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Abstract Zinc finger proteins (ZNFs) have been demonstrated to participate extensively in breast cancer progression by functioning as transcription factors, but there are still a variety of ZNFs whose biological mechanisms remain unknown. Here, we show that zinc finger protein 276 (ZNF276) is highly expressed in breast cancer tissues and cell lines. Higher level of ZNF276 correlated with poor prognosis. Gain-of and loss-of function suggested that ZNF276 is essential for the proliferation, migration and invasion of breast cancer cells in vitro and metastasis in vivo. RNA-sequencing and CUT&Tag assay revealed that ZNF276 controlled a variety of growth and metastasis-related genes expression. ZNF276 transcriptionally promoted the expression of CYP1B1 by directly binds to the promoter region of the CYP1B1 through its C2H2 domain. ZNF276 facilitated the translocation of β-catenin from cytoplasm to nucleus through CYP1B1, leading to the upregulation of cyclin D1 and c-Myc, and the activation of the Wnt/β-catenin pathway. Knockdown of CYP1B1 significantly blocked the ZNF276-mediated effects on cell proliferation, migration and invasion. Lastly, ZNF276 interacted with MAGEB2 which enhanced the binding of ZNF276 at the CYP1B1 promoter, promoted CYP1B1 expression and Wnt signaling activation. Collectively, these findings highlight the oncogenic role of ZNF276 on breast cancer cell proliferation and metastasis. Targeting ZNF276/MAGEB2 axis may serve as a potential therapeutic strategy for breast cancer patients.

Abstract Background Glutamate metabotropic receptors (GRM) play a variety of roles in neuronal cells. However, their clinical significance and biological functions in breast cancer remain unknown. Methods RNA sequencing data of breast cancer was obtained from the TCGA dataset (v2) and mined for the expression profiles of GRM family according to cancer subtypes. mRNA expression of GRM family in breast cancer tissues and para-cancerous tissue samples as well as breast cancer cell lines were measured by qPCR. The effects of over- and under-expression of GRM4 on cell capabilities to survive, migrate and invade were determined by colony formation, transwell migration and invasion assays. To explore the upstream regulation pattern of GRM4, miRNAs that target GRM4 were predicted and validated by dual luciferase reporter assay. In addition, the mRNA and protein expression of GRM4 regulated by these miRNAs were further measured by qPCR and western blot assay. Results GRM4 was the only GRM member that expressed in breast cancer tissues. Ectopic expression of GRM4 was correlated with better prognosis of breast cancer patients. Overexpression of GRM4 could significantly inhibit cell proliferation, migration and invasion capacity in MDA-MB-231, while knockdown of GRM4 could promote these processes. miR-328-3p and miR-370-3p were predicted to regulate the expression of GRM4 and dual luciferase reporter assay demonstrated that miR-328-3p and miR-370-3p directly bound to the 3′ UTR of GRM4 and mutations on the binding regions on GRM4 significantly decreased the luciferase activity. qPCR demonstrated that expression of miR-328-3p and miR-370-3p was significantly decreased in breast cancer tissues and cells compared with that in control samples. However, there were no correlations between the expression of miR-328-3p and GRM4, as well as the expression of miR-370-3p and GRM4. Moreover, overexpression of miR-328-3p and miR-370-3p counteracted the inhibitory effect of GRM4-induced cell proliferation, migration and invasion. Conclusions Our results suggest that GRM4 might be a tumor suppressor gene in breast cancer under the direct regulation of miR-328-3p and miR-370-3p.

Abstract Background The Orf virus (ORFV) is the causative agent of orf, a globally-occurring, acute, pustular, contagious disease affecting sheep, goats and humans with a worldwide distribution. Currently, the genomic analysis of four ORFV strains from the Fujian province in southern China and a NA1/11 strain isolated from the Jilin province in northeast China have been reported. However, little is known about the genomic information of ORFV strains from central China. Results From a recent outbreak in a sheep herd in the Henan province of central China, a novel ORFV strain (HN3/12) was isolated and cultured in ovine fetal turbinate (OFTu) cells. The strain was identified as HN3/12 and verified by PCR based on the DNA sequences of 011 and 059 genes. The whole genomic sequence of this isolate was determined by Next Generation Sequencing technology. To determine the genetic characteristics of the HN3/12 strain, phylogenetic analysis of the 011 and 059 genes and amino acid sequence alignment of the HN3/12 strain were performed and compared with reference parapoxvirus strains. Conclusions The HN3/12 genome is 136,643 bp in length, contains 63.67% G + C and encodes 132 putative genes. Phylogenetic analysis of the 011 and 059 nucleotide sequences showed that this viral strain was similar to the NA1/11 isolate. The homology analysis indicates that HN3/12 has 93% to 98% identity with published ORFV strains at amino acid level. When open reading frames (ORFs) were aligned among the HN3/12 and four Fujian ORFV strains, most of them have identities greater than 90% and only a few less than 60%. The availability of the whole genomic sequence of HN3/12 aids in our understanding of, and provides new insights into, the genetic diversity of ORFV.

Apoptosis, a significant form of cell death, has a leading role in the host cell defense against virus infection. Viruses have evolved a series of strategies that block apoptosis during the early stage of viral infection to enhance viral replication, and induce apoptosis in the late stages to facilitate viral particle release from the cells. Here we show that orf virus (ORFV), the causative agent of orf, encodes an apoptosis-inducing protein ORFV119. ORFV119 targets the mitochondria in host cells, inhibits cell proliferation, and induces cell apoptosis. Protein array data indicated that ORFV119 could induce apoptosis via up-regulation of Smac, Bak, and Bax and down-regulation of anti-apoptotic proteins Bcl-2 and cIAP-2. Activation of caspase-9 and caspase-3, and consequent PARP cleavage, ultimately lead to apoptosis. ORFV119 could also directly activate caspase-8 and induce Bid, involved in the extrinsic pathway, to achieve cell death. Furthermore, sequence analysis and experiments with mutants of ORFV119 introduced revealed that ORFV119 contains a key N-terminal domain that is necessary and sufficient to direct the protein to the mitochondria. Together, we report, for the first time, the identification of the novel apoptosis-inducing protein ORFV119 encoded by a parapoxvirus. This provides an important reference for the study of pathogenesis, identification of immunomodulation mechanisms of ORFV, and may lead to new strategies for orf disease control.

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1. [ABSTRACT FROM AUTHOR]