LncRNA HOTTIP promotes the proliferation and invasion of ovarian cancer cells by activating the MEK/ERK pathway.

Recent studies have revealed that long non­coding RNAs (lncRNAs) serve important roles in carcinogenesis and that this type of gene may be used as biomarkers in cancer. A high level of lncRNA HOXA distal transcript antisense RNA (HOTTIP) is associated with unfavorable prognosis for patients with ovarian cancer (OC), but the mechanism of HOTTIP involved in OC development remains to be elucidated. The present study aimed to investigate the mechanism of HOTTIP in metastasis­associated OC cell behaviors. HOTTIP levels in ovarian cells were quantified by reverse transcription­quantitative PCR, cell proliferation was analyzed by colony formation assay, and apoptosis was assessed by flow cytometry. Cell migratory and invasive abilities were evaluated by wound healing and Transwell assays, respectively. The expression levels of mitogen­activated protein kinase kinase (MEK)/ERK pathway­associated proteins were detected by western blotting. The results demonstrated that knockdown of HOTTIP in OC cells significantly reduced the phosphorylation levels of MEK and ERK, inhibited the proliferation and invasion of OC cells and promoted their apoptosis. Furthermore, the effects of HOTTIP on cell migration and invasion were partly associated with the epithelial­mesenchymal transition (EMT) process. Proliferation, invasion and EMT of OC cells were enhanced following overexpression of HOTTIP; however, these effects were reversed by the MEK/ERK pathway inhibitor U0126. In conclusion, HOTTIP was demonstrated to promote the proliferation, migration and invasion of OC cells by activating the MEK/ERK pathway. Therefore, HOTTIP may serve as a potential therapeutic target for OC.

Herpes Simplex Virus 1 UL2 Inhibits the TNF-α-Mediated NF-κB Activity by Interacting With p65/p50.

Herpes simplex virus 1 (HSV-1) is a large double-stranded DNA virus that encodes at least 80 viral proteins, many of which are involved in the virus-host interaction and are beneficial to the viral survival and reproduction. However, the biological functions of some HSV-1-encoded proteins are not fully understood. Nuclear factor κB (NF-κB) activation is the major antiviral innate response, which can be triggered by various signals induced by cellular receptors from different pathways. Here, we demonstrated that HSV-1 UL2 protein could antagonize the tumor necrosis factor α (TNF-α)-mediated NF-κB activation. Co-immunoprecipitation assays showed that UL2 could interact with the NF-κB subunits p65 and p50, which also revealed the region of amino acids 9 to 17 of UL2 could suppress the NF-κB activation and interact with p65 and p50, and UL2 bound to the immunoglobulin-like plexin transcription factor functional domain of p65. However, UL2 did not affect the formation of p65/p50 dimerization and their nuclear localizations. Yet, UL2 was demonstrated to inhibit the NF-κB activity by attenuating TNF-α-induced p65 phosphorylation at Ser536 and therefore decreasing the expression of downstream inflammatory chemokine interleukin 8. Taken together, the attenuation of NF-κB activation by UL2 may contribute to the escape of host's antiviral innate immunity for HSV-1 during its infection.

The Interaction Mechanism Between Herpes Simplex Virus 1 Glycoprotein D and Host Antiviral Protein Viperin.

Viperin is an interferon-inducible protein that responsible for a variety of antiviral responses to different viruses. Our previous study has shown that the ribonuclease UL41 of herpes simplex virus 1 (HSV-1) can degrade the mRNA of viperin to promote HSV-1 replication. However, it is not clear whether other HSV-1 encoded proteins can regulate the function of viperin. Here, one novel viperin associated protein, glycoprotein D (gD), was identified. To verify the interaction between gD and viperin, gD and viperin expression plasmids were firstly co-transfected into COS-7 cells, and fluorescence microscope showed they co-localized at the perinuclear region, then this potential interaction was confirmed by co-immunoprecipitation (Co-IP) assays. Moreover, confocal microscopy demonstrated that gD and viperin co-localized at the Golgi body and lipid droplets. Furthermore, dual-luciferase reporter and Co-IP assays showed gD and viperin interaction leaded to the increase of IRF7-mediated IFN-ß expression through promoting viperin and IRAK1 interaction and facilitating K63-linked IRAK1 polyubiquitination. Nevertheless, gD inhibited TRAF6-induced NF-κB activity by decreasing the interaction of viperin and TRAF6. In addition, gD restrained viperin-mediated interaction between IRAK1 and TRAF6. Eventually, gD and viperin interaction was corroborated to significantly inhibit the proliferation of HSV-1. Taken together, this study would open up new avenues toward delineating the function and physiological significance of gD and viperin during HSV-1 replication cycle.

Heat Shock Protein 60: Identification of an Undetected Allergen from Dermatophagoides farinae.

BACKGROUND: Recently, the incidence of allergic diseases has been on the rise; Dust mite is the major indoor allergen which needs a special consideration. OBJECTIVES: This study was carried out to identify and investigate the molecular properties of a new allergen named Hsp60 and to afford a foundation for future research of the allergic diseases caused by Dermatophagoides farinae. MATERIALS AND METHODS: Using polymerase chain reaction (PCR) with degenerate primer, the cDNA of Dermatophagoides farinae Hsp60 was amplified and sequenced. Next, the cDNA fragment was cloned into the prokaryotic expression vector pET-32a for the expression of the Hsp60. Then, it was further characterized by Elisa and Western Blot analysis. RESULTS: The partial cDNA sequence of the Dermatophagoides farinae Hsp60 was determined, and the recombinant Hsp60 was successfully expressed in Escherichia coli BL21. ELISA and Western blot analysis showed that the recombinant protein could be specifically recognized by SIgE from sera of the Dermatophagoides farina-allergic patients. CONCLUSIONS: Our group has, for the first time, demonstrated the fact that there is an Hsp60 family of Dermatophagoides farinae and analyzed the allergenicity of the Hsp60 with immunological method. These results provide a foundation for further allergological research of the Dermatophagoides farinae Hsp60.

Characterization of the subcellular localization of Epstein-Barr virus encoded proteins in live cells.

Epstein-Barr virus (EBV) is the pathogenic factor of numerous human tumors, yet certain of its encoded proteins have not been studied. As a first step for functional identification, we presented the construction of a library of expression constructs for most of the EBV encoded proteins and an explicit subcellular localization map of 81 proteins encoded by EBV in mammalian cells. Viral open reading frames were fused with enhanced yellow fluorescent protein (EYFP) tag in eukaryotic expression plasmid then expressed in COS-7 live cells, and protein localizations were observed by fluorescence microscopy. As results, 34.57% (28 proteins) of all proteins showed pan-nuclear or subnuclear localization, 39.51% (32 proteins) exhibitted pan-cytoplasmic or subcytoplasmic localization, and 25.93% (21 proteins) were found in both the nucleus and cytoplasm. Interestingly, most envelope proteins presented pan-cytoplasmic or membranous localization, and most capsid proteins displayed enriched or complete localization in the nucleus, indicating that the subcellular localization of specific proteins are associated with their roles during viral replication. Taken together, the subcellular localization map of EBV proteins in live cells may lay the foundation for further illustrating the functions of EBV-encoded genes in human diseases especially in its relevant tumors.

Characterization of the subcellular localization and nuclear import molecular mechanisms of herpes simplex virus 1 UL2.

As a crucial protein, the herpes simplex virus 1 (HSV-1) UL2 protein has been shown to take part in various stages of viral infection, nonetheless, its exact subcellular localization and transport molecular determinants are not well known thus far. In the present study, by using live cells fluorescent microscopy assay, UL2 tagged with enhanced yellow fluorescent protein was transiently expressed in live cells and showed a completely nuclear accumulation without the presence of other HSV-1 proteins. Moreover, the nuclear transport of UL2 was characterized to be assisted by multiple transport pathways through Ran-, importin α1-, α5-, α7-, ß1- and transportin-1 cellular transport receptors. Consequently, these results will improve understanding of UL2-mediated biological functions in HSV-1 infection cycles.