Ninghan Feng

MiR-146a suppresses tumor growth and progression by targeting EGFR pathway and in a p-ERK-dependent manner in castration-resistant prostate cancer†‡

Castration‐resistant prostate cancer (CRPC) is a leading cause of cancer‐related deaths in elder men. This disease has limited therapeutic options and poor prognosis as the underlying molecular mechanisms are not clearly understood. Given the emerging roles of microRNA (miRNA) as a key regulator, we postulated that miRNA may play a significant role in CRPC formation.

HIV-1 Tat Promotes Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) vIL-6-Induced Angiogenesis and Tumorigenesis by Regulating PI3K/PTEN/AKT/GSK-3β Signaling Pathway

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is etiologically associated with KS, the most common AIDS-related malignancy. KS is characterized by vast angiogenesis and hyperproliferative spindle cells. We have previously reported that HIV-1 Tat can trigger KSHV reactivation and accelerate Kaposin A-induced tumorigenesis. Here, we explored Tat promotion of KSHV vIL-6-induced angiogenesis and tumorigenesis. Tat promotes vIL-6-induced cell proliferation, cellular transformation, vascular tube formation and VEGF production in culture. Tat enhances vIL-6-induced angiogenesis and tumorigenesis of fibroblasts and human endothelial cells in a chicken chorioallantoic membrane (CAM) model. In an allograft model, Tat promotes vIL-6-induced tumorigenesis and expression of CD31, CD34, SMA, VEGF, b-FGF, and cyclin D1. Mechanistic studies indicated Tat activates PI3K and AKT, and inactivates PTEN and GSK-3β in vIL-6 expressing cells. LY294002, a specific inhibitor of PI3K, effectively impaired Tat’s promotion of vIL-6-induced tumorigenesis. Together, these results provide the first evidence that Tat might contribute to KS pathogenesis by synergizing with vIL-6, and identify PI3K/AKT pathway as a potential therapeutic target in AIDS-related KS patients.

Activation of PI3K/AKT and ERK MAPK signal pathways is required for the induction of lytic cycle replication of Kaposi's Sarcoma-associated herpesvirus by herpes simplex virus type 1

BackgroundKaposis sarcoma-associated herpesvirus (KSHV) is causally linked to several acquired immunodeficiency syndrome-related malignancies, including Kaposis sarcoma (KS), primary effusion lymphoma (PEL) and a subset of multicentric Castlemans disease. Regulation of viral lytic replication is critical to the initiation and progression of KS. Recently, we reported that herpes simplex virus type 1 (HSV-1) was an important cofactor that activated lytic cycle replication of KSHV. Here, we further investigated the possible signal pathways involved in HSV-1-induced reactivation of KSHV.ResultsBy transfecting a series of dominant negative mutants and protein expressing constructs and using pharmacologic inhibitors, we found that either Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) or JAK1/STAT6 signaling failed to regulate HSV-1-induced KSHV replication. However, HSV-1 infection of BCBL-1 cells activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, also called AKT) pathway and inactivated phosphatase and tensin homologue deleted on chromosome ten (PTEN) and glycogen synthase kinase-3β (GSK-3β). PTEN/PI3K/AKT/GSK-3β pathway was found to be involved in HSV-1-induced KSHV reactivation. Additionally, extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinase (MAPK) pathway also partially contributed to HSV-1-induced KSHV replication.ConclusionsHSV-1 infection stimulated PI3K/AKT and ERK MAPK signaling pathways that in turn contributed to KSHV reactivation, which provided further insights into the molecular mechanism controlling KSHV lytic replication, particularly in the context of HSV-1 and KSHV co-infection.

Cellular MicroRNAs 498 and 320d Regulate Herpes Simplex Virus 1 Induction of Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication by Targeting RTA

Kaposi’s sarcoma-associated herpesvirus (KSHV) infection was necessary but not sufficient for KS development without other cofactors. We have previously reported that herpes simplex virus (HSV)-1 was an important cofactor that reactivated KSHV from latency by inducing the expression of KSHV replication and transcription activator (RTA), the lytic switch protein. Here, we further investigated the possible cellular microRNAs (miRNAs) involved in regulation of RTA during HSV-1-induced KSHV replication. The differential profiles of miRNAs expression between Mock- and HSV-1-infected body cavity-based lymphoma (BCBL-1) cells were identified by miRNA microarray analysis. Bioinformatics and luciferase reporter analyses showed that two of the HSV-1-downregulated cellular miRNAs, miR-498 and miR-320d, directly targeted the 3′ untranslated region (UTR) of KSHV RTA. As a result, overexpression of these two miRNAs significantly inhibited HSV-1-induced KSHV replication, whereas repression of these miRNAs with specific suppressors enhanced HSV-1-mediated KSHV replication. In addition, miR-498 or miR-320d alone, without HSV-1 infection, regulated KSHV replication in BCBL-1 cells. Finally, bioinformatics Gene Ontology (GO) analysis indicated that targets of HSV-1-regulated miRNAs were enriched for proteins, whose roles were involved in protein binding, enzyme activity, biological regulation, and several potential signaling pathways including transforming growth factor (TGF)-β were likely to participate in HSV-1-induced KSHV replication. Collectively, these novel findings demonstrated that host-encoded miR-498 and miR-320d regulated HSV-1 induction of KSHV lytic replication by targeting RTA, which provided further insights into the molecular mechanisms controlling KSHV lytic replication.

Inhibition of Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by HIV-1 Nef and Cellular MicroRNA Hsa-miR-1258

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is causally linked to several AIDS-related malignancies, including Kaposis sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castlemans disease. The interaction of human immunodeficiency virus type 1 (HIV-1) and KSHV has a central role in promoting the aggressive manifestations of AIDS-KS. We have previously shown that negative factor (Nef), a secreted HIV-1 protein, synergizes with KSHV viral interleukin-6 (vIL-6) to promote angiogenesis and tumorigenesis by activating the AKT pathway (X. Zhu, et al., Oncogene, 22 April 2013, http://dx.doi.org/10.1038/onc.2013.136). Here, we further demonstrated the role of soluble and ectopic Nef in the regulation of KSHV latency. We found that both soluble Nef protein and ectopic expression of Nef by transfection suppressed the expression of KSHV viral lytic mRNA transcripts and proteins and the production of infectious viral particles. MicroRNA (miRNA) microarray analysis identified a number of Nef-regulated miRNAs. Bioinformatics and luciferase reporter analyses showed that one of the Nef-upregulated miRNAs, cellular miRNA 1258 (hsa-miR-1258), directly targeted a seed sequence in the 3′ untranslated region (UTR) of the mRNA encoding the major lytic switch protein (RTA), which controls KSHV reactivation from latency. Ectopic expression of hsa-miR-1258 impaired RTA synthesis and enhanced Nef-mediated inhibition of KSHV replication, whereas repression of hsa-miR-1258 has the opposite effect. Mutation of the seed sequence in the RTA 3′UTR abolished downregulation of RTA by hsa-miR-1258. Collectively, these novel findings demonstrate that, by regulating cellular miRNA, Nef may inhibit KSHV replication to promote viral latency and contribute to the pathogenesis of AIDS-related malignancies. IMPORTANCE This study found that Nef, a secreted HIV-1 protein, suppressed KSHV lytic replication to promote KSHV latency. Mechanistic studies indicated that a Nef-upregulated cellular miRNA, hsa-miR-1258, inhibits KSHV replication by directly targeting a seed sequence in the KSHV RTA 3′UTR. These results illustrate that, in addition to viral miRNAs, cellular miRNAs also play an important role in regulating the life cycle of KSHV. Overall, this is the first study to report the involvement of Nef in KSHV latency, implying its likely important role in the pathogenesis of AIDS-related malignancies.

Herpes Simplex Virus Type 2 Triggers Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency and Collaborates with HIV-1 Tat

Kaposis sarcoma-associated herpesvirus (KSHV) infection was necessary but not sufficient for Kaposis sarcoma (KS) development without other cofactors. Previously, we identified that both human immunodeficiency type 1 (HIV-1) Tat and herpes simplex virus 1 (HSV-1) were important cofactors reactivating KSHV from latency. Here, we further investigated the potential of herpes simplex virus 2 (HSV-2) to influence KSHV replication and examined the role of Tat in this procedure. We demonstrated that HSV-2 was a potentially important factor in the pathogenesis of KS, as determined by production of lytic phase mRNA transcripts, viral proteins and infectious viral particles in BCBL-1 cells. These results were further confirmed by an RNA interference experiment using small interfering RNA targeting KSHV Rta and a luciferase reporter assay testing Rta promoter-driven luciferase activity. Mechanistic studies showed that HSV-2 infection activated nuclear factor-kappa B (NF-κB) signaling pathway. Inhibition of NF-κB pathway enhanced HSV-2-mediated KSHV activation, whereas activation of NF-κB pathway suppressed KSHV replication in HSV-2-infected BCBL-1 cells. Additionally, ectopic expression of Tat enhanced HSV-2-induced KSHV replication. These novel findings suggest a role of HSV-2 in the pathogenesis of KS and provide the first laboratory evidence that Tat may participate HSV-2-mediated KSHV activation, implying the complicated pathogenesis of acquired immunodeficiency syndrome (AIDS)-related KS (AIDS-KS) patients.

A Meta-Analysis of the Effects of the 5-Hydroxytryptamine Transporter Gene-Linked Promoter Region Polymorphism on Susceptibility to Lifelong Premature Ejaculation

Objective Premature ejaculation (PE) has been reported as the most common male sexual dysfunction with global prevalence rates estimated at approximately 30%. The neurobiogenesis of ejaculation is very complex and involves the serotoninergic (5-hydroxytryptamine, 5-HT) system. Recently, genetic polymorphisms located on SLC6A4 gene codifying for 5-HT transporter (5-HTT), the major regulator of serotonic neurotransmission, have been linked with the pathogenesis and risk of PE. Apparently studies of this type of polymorphism in PE have show conflicting results. Methods A meta-analysis was performed that are available in relation with 5-HTT gene-linked promoter region (5-HTTLPR) polymorphism and the risk of lifelong PE (LPE) in men to clarify this relationship. We searched Pubmed and Embase (last search updated on Aug 2012) using ‘premature ejaculation’, ‘polymorphism or variant’, ‘genotype’, ‘ejaculatory function’, and ‘rapid ejaculation’ as keywords and reference lists of studies corresponded to the inclusion criteria for meta-analysis. These studies involved the total number of 481 LPE men and 466 health control men subjects. Odds ratio (OR) and 95% confidence intervals (CIs) were used to evaluate this relationship. Results In the overall analysis, significant associations between LPE risk and 5-HTTLPR polymorphism were found (L-allele vs. S-allele OR = 0.86, 95% CI = 0.79–0.95, P = 0.002; LL vs. SS: OR = 0.80, 95% CI = 0.68–0.95, P = 0.009; LS vs. SS: OR = 0.85, 95% CI = 0.76–0.97, P = 0.012 and LL+LS vs. SS: OR = 0.88, 95% CI = 0.81–0.95, P = 0.002). Moreover, in subgroup analysis based on ethnicity, similar significant associations were detected. The Egger’s test did not reveal presence of a publication bias. Conclusions Our investigations demonstrate that 5-HTTLPR (L>S) polymorphism might protect men against LPE risk. Further studies based on larger sample size and gene-environment interactions should be conducted the role of 5-HTTLPR polymorphism and LPE risk.

Differential expression of the Wnt/β-catenin pathway in the genital tubercle (GT) of fetal male rat following maternal exposure to di-n-butyl phthalate (DBP)

Di-n-butyl phthalate (DBP) is one of the most abundantly produced endocrine disruptors that leaches out from polyvinyl chloride plastics and can cause hypospadias in male rats during maternal exposure. The objective of this study was to first explore the roles of Wnt/β-catenin pathway in the fetal rat genital tubercle (GT) following in-utero exposure to DBP. Timed-pregnant rats were given DBP by gastric intubation at a dose of 750 mg/kg body weight (bw)/day from gestation day (GD) 14 to GD18 to establish a rat model of hypospadias. On GD19, genital tubercle down-regulation of β-catenin, Phospho-GSK-3β, and up-regulation of GSK-3β (glycogen synthase kinase-3β), NFκB in fetal male rats was observed by western blot analysis. β-catenin was located in the urethral plate epithelium (UPE). Immunochemistry showed that the relative expression of β-catenin decreased in the DBP-treated fetal rat GT compared to the normal control. These findings, for the first time, indicate that DBP may affect the development of GT by down-regulating the Wnt/β-catenin pathway in fetal male rats.

Construction and identification of recombinant lentiviral vector containing HIV-1 Tat gene and its expression in 293T cells

Objective To construct a lentiviral vector expressing HIV-1 Tat and identify its expression in 293T cells. Methods The gene fragment of HIV-1 Tat101 was subcloned to lentiviral transfer vector pHAGE-CMV-MCS-IZsGreen, which was named pHAGE-Tat. Then the constructed pHAGE-Tat was used to co-transfect the packing 293T cells, together with the packaging plasmids pMD2.G and psPAX2. The packaged viral particles designated LV-Tat were used to infect the 293T cells and the viral titer was calculated. The expression of HIV-1 Tat in 293T cells was confirmed using RT-PCR and western blot. Results The recombinant lentiviral vector was successfully constructed and could express HIV-1 Tat in 293T cells. The virus titer was 5.73×106 ifu/ml. Conclusion The successfully constructed recombinant lentiviral vector makes a strong foundation for further exploring the possible role of HIV-1 Tat in the development of prostate cancer.

Association between p53 Pro72Arg polymorphism and prostate cancer risk: a meta-analysis.

The tumor suppressor gene p53 appears to be important in the development of many human cancers, such as prostate cancer. The association of p53 codon72 polymorphism with prostate cancer has been widely reported; however, the results are inconsistent. To derive a more precise estimation of this relationship, we performed an updated meta-analysis from 10 case-control studies. We conducted a search in the PubMed database without a language limitation, covering all papers published until July 2010. Risk ratios (RR) with 95% confidence intervals (CIs) were used to assess the strength of the association. Ten studies including 1,196 cases and 1,704 controls were selected. Overall, no significant differences of total prostate cancer risk and p53 codon polymorphism was found (Pro/Pro vs Arg/Arg, RR = 1.12, 95%CI=0.74-1.70, Pheterogeneity = 0.016, I2 = 55.8%; Pro/Pro+Pro/Arg vs Arg/Arg, RR = 1.05, 95%CI=1.00-1.11, Pheterogeneity = 0.077, I2 = 51.1%). In the stratified analysis by ethnicity, the same results were found. However, in the control subgroup, there was a modest decreased association between prostate cancer risk and population-based control subjects under the recessive genetic model (RR = 0.31, 95%CI=0.10-0.91, Pheterogeneity = 0.110, I2 =60.8%). This meta-analysis suggested that p53 codon Pro72Arg polymorphism could be weakly associated with prostate cancer risk.