CircRNA ARFGEF1 functions as a ceRNA to promote oncogenic KSHV-encoded viral interferon regulatory factor induction of cell invasion and angiogenesis by upregulating glutaredoxin 3.

Circular RNAs (circRNAs) are novel single-stranded noncoding RNAs that can decoy other RNAs to inhibit their functions. Kaposi's sarcoma (KS), caused by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), is a highly angiogenic and invasive vascular tumor of endothelial origin commonly found in AIDS patients. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) induces cell invasion, angiogenesis and cellular transformation; however, the role of circRNAs is largely unknown in the context of KSHV vIRF1. Herein, transcriptome analysis identified 22 differentially expressed cellular circRNAs regulated by vIRF1 in an endothelial cell line. Among them, circARFGEF1 was the highest upregulated circRNA. Mechanistically, vIRF1 induced circARFGEF1 transcription by binding to transcription factor lymphoid enhancer binding factor 1 (Lef1). Importantly, upregulation of circARFGEF1 was required for vIRF1-induced cell motility, proliferation and in vivo angiogenesis. circARFGEF1 functioned as a competing endogenous RNAs (ceRNAs) by binding to and inducing degradation of miR-125a-3p. Mass spectrometry analysis demonstrated that glutaredoxin 3 (GLRX3) was a direct target of miR-125a-3p. Knockdown of GLRX3 impaired cell motility, proliferation and angiogenesis induced by vIRF1. Taken together, vIRF1 transcriptionally activates circARFGEF1, potentially by binding to Lef1, to promote cell oncogenic phenotypes via inhibiting miR-125a-3p and inducing GLRX3. These findings define a novel mechanism responsible for vIRF1-induced oncogenesis and establish the scientific basis for targeting these molecules for treating KSHV-associated cancers.

Protection via a ROM4 DNA vaccine and peptide against Toxoplasma gondii in BALB/c mice.

BACKGROUND: Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite with a broad host range including most warm-blooded animals, including humans. T. gondii surface antigen 1 (SAG1) is a well-characterized T. gondii antigen. T. gondii expresses five nonmitochondrial rhomboid intramembrane proteases, TgROM1-5. TgROM4 is uniformly distributed on the surface of T. gondii and involved in regulating MIC2, MIC3, MIC6, and AMA1 during T. gondii invasion of host cells. Bioinformatics have predicted ROM4 B-cell and T-cell epitopes. Immunization strategy is also a key factor in determining the effectiveness of the immune response and has gained increasing attention in T. gondii vaccine research. In this study, we used a DNA prime-peptide boost vaccination regimen to assess the protective efficacy of various vaccination strategies using TgROM4. METHODS: We identified a polypeptide (YALLGALIPYCVEYWKSIPR) using a bioinformatics approach, and immunized mice using a DNA-prime and polypeptide-boost regimen. BALB/c mice were randomly divided into six groups, including three experimental groups (peptide, pROM4 and pROM4/peptide) and three control groups (PBS, pEGFP-C1 and pSAG1). Mice were then immunized intramuscularly four times. After immunization, IgG and cytokine productions were determined using enzyme-linked immunosorbent assays. The survival time of mice was evaluated after challenge with tachyzoites of T. gondii RH strain. Additionally, the number of cysts in the brain was determined after intragastric challenge with cysts of T. gondii PRU strain. RESULTS: Mice vaccinated with different immunization regimens (peptide, pROM4 and pROM4/peptide) elicited specific humoral and cellular responses, with high levels of IgG, IgG2a, and interferon (IFN)-γ. Moreover, IgG, IgG2a and IFN-γ levels were highest in the pROM4/peptide group. Immunized mice, especially those in the pROM4/peptide group, had prolonged survival times after challenge with tachyzoites and reduced numbers of brain cysts after infection compared with negative controls. CONCLUSION: A DNA prime-peptide boost regimen based on ROM4 elicited the highest level of humoral and cellular immune responses among immunization regimens, and may be a promising approach to increase the efficacy of DNA immunization.

Vaccination with toxofilin DNA in combination with an alum-monophosphoryl lipid A mixed adjuvant induces significant protective immunity against Toxoplasma gondii.

BACKGROUND: A widely prevalent disease, toxoplasmosis poses serious health threats to both humans and animals; therefore, development of an ideal DNA vaccine against Toxoplasma gondii is needed eagerly. The purpose of the present study is to assess the protective efficacy of a DNA vaccine encoding the T. gondii toxofilin gene (pEGFP-toxofilin). In addition, toxofilin DNA vaccine combined with the individual adjuvants, alum or monophosphoryl lipid A (MPLA), or a mixture of alum-MPLA adjuvant were screened for their ability to enhance antibody responses. METHODS: Using bioinformatics, we analyzed the gene and amino acid sequences of the toxofilin protein, recognizing and identifying several potential linear B and T helper (Th)-1 cell epitopes. BALB/c mice were immunized three times with either toxofilin DNA vaccine alone or in combination with the adjuvants such as alum, MPLA or an alum-MPLA mixture. The systemic immune response was evaluated by cytokine, the percentage of CD4 (+) and CD8 (+) T cells and specific antibody measurement. Two weeks after the last immunization, protective efficacy was evaluated by challenging intraperitoneally with 1 × 104 tachyzoites of T. gondii or intragastrically with 20 cysts of T. gondii PRU strain. RESULTS: All experimentally immunized mice developed strong humoral and cellular immune responses compared with the control groups. Moreover, by comparison with the non-adjuvant toxofilin DNA vaccine group, adding alum adjuvant to toxofilin DNA vaccine resulted in an increase in humoral response and a skewed Th2 response. However, the MPLA adjuvant with toxofilin DNA vaccine induced significantly enhanced humoral and Th1-biased immune responses. Importantly, the co-administration of alum-MPLA adjuvant in combination with the toxofilin DNA vaccine shifted the Th2 immune response to a Th1 response compared with the alum-toxofilin group, and elicited the strongest humoral and Th1 responses among all the groups. At the same time, a longer survival time and less cyst amounts against T. gondii infection were also observed in the alum-MPLA-toxofilin group in comparison with single or no adjuvant groups. CONCLUSIONS: Toxoplasma gondii toxofilin is a promising vaccine candidate that warrants further development. Co-administration of the alum-MPLA adjuvant mixture with DNA vaccine could effectively enhance immunogenicity and strongly skew the cellular immune response towards a Th1 phenotype.

Down-regulation of HPGD by miR-146b-3p promotes cervical cancer cell proliferation, migration and anchorage-independent growth through activation of STAT3 and AKT pathways.

While the application of early screening and HPV vaccines has reduced the incidence and mortality rates of cervical cancer, it remains the third most common carcinoma and fourth leading cause of cancer-associated death among women worldwide. The precise mechanisms underlying progression of cervical cancer are not fully understood at present. Here, we detected significant down-regulation of 15-hydroxyprostaglandin dehydrogenase (HPGD) in cervical cancer tissues. Overexpression of HPGD inhibited cervical cancer cell proliferation, migration and anchorage-independent growth to a significant extent. To clarify the mechanisms underlying HPGD down-regulation in cervical cancer, miRNA microarray, bioinformatics and luciferase reporter analyses were performed. HPGD was identified as a direct target of miR-146b-3p displaying up-regulation in cervical cancer tissues. Similar to the effects of HPGD overexpression, down-regulation of miR-146b-3p strongly suppressed proliferation, migration and anchorage-independent growth of cervical cancer cells. Furthermore, HPGD negatively regulated activities of STAT3 and AKT that promote cervical cancer cell proliferation. Notably, HPV oncogenes E6 and E7 were determined as potential contributory factors to these alterations. Our results collectively suggest that the HPGD/miR-146b-3p axis plays a significant role in cervical cancer and may serve as a potentially effective therapeutic target.

Structural predication and antigenic analysis of Toxoplasma gondii ROP20.

Toxoplasma gondii infects almost all the warm-blooded animals. ROP20 protein is expressed in the rhoptry of Toxoplasma gondii. In this study, the secondary structure of ROP20 was analyzed using SMART software. We constructed and analyzed the 3D model of ROP20 protein using SWISS-MODEL online procedure and Visual Molecular Dynamics (VMD) software. The structure analysis fully indicated that ROP20 protein is an important member of the ROP family. Furthermore, We used DNASTAR software and Epitope Database online service to analyze liner-B cell epitopes and T-cell epitopes of ROP20 protein. All the analysis results of ROP20 protein can provide positive information on treatment and vaccine for toxoplasmosis. Moreover, ROP20 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-C1-ROP20) was constructed in the following study. After restriction enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result indicated that the recombinant plasmid could transcribe successfully in HEK 293-T cell. The results of western blotting indicated the expressed proteins can be recognized by anti-STAg mouse sera.

SAG5B and SAG5C combined vaccine protects mice against Toxoplasma gondii infection.

Infections with the protozoan parasite Toxoplasma gondii, which are common around the world, can lead to congenital infections in humans. T. gondii surface antigen protein 5B (SAG5B) and SAG5C are potential stimulators of humoral and cellular immune responses. In this study, a multi-antigenic DNA vaccine constructed to express T. gondii SAG5B and SAG5C proteins simultaneously was used to immunize BALB/c mice to evaluate the protective efficacy of the vaccine. IgG antibody and gamma interferon (IFN-γ) cytokine production in the pSAG5B/SAG5C DNA vaccine group were significantly higher (0.853±0.103 and 915.2±106.9, respectively) than in the single DNA vaccine groups (pSAG5B, 0.667±0.109 and 598.3±74.9, respectively; pSAG5C, 0.696±0.092 and 623.7±95.5, respectively). After a lethal challenge with 1×104 RH strain tachyzoites, the survival time of the mice (17days) immunized with pSAG5B/SAG5C was longer than that of the single-gene-immunized mice (12days) or the control mice (6days). Moreover, after intragastric infection with 20 T. gondii PRU (low virulence) strain cysts, the number of brain cysts in the pSAG5B/SAG5C-vaccinated mice was only 25% of the number for the PBS-injected mice. Our findings indicate that, in comparison with the other mouse groups, the multi-antigenic DNA vaccine (pSAG5B/SAG5C) significantly induced immune responses and improved the protection against challenge with T. gondii in the host animals.

Structuraland antigenic analysis of a new Rhoptry Pseudokinase Gene (ROP54) in Toxoplasma gondii.

Toxoplasma gondii is defined as an obligate intracellular apicomplexan parasite and influences approximatelyone-third of the human all over the world. ROP54 protein is expressed in the rhoptry of Toxoplasma gondii. In the present study, we used SMART software to analyzethe secondary structure of ROP54. The 3D model of ROP54 protein was constructed and analyzed using SWISS-MODEL server and VMD software. The structure results fully showed that ROP54 proteinis an importantmember from the ROP family. Moreover, DNAMAN software and Epitope Database online service were used to analyze liner-B cell epitopes and Th-cell epitopes of the protein. The bioinformatics prediction of ROP54 protein could provide positive information on treatment and vaccine for toxoplasmosis. Furthermore, ROP54 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-ROP54) was constructed in the following study. After identification of enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result suggested that the recombinant plasmid could transcribe successfully in HEK 293-T cell.

Epitope analysis, expression and protection of SAG5A vaccine against Toxoplasma gondii.

Bioinformatics approaches were used to identify B-cell epitopes and T-cell epitopes on SAG5A protein. Compared to SAG1, SAG5A with good B-cell epitopes and T-cell epitopes had a potentiality to become a more successful vaccine against Toxoplasma gondii. Thereafter, SAG5A DNA vaccine was constructed successfully and was injected into mice with peptide to evaluate the immunoprotection. Compared to the control groups, the vaccine (DNA/peptide) could induce more effective cellular and humoral immune responses in immunized mice. Furthermore, a significant reduction of brain cyst was detected in the mice vaccinated with peptide (732±160), pSAG5A (815±197), or pSAG5A/peptide (436±174) compared by the mice injected by PBS (1260±241) or pEGFP-C1 (1350±268). The number of cysts in brains was 35% reduced in the mice immunized with DNA/peptide than in the control mice treated by PBS. The results indicated that the DNA vaccine encoding SAG5A significantly induced immune responses and enhanced protection against cysts of PRU strain, especially with the help of peptide.