein. Subsequently, cellular localization analysis was performed and the results showed that the majority of PEDV E protein distributed at cytoplasm and localized in endoplasmic reticulum (ER). Over-expression of PEDV E protein significantly inhibited poly(I:C)-induced IFN-ß and IFN-stimulated genes (ISGs) productions. We also found that PEDV E protein remarkably suppressed the protein expression of RIG-I signaling-associated molecules, but all their corresponding mRNA levels remained unaffected and unchanged. Furthermore, PEDV E protein obviously interfered with the translocation of IRF3 from cytoplasm to nucleus through direct interaction with IRF3, which is crucial for the IFN-ß production induced by poly(I:C). Taken together, our results suggested that PEDV E protein acts as an IFN-ß antagonist through suppression of the RIG-I-mediated signaling. This study will pave the way for the further investigation into the molecular mechanisms by which PEDV E protein evades host innate immune response.

Lipid raft-associated PI3K/Akt/SREBP1 signaling regulates coxsackievirus A16 (CA16) replication.

Coxsackievirus A16 (CA16) is one of predominant Enterovirus that possesses high pathogenicity. Lipid rafts, as cholesterol - and sphingolipid - enriched membrane nanodomains, are involved into many aspects of the virus life cycle. Our previous study found that lipid rafts integrity was essential for CA16 replication, but how lipid rafts regulate CA16 replication through activating downstream signaling remains largely unknown. Thus, in this study, we revealed that lipid rafts were required for activation of PI3K/Akt signaling at early stage of CA16 infection. Treatment with wortmannin significantly reduced the expression of virus protein, indicating PI3K/Akt signaling was beneficial for early stage of virus infection. In addition, lipid rafts integrity was also indispensable for PI3K/Akt activation during the late stage of CA16 infection, which played critical functions in mediating sterol regulatory element-binding proteins 1 (SREBP1) maturation. Whereas, over-expression of SREBP1 exhibited inhibition on virus replication, suggesting that PI3K/Akt signaling and SREBP1 might positively and negatively influence virus replication in two different stages of infection, respectively. Taken together, our study demonstrates an important role of the lipid raft-associated PI3K/Akt/SREBP1 signaling in modulating CA16 replication, which will deepen our understanding mechanism of CA16 infection.

The accessory protein ORF3 of porcine epidemic diarrhea virus inhibits cellular interleukin-6 and interleukin-8 productions by blocking the nuclear factor-κB p65 activation.

Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded positive-sense RNA virus that belongs to a porcine entero-pathogenic alphacoronavirus, causing lethal watery diarrhea in piglets. Despite existing study reports the sole accessory protein ORF3 identified as NF-κB antagonist, the contribution of PEDV ORF3 to production of the pro-inflammatory cytokines mediated by NF-κB signaling remains largely unknown. Thus in this present study, we showed that PEDV ORF3 protein significantly inhibited the productions of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8. The phosphorylation of IκBα was inhibited by ORF3 protein, but no degradation of IκBα was induced in ORF3-expressing cells. Furthermore, PEDV ORF3 inhibited NF-κB activation through preventing nuclear factor p65 phosphorylation and down-regulating p65 expression level, as well as interfering nuclear translocation of p65, eventually resulting into the inhibition of IL-6 and IL-8 production. Our study definitely links PEDV ORF3 to inhibition of pro-inflammatory cytokines production, which will provide new insight into the molecular mechanisms of NF-κB activity inhibited by PEDV proteins to facilitate virus evasion of host innate immune.

The effectiveness of various cytotherapeutic strategies for the treatment of ischemic stroke: a Bayesian network meta-analysis of randomized controlled trials.

BACKGROUND: Cytotherapy is a potential treatment for ischemic stroke (IS) patients but lacks uniform procedures. We aimed to assess the impact of the time of intervention, cell type, dose, and route of administration on the clinical effects by network meta-analysis. METHODS: We searched public electronic databases through July 7, 2019. Bayesian network meta-analyses were performed to compare differences among different cytotherapeutic strategies. RESULTS: Cytotherapy can significantly improve patients' activity of daily living according to the modified Rankin Scale (standard mean difference (SMD) - 0.81; 95% confidence interval (CI) - 1.58, - 0.03; p = 0.0417) and Barthel Index (SMD 0.67; 95% CI 0.05, 1.30; p = 0.036) results as well as improve neurological recovery (SMD - 0.93; 95% CI - 1.29, - 0.57; p < 0.001). Network meta-analysis showed that the intra-arterial injection of large amounts of mononuclear cells (NCs) or aldehyde dehydrogenase (ALDH)-positive cells was beneficial for improving patients' activity of daily living, while CD34+ cells through intracerebral injection had an advantage in the recovery of injured nerve function. Intravenous injection of mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs) was beneficial in reducing mortality and serious adverse event (SAE) onset. CONCLUSIONS: In the subacute stage, the intra-arterial injection of NCs or ALDH cells improves patients' activity of daily living. Additionally, CD34+ cells through intracerebral injection had an advantage in the recovery of injured nerve function even in the chronic stage. Intravenous injection of MSCs or EPCs is a safety delivery route that can reduce mortality and SAE onset. However, further clinical studies are still needed to confirm these results.

Aberrant alterations of the expressions and S-nitrosylation of calmodulin and the downstream factors in the brains of the rodents during scrapie infection.

The aberrant alterations of calmodulin (CaM) and its downstream substrates have been reported in some neurodegenerative diseases, but rarely described in prion disease. In this study, the potential changes of Ca2+/CaM and its associated agents in the brains of scrapie agent 263K-infected hamsters and the prion infected cell line SMB-S15 were evaluated by various methodologies. We found that the level of CaM in the brains of 263K-infected hamsters started to increase at early stage and maintained at high level till terminal stage. The increased CaM mainly accumulated in the regions of cortex, thalamus and cerebellum of 263K-infected hamsters and well localization of CaM with NeuN positive cells. However, the related kinases such as total and phosphorylated forms of CaMKII and CaMKIV, as well as the downstream proteins such as CREB and BDNF in the brain of 263K-infected hamsters were decreased. Further analysis showed a remarkable increase of S-nitrosylated (SNO) form of CaM in the brains of 263K-infected hamsters. Dynamic analysis of S-nitrosylated CaM showed the SNO form of CaM abnormally increases in a time-dependent manner during prion infection. Compared with that of the normal partner cell line SMB-PS, the CaM level in SMB-S15 cells was increased, meanwhile, the downstream proteins, such as CaMKII, p-CaMKII, CREB, as well as BDNF, were also increased, especially in the nucleic fraction. No SNO-CaM was detected in the cell lines SMB-S15 and SMB-PS. Our data indicate an aberrant increase of CaM during prion infection in vivo and in vitro.

Enterovirus 71 inhibits cellular type I interferon signaling by inhibiting host RIG-I ubiquitination.

Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease (HFMD) and fatal neurological diseases in young children and infants. Pathogenicity of EV71 is likely related to its ability to evade host innate immunity through inhibiting cellular type I interferon signaling. However, it is less well understood the molecular events governing this process. In this study, we found that EV71 infection suppressed the induction of antiviral immunity by inhibiting the expression levels of IFN-ß and IFN-stimulated genes (ISGs), such as ISG54 and ISG56, at the late stage of viral infection. At the same time, our results showed that EV71 infection significantly inhibited ubiquitination of RIG-I. In contrast, up-regulation of RIG-I ubiquitination promoted expression of IFN-ß and ISGs, suggesting that inhibition of cellular type I interferon signaling was caused by down-regulation of RIG-I ubiquitination during EV71 infection. These results suggest that inhibition of RIG-I-mediated type I IFN responses by EV71 may contribute to the pathogenesis of viral infection.

The nuclear protein Sam68 is recruited to the cytoplasmic stress granules during enterovirus 71 infection.

Our previous study found that the nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), is translocated to the cytoplasm and forms punctate pattern during enterovirus 71 (EV71) infection [Virus Research, 180 (2014), 1-11]. However, the exact function of this punctate pattern in cytoplasm during EV71 infection remains unknown. In this study, we firstly have examined this punctate pattern of Sam68 re-localization in the cytoplasm, and observed the obvious recruitments of Sam68 to the EV71-induced stress granules (SGs). Sam68, belongs to the KH domain family of RNA binding proteins (RBPs), was then confirmed that its KH domain was essential for this recruitment. Nevertheless, Knockdown of Sam68 expression using ShRNA had no effects on SGs assembly, indicating that Sam68 is not a constitutive component of the SGs during EV71 infection. Lastly, we investigated the importance of microtubulin transport to SGs aggregation, and revealed that microtubule depolymerization inhibited SGs formation, suggesting that EV71-induced SGs move throughout the cytoplasm in a microtubule-dependent manner. Taken together, these results illuminated that EV71 infections can induce SGs formation, and Sam68, as a SGs component, migrates alone with SGs dependent on intact microtubule upon the viral infections. These findings may provide novel underlying mechanism for delineating the role of SGs during EV71 infection.

CX3CL1 increases invasiveness and metastasis by promoting epithelial-to-mesenchymal transition through the TACE/TGF-α/EGFR pathway in hypoxic androgen-independent prostate cancer cells.

Epithelial-to-mesenchymal transition (EMT) endows cancer cells with enhanced invasive and metastatic potential during cancer progression. Fractalkine, also known as chemokine (C-X3-C motif) ligand 1 (CX3CL1), the only member recognized so far that belongs to the CX3C chemokine subfamily, was reported to participate in the molecular events that regulate cell adhesion, migration and survival of human prostate cancer cells. However, the relationship between CX3CL1 and EMT remains unknown. We treated DU145 and PC-3 cells with CX3CL1 under hypoxic conditions. The migration and invasion abilities of DU145 and PC-3 cells were detected by Transwell assays. Induction of EMT was verified by morphological changes in the DU145 and PC-3 cells and analysis of protein expression of EMT markers such as E-cadherin and vimentin. To identify the involved signaling pathway in CX3CL1-induced EMT, activation of epidermal growth factor receptor (EGFR) was measured using western blot analysis, and Slug expression was detected with or without an EGFR inhibitor prior to CX3CL1 treatment. Concentrations of soluble and total TGF-α in the CX3CL­treated DU145 cells were detected by ELISA. Additionally, we determined the involvement of the TACE/TGF-α/EGFR pathway in CX3CL1­induced EMT using RNA interference and specific inhibitors. CX3CL1 increased the migration and invasiveness of the DU145 and PC-3 cells, and resulted in characteristic alterations of EMT. Our results demonstrated that TACE/TGF-α/EGFR pathway activation and subsequent upregulation of Slug expression were responsible for CX3CL1­induced EMT, and contributed to the migration and inva-sion of prostate cancer cells. Inhibition of TACE/TGF-α/EGFR signaling reversed EMT and led to reduced migration and invasion abilities of the prostate cancer cells. We provide initial evidence that CX3CL1 exposure resulted in EMT occurrence and enhancement of cell migration and invasion through a mechanism involving activation of TACE/TGF-α/EGFR signaling. These findings revealed that CX3CL1 may serve as a new target for the treatment of prostate cancer.

Upregulation of fractalkine contributes to the proliferative response of prostate cancer cells to hypoxia via promoting the G1/S phase transition.

Hypoxia is a common phenomenon in prostate cancer, which leads to cell proliferation and tumor growth. Fractalkine (FKN) is a membrane­bound chemokine, which is implicated in the progression of human prostate cancer and skeletal metastasis. However, the association between FKN and hypoxia­induced prostate cancer cell proliferation remains to be elucidated. The present study demonstrated that hypoxia induced the expression and secretion of FKN in the DU145 prostate cancer cell line. Furthermore, inhibiting the activity of FKN with the anti­FKN FKN­specific antibody markedly inhibited hypoxia­induced DU145 cell proliferation. Under normoxic conditions, DU145 cell proliferation markedly increased following exogenous administration of human recombinant FKN protein, and the increase was significantly alleviated by anti­FKN, indicating the importance of FKN in DU145 cell proliferation. In addition, subsequent determination of cell cycle distribution and expression levels of two core cell cycle regulators, cyclin E and cyclin­dependent kinase (CDK)2, suggested that FKN promoted the G1/S phase transition by upregulating the expression levels of cyclin E and CDK2. The results of the present study demonstrated that hypoxia led to the upregulation of the secretion and expression of FKN, which enhanced cell proliferation by promoting cell cycle progression in the prostate cancer cells. These findings provide evidence of a novel function for FKN, and suggest that FKN may serve as a potential target for treating androgen­independent prostate cancer.

Hypoxia increases CX3CR1 expression via HIF-1 and NF­κB in androgen-independent prostate cancer cells.

The unique CX3C chemokine CX3CL1 and its cognate receptor CX3CR1 have been implicated in organ-specific metastasis of various types of tumors. Hypoxia, a common phenomenon in solid tumors, is associated with a malignant cancer phenotype. Previous studies have proved that hypoxia facilitates cancer cell metastasis through upregulation of specific chemokine receptors. We hypothesized that hypoxia could upregulate CX3CR1 expression and lead to an increased chemotactic response to CX3CL1 in prostate cancer cells. In the present study, we found that CX3CR1 expression was significantly increased in androgen-independent prostate cancer cells, including DU145, PC-3 and PC-3M, following exposure to hypoxia. This upregulation of CX3CR1 corresponded to a significant increase in migration and invasion of prostate cancer cells under hypoxic conditions, which was attenuated after knocking down CX3CR1 expression. In addition, we examined the possible role of HIF-1 and NF-κB in the process of hypoxia-induced CX3CR1 expression and hypoxia-mediated metastasis. Attenuation of HIF-1 and NF-κB transcriptional activity by siRNAs or pharmacological inhibitors, abrogated hypoxia-induced upregulation of CX3CR1, and also prevented the migration and invasion of DU145 cells under a hypoxic environment. In summary, our study demonstrated that HIF-1 and NF-κB are essential for hypoxia-regulated CX3CR1 expression, which is associated with increased migratory and invasive potential of prostate cancer cells. CX3CR1 signaling is a potential therapeutic target in the adjuvant treatment of prostate cancer.

ADAM17 targets MMP-2 and MMP-9 via EGFR-MEK-ERK pathway activation to promote prostate cancer cell invasion.

ADAM17, also known as tumor necrosis factor-α converting enzyme (TACE), is involved in proteolytic ectodomain shedding of cell surface molecules and cytokines. Although aberrant expression of ADAM17 has been shown in various malignancies, the function of ADAM17 in prostate cancer has not been clarified. In the present study, we sought to elucidate whether ADAM17 contributes to prostate cancer cell invasion, as well as the mechanism involved in the process. The expression pattern of ADAM17 was investigated in human prostate cancer cells. The results showed that ADAM17 expression levels are correlated with the invasive ability of androgen-independent prostate cancer cell lines. Further, ADAM17 was overexpressed in cells showing high invasion characteristics, activation of the EGFR-MEK-ERK pathway, up-regulation of MMP-2, MMP-9, and an increased TGF-α release into the supernatant. However, AG1478, PD98059 and antibody against TGF-α deactivating the EGFR-MEK-ERK signaling pathway, abolished up-regulation of MMP-2, MMP-9 and prevented cell invasion. In addition, cells with knockdown of ADAM17 by siRNA exhibited low invasive ability, deactivated EGFR-MEK-ERK signaling pathway, reduced TGF-α released and down-regulation of MMP-2, MMP-9. However, these effects could be reversed by simultaneous addition of TGF-α. These data demonstrated that ADAM17 contributes to androgen-independent prostate cancer cell invasion by shedding of EGFR ligand TGF-α, which subsequently activates the EGFR-MEK-ERK signaling pathway, leading finally to overexpression of MMP-2 and MMP-9. This study suggests that the ADAM17 expression level may be a new predictive biomarker of invasion and metastasis of prostate cancer, and ADAM17 could provide a target for treating metastatic PCa.