Xinxing Wu

HSV-2 induces TLRs and NF-κB-dependent cytokines in cervical epithelial cells

Herpes simplex virus type 2 (HSV-2) is one of the most common sexually transmitted pathogens worldwide. The host immune response induced by viral infection is cell-type specific. Little is known about the innate immune response to this virus in its natural host cells. In this study, we established an in vitro HSV-2 infection model with human cervical epithelial (HCE) cells. The viral infection was sufficient to induce expression of Toll-like receptors (TLRs), and Western blot and reporter assays suggest that HSV-2 infection leads to dramatic activation of the NF-kappaB signaling pathway. More importantly, our data provide direct evidence that the activation of NF-kappaB is required for the production of both IL-6 and IFN-beta induced by HSV-2 in HCE cells. Taken together, our results suggest the potential contributions of TLRs and a critical role of NF-kappaB in the innate immune response to HSV-2 in HCE cells.

TLR4-MyD88/Mal-NF-kB Axis Is Involved in Infection of HSV-2 in Human Cervical Epithelial Cells

We have established an in vitro HSV-2 acute infection model with Human cervical epithelial (HCE cells, the primary target and natural host cells for HSV-2) to investigate the role of TLRs-mediated innate immune response to HSV-2. In current study, we found that HSV-2 infection induced activity of NF-kB reporter and expression of cytokines are TLR4-dependent using approaches with shRNA and TLR4 antagonist. Knockdown experiments demonstrated that the adaptor molecules MyD88 and Mal of the TLRs signaling pathway are required in the HSV-2 induced TLR4-dependent NF-kB activation in HCE cells. Western blot assay suggested that knockdown of TLR4 decreased the phosphorylation of IRAK1 and inhibitor of NF-kB (IkB-α) upon HSV-2 infection. Finally, decreased expression of either TLR4 or MyD88/Mal alone or both significantly abolished productions of IL-6 and IFN-β by ELISA analysis. Taken together, our results from the in vitro infection model reveal for the first time that there exists the pathway via TLR4-Mal/MyD88-IRAK1-NF-kB axis in human cervical epithelial cells in response to HSV-2 infection.

Antifungal efficacy of itraconazole-loaded TPGS-b-(PCL-ran-PGA) nanoparticles.

This research was conducted to formulate biodegradable itraconazole (ITZ)-loaded d-a-tocopheryl polyethylene glycol 1000 succinate-b-poly(e-caprolactone-ran-glycolide) (TPGS-b-(PCL-ran-PGA); TPP) nanoparticles (NPs) (designed as ITZ-loaded TPP NPs) to improve antifungal efficacy. ITZ-loaded TPP NPs were prepared by a modified double-emulsion method, and their size distribution, morphology, zeta potential, drug encapsulation efficiency, drug-release profile, and antifungal effects were characterized. The cytotoxicity of ITZ-loaded-TPP NPs on HeLa cells and fibroblasts was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The in vivo antifungal activity of ITZ-loaded-TPP NPs was examined in mice by administrating 5×105 colony forming units of Candida albicans through the tail vein. The survival rate and survival time of the mice was observed. The fungal count and pathology of lung tissue was analyzed. The data showed that ITZ-loaded-TPP NPs have size of 265±5.8 nm, zeta potential of −31±0.5 mV, high encapsulation efficiency (95%), and extended drug-release profile. ITZ-loaded-TPP NPs at a high concentration of 25 mg/mL had no cytotoxicity on HeLa cells and fibroblasts. Furthermore, ITZ-loaded-TPP NPs achieved a higher level of antifungal activity both in vitro and in vivo. The survival rate and duration was higher in mice treated by ITZ-loaded-TPP NPs than in the other groups (P<0.05). In conclusion, ITZ-loaded-TPP NPs significantly improved ITZ bioavailability by increasing its aqueous dispersibility and extending the duration of drug release, thereby improving the antifungal efficacy of the ITZ agent.

A risk evaluation model of cervical cancer based on etiology and human leukocyte antigen allele susceptibility

BACKGROUND There are no reliable risk factors to accurately predict progression to cervical cancer in patients with chronic cervicitis infected with human papillomavirus (HPV). The aim of this study was to create a validated predictive model based on the risk factors for cervical cancer. A model to estimate the risk of cervical cancer may help select patients for intervention therapy in order to reduce the occurrence of cervical cancer after HPV infection. METHODS This retrospective analysis included 68 patients with cervical cancer and 202 healthy female controls. HPV infection and human leukocyte antigen (HLA) class II alleles in HLA-DRB1, 3-7, and 9 were detected. Other information was collected, including level of education and age at first parturition. Multiple regression analysis and an artificial neural network (ANN) were performed to identify the independent risk factors for cervical cancer, and based on these, an evaluation model for the prediction of the incidence of cervical cancer was formed. RESULTS This model showed HPV to be a pivotal player in cervical cancer that increased the risk by 7.6-fold. The presence of the HLA-DRB1*13-2 and HLA-DRB1*3(17) alleles was associated with an increased risk of developing cervical cancer. Conversely, the HLA-DRB1*09012 and HLA-DRB1*1201 alleles were found to be associated with a reduced cervical cancer risk. In addition, other factors, such as age at first parturition and education level, had significant effects on cervical cancer risk. The model was applied to conduct a risk assessment of women in the mountain area of Wufeng County, Hubei Province in China. The sensitivity and specificity of our model both exceeded 95%. CONCLUSIONS This model, based on etiology and HLA allele susceptibility, can estimate the risk of cervical cancer in chronic cervicitis patients after HPV infection. It combines genetic and environmental factors and significantly enhances the accuracy of risk evaluation for cervical cancer. This model could be used to select patients for intervention therapy and to guide patient classification management.

Hepatitis C virus NS4B protein induces epithelial-mesenchymal transition by upregulation of Snail

BackgroundChronic hepatitis C virus (HCV) infection is an important cause of hepatocellular carcinoma (HCC). Epithelial to mesenchymal transition (EMT) is a key process associated with tumor metastasis and poor prognosis. HCV infection, HCV core and NS5A protein could induce EMT process, but the role of NS4B on EMT remains poorly understood.MethodsWe overexpressed HCV NS4B protein in HepG2 cells or Huh7.5.1 cells infected by HCVcc, the E-cadherin expression, N-cadherin expression and the EMT-associated transcriptional factor Snail were determined. The migration and invasion capabilities of the transfected cells were evaluated using wound-healing assay. Additionally, we used Snail siRNA interference to confirm the relation of HCV NS4B and Snail on EMT promotion.ResultsHCV NS4B increased the expression of EMT related markers and promoted cell migration and invasion. Snail knock-down almost completely eliminated the function of NS4B protein in EMT changes and reversed cell migration capacity to lower level. HCV NS4B protein could reduce the expression of Scribble and Hippo signal pathway were subsequently inactivated, resulting in the activation of PI3K/AKT pathway, which may be the reason for the up-regulation of Snail.ConclusionsThis study demonstrates that HCV NS4B protein induces EMT progression via the upregulation of Snail in HCC, which may be a novel underlying mechanism for HCV-associated HCC development, invasion and metastasis.

A-to-I RNA editing of BLCAP lost the inhibition to STAT3 activation in cervical cancer

Bladder cancer-associated protein (BLCAP) gene is a highly conserved gene with tumor-suppressor function in different carcinomas. It is also a novel ADAR-mediated editing substrate undergoes multiple A-to-I RNA editing events. Although the anti-tumorigenic role of BLCAP has been examined in preliminarily studies, the relationship between BLCAP function and A-to-I RNA editing in cervical carcinogenesis still require further exploration. Herein, we analyzed the coding sequence of BLCAP transcripts in 35 paired cervical cancer samples using high-throughput sequencing. Of note, editing levels of three novel editing sites were statistically different between cancerous and adjacent cervical tissues, and editing of these three sites was closely correlated. Moreover, two editing sites of BLCAP coding region were mapped-in the key YXXQ motif which can bind to SH2 domain of STAT3. Further studies revealed that BLCAP interacted with signal transducer and activator of transcription 3 (STAT3) and inhibited its phosphorylation, while A-to-I RNA editing of BLCAP lost the inhibition to STAT3 activation in cervical cancer cell lines. Our findings reveal that A-to-I RNA editing events alter the genetically coded amino acid in BLCAP YXXQ motif, which drive the progression of cervical carcinogenesis through regulating STAT3 signaling pathway.

BLCAP arrests G1/S checkpoint and induces apoptosis through downregulation of pRb1 in HeLa cells

BLCAP (bladder cancer-associated protein) gene exhibited tumor suppressor function in different tumors and is regarded as a candidate tumor suppressor gene; however, the mechanism by which BLCAP exerts its function remains elusive. This study investigated the functional association between BLCAP and proliferation or apoptosis in cervical cancer cells, to identify the functional motifs of BLCAP. The BLCAP-shRNA expression vector based on pRNA-U6.1/Hygro plasmid was used to specifically inhibit BLCAP activity in HeLa cells. The optimal shRNA plasmid was selected to knock down BLCAP expression and the biological effects were investigated. The effects on cell cycle and apoptosis were detected by flow cytometric or Annexin V-FITC staining analysis. The gene expression profiles of HeLa cells transfected with blcap-wt and BLCAP-shRNA were analyzed using human signal pathway gene Oligochips. The levels of protein expression and interaction of BLCAP with Rb1 proteins were determined by western blotting and Co-IP assays. The site-specific mutagenesis assay was used to identify amino acid residues important for BLCAP. Significantly differentially expressed genes were found by gene Oligo chips analysis. These genes were all correlated with proliferation, cell cycle and apoptosis. The results of western blotting and Co-IP assays confirmed that overexpression of BLCAP could interact with Rb1 and inhibit Rb1 phosphorylation. Further investigation revealed that SAXX mutation in the key regions of BLCAP suppressed the function of BLCAP and significantly increased the level of phosphorylated Rb1 protein. Here our findings suggested that the functional association of BLCAP and Rb1 might play important roles in proliferation and apoptosis of HeLa cells. It suggested that BLCAP could be a novel therapeutic target for cervical cancer.