Identification of Two Isoforms of Canine Tetherin in Domestic Dogs and Characterization of Their Antiviral Activity against Canine Influenza Virus.

Canine influenza virus (CIV) significantly threatens the canine population and public health. Tetherin, an innate immune factor, plays an important role in the defense against pathogen invasion and has been discovered to restrict the release of various enveloped viruses. Two isoforms of canine tetherin (tetherin-X1 and tetherin-X2) were identified in peripheral blood leukocytes of mixed-breed dogs using reverse transcription polymerase chain reaction (RT-PCR). Amino acid alignment revealed that relative to full-length tetherin (tetherin-X1) and truncated canine tetherin (tetherin-X2) exhibited deletion of 34 amino acids. The deletion occurred at the C-terminus of the coiled-coiled ectodomain and the N-terminus of the glycosylphosphatidylinositol (GPI)-anchor domain. Tetherin-X2 was localized subcellularly at the cell membrane, which was consistent with the localization of tetherin-X1. In addition, canine tetherin-X1 and tetherin-X2 restricted the release of H3N2 CIV. However, canine tetherin-X1 had higher antiviral activity than canine tetherin-X2, indicating that the C-terminus of the coiled-coiled ectodomain and the N-terminus of the GPI-anchor domain of canine tetherin (containing the amino acids deleted in tetherin-X2) are critical for its ability to restrict H3N2 CIV release. This study provides insights for understanding the key functional domains of tetherin that restrict CIV release.

Upregulated circ_0002141 facilitates oral squamous cell carcinoma progression via the miR-1231/EGFR axis.

OBJECTIVES: The dysregulation of circular RNAs (circRNAs) is implicated in the progression of various cancers. This study was aimed at expounding the role and mechanism of hsa_circ_0002141 in the OSCC progression. MATERIALS AND METHODS: Circ_0002141 expression was examined in 52 pairs of OSCC tissues and matched adjacent tissue samples by quantitative real-time polymerase chain reaction (qRT-PCR) assay. After circ_0002141 was overexpressed or knocked down in OSCC cell lines, cell counting kit-8 (CCK-8) assay, Transwell assay, flow cytometry, and Western blotting were conducted to detect the changes in the growth, migration, invasion and apoptosis of OSCC cells. Western blot assay, qRT-PCR and dual-luciferase reporter assay were performed to clarify the interplay among circ_0002141, miR-1231, and epidermal growth factor receptor (EGFR). RESULTS: Circ_0002141 expression was significantly upregulated in OSCC tissues and cell lines. Circ_0002141 overexpression markedly promoted the proliferation, migration, and invasion of OSCC cells whereas reduced the apoptotic of OSCC cells. Also, circ_0002141 knockdown suppressed the malignant characteristics of OSCC cells. EGFR was validated as the target of miR-1231. Besides, circ_0002141 could sponge miR-1231 and upregulate EGFR expression in OSCC cells. CONCLUSION: Circ_0002141/miR-1231/EGFR axis is involved in the progression of OSCC.

microRNA-17 is a tumor suppressor in oral squamous cell carcinoma and is repressed by LSD1.

OBJECTIVE: The effects of epigenetic modifiers have been uncovered on cellular reprogramming and, specifically, on sustaining characteristics of cancer stem cells. We here aim to investigate whether lysine-specific demethylase 1 (LSD1) affects the development of oral squamous cell carcinoma (OSCC) by sustaining the cancer stem cells from OSCC (OSCSCs). METHODS: RT-qPCR detection was firstly conducted to screen out research gene by determining differential expression of histone demethylases and methylases in identified OSCSCs. Then, microarray analysis was carried out in cells with poor expression of LSD1. RESULTS: OSCSCs expressed high levels of LSD1, and LSD1 inhibition reduced cell viability, migration, invasion, and sphere formation of OSCSCs. Later mechanistic studies suggested that LSD1 inhibited microRNA (miR)-17 expression through histone demethylation. miR-17 bound to KPNA2, and LSD1 downstream genes were mainly enriched in the PI3K/AKT pathway. Importantly, miR-17 inhibitor reversed the inhibitory effect of si-LSD1 on cell activity, while si-KPNA2 abolished the promotive effect of miR-17 inhibitor on cell activity both in vitro and in vivo. CONCLUSION: Overall, LSD1 functions as a cancer stem cell supporter in OSCC by catalyzing demethylation of miR-17 and activating the downstream KPNA2/PI3K/AKT pathway, which contributes to understanding of the mechanisms associated with epigenetic regulation in OSCC.

Identification and genetic characterization of bovine hepacivirus in China: A large scale epidemiological study.

Bovine hepacivirus (BovHepV) is a novel virus that was recently discovered in Ghana and Germany in 2015. Until now, this virus has been identified in cattle population worldwide and is classified into subtypes A-G. To fully understand the epidemic situation and genetic characteristic of BovHepV in China, a total of 612 cattle serum samples were collected from 20 farms in seven provinces and municipality in China between 2018 and 2020 and were tested for the presence of BovHepV RNA via semi-nested PCR. The results demonstrated that 49 (8.0%) samples were BovHepV RNA-positive. It is noted that BovHepV infection in yak was confirmed for the first time. BovHepV was detected in all the seven provinces, with the positive rate ranging from 3.1% to 13.3%, which indicates a wide geographical distribution pattern of BovHepV in China. Sequencing results revealed that 5' UTR of the 49 field BovHepV strains have a nucleotide similarity of 96.3%-100% between each other and 93.9%-100% with previously reported BovHepV strains. In addition, genetic analysis identified five critical nucleotide sites in 5' UTR to distinguish different subtypes, which was further verified by genomic sequencing and nucleotide similarity analysis. All the 49 Chinese field BovHepV strains were classified into subtype G and this subtype is only determined in cattle in China currently. This study will provide insights for us to better understand the epidemiology and genetic diversity of BovHepV.

Swine Interferon-Inducible Transmembrane Proteins Potently Inhibit African Swine Fever Virus Replication.

African swine fever virus (ASFV) causes an acute, hemorrhagic, and highly contagious disease in domestic swine, leading to significant economic losses to the global porcine industry. Restriction factors of innate immunity play a critical in host antiviral action. However, function of swine restriction factors of innate immunity on ASFV has been seldomly investigated. In this study, we determined five homologues of swine interferon-induced transmembrane proteins (SwIFITM [named SwIFITM1a, -1b, -2, -3, and -5]), and we found that they all exhibit potent antiviral activity against ASFV. Expression profile analysis indicated that these SwIFITMs are constitutively expressed in most porcine tissues. Whether infected with ASFV or treated with swine interferon, the expression levels of SwIFITMs were induced in vitro. The subcellular localization of SwIFITMs was similar to that of their human homologues. SwIFITM1a and -1b localized to the plasma membrane, SwIFITM2 and -3 focused on the cytoplasm and the perinuclear region, while SwIFITM5 accumulated in the cell surface and cytoplasm. The overexpression of SwIFITM1a, -1b, -2, -3, or -5 could significantly inhibit ASFV replication in Vero cells, whereas knockdown of these genes could enhance ASFV replication in PAMs. We blocked the constitutive expression of endogenous IFITMs in Vero cells using a CRISPR-Cas9 system and then infected them with ASFV. The results indicated that the knockout of endogenous IFITMs could enhance ASFV replication. Finally, we expressed five SwIFITMs in knockout Vero cell lines and then challenged them with ASFV. The results showed that all of the SwIFITMs had a strong antiviral effect on ASFV. This research will further expand the understanding of the anti-ASFV activity of porcine IFITMs.

Canine Interferon-Inducible Transmembrane Protein Is a Host Restriction Factor That Potently Inhibits Replication of Emerging Canine Influenza Virus.

Canine influenza virus (CIV) is an emerging virus that is associated with major hidden hazards to the canine population and public health. Until now, how canine uses its innate immunity to restrict CIV replication is seldomly investigated. Recently, studies on interferon-inducible transmembrane (IFITM) of several major hosts of influenza virus (human, chicken, duck, pig) indicated it can potently restrict the viral replication. Here, the gene locus of five previously annotated canine IFITM (caIFITM) genes was determined on chromosome 18 using multiple bioinformatics strategies, provisionally designated as caIFITM1, caIFITM2a, caIFITM2b, caIFITM3, and caIFITM5. An analysis on protein sequences between caIFITM and its homologs indicated they shared the same conserved amino acids important for the antiviral activity. Expression profile analysis showed that caIFITM was constitutively expressed in tissues and MDCK cell line. After treatment with interferon or infection with influenza virus, the expression level of caIFITM increased with different degrees in vitro. An animal challenge study demonstrated CIV infection resulted in upregulation of caIFITM in beagles. caIFITMs had a similar subcellular localization to their human homologs. caIFITM1 was present at the cell surface and caIFITM3 was present perinuclearly and colocalized with LAMP1-containing compartments. Finally, we generated A549 cell lines stably expressing caIFITM and challenged them with influenza virus. The result demonstrated caIFITM1, caIFITM2a, caIFITM2b, and caIFITM3 had a potent antiviral activity against influenza virus. Our study will help better understand the evolutional pattern of IFITM and its role in the host's defense against virus infection.

First identification and genomic characterization of equine hepacivirus subtype 2 in China.

Equine hepacivirus (EqHV) is a newly discovered hepatitis C virus-like virus that can infect equines. EqHV strains circulating worldwide have been classified into subtypes 1-3. In previous studies, we detected the presence of EqHV strains of subtype 1 and 3 in China. To determine whether EqHV strains of subtype 2 are prevalent in China, serum samples were collected from 133 racehorses in Guangdong province in 2021 and were tested for EqHV RNA by RT-PCR, and the positive rate was 9% (12/133). Sequencing of the NS3 gene revealed that one field strain (GD2021) had a high degree of genetic similarity to EqHV strains of subtype 2. Subsequent genome sequencing and analysis demonstrated that strain GD2021 belongs to subtype 2. The present study enriches our knowledge about the genetic diversity of EqHV in China.

Novel parvovirus in cats, China.

Parvovirus is a common element of the feline virus group and usually causes gastroenteritis and leukopenia in cats. In this study, we identified a novel protoparvovirus from the Chinese domestic cats, which is genetically similar to canine bufavirus (98.0%-99.8%), but sharing low amino acid identities in the viral structural proteins 2 (VP2) (36.1-37.2%) to the well-known canine parvovirus type 2 and feline panleukopenia virus. This virus was provisionally designated as feline bufavirus (FBuV). Screening of fecal samples revealed a prevalence of 7.4% (19/257) in domestic cats. Diarrhea was present in 52.6% (10/19) of cats positive for FBuV. However, statistical analysis showed no association between FBuV and clinical signs. VP2 gene of the 19 field FBuV was sequenced and phylogenetic analysis demonstrated that FBuV determined from China had a genetic diversity. This study will strengthen the understanding of the epidemiology and genetic diversity of bufavirus and provide a foundation for further studies.

Circular RNA ITCH Suppresses Cell Proliferation but Induces Apoptosis in Oral Squamous Cell Carcinoma by Regulating miR-421/PDCD4 Axis.

BACKGROUND: Circular RNAs (circRNAs), a group of covalently closed non-coding RNAs, serve critical regulatory roles in many human cancers, including oral squamous cell carcinoma (OSCC). The purpose of this study was to investigate the functional role of circular RNA ITCH (circ-ITCH) in OSCC and the underlying mechanisms. METHODS: RT-qPCR analysis was applied to detect the expression levels of circ-ITCH in OSCC tissues and cell lines. MTT assay and flow cytometer analysis were used to evaluate the effects of circ-ITCH overexpression on the proliferation and apoptosis of OSCC cells. Bioinformatics analysis and dual-luciferase reporter assay were applied to determine the binding relation between circ-ITCH and miR-421 as well as PDCD4 mRNA and miR-421. RESULTS: Our results showed that circ-ITCH expression was remarkably decreased in OSCC tissues and cell lines. Low circ-ITCH expression was strongly associated with adverse clinicopathological characteristics of OSCC patients. Moreover, functional assays demonstrated that circ-ITCH overexpression significantly inhibited OSCC cell proliferation and induced cell apoptosis. Our data further uncovered that circ-ITCH could bind directly to miR-421 and block its repression on PDCD4 in OSCC. MiR-421 expression was significantly increased in OSCC tissues and was inversely correlated with circ-ITCH expression. Notably, miR-421 restoration blocked the tumor-suppressive role of circ-ITCH in OSCC cells. CONCLUSION: In conclusion, our study reveals that circ-ITCH serves as a tumor suppressor in OSCC partly by regulating miR-421/PDCD4 axis.

LncRNA MAFG-AS1 regulates human periodontal ligament stem cell proliferation and Toll-like receptor 4 expression.

LncRNA MAFG-AS1 is predicted to interact with miR-146a, which can target Toll-like receptor 4 (TLR4), a key player in periodontitis. This study aimed to investigate the roles of MAFG-AS1 in periodontitis. It was observed that MAFG-AS1 was downregulated in the human periodontal ligament stem cells (PDLSCs) derived from periodontitis-affected teeth. Dual-luciferase assay revealed that co-transfection of MAFG-AS1 expression vector and miR-146a mimic showed significantly lower relative luciferase activity comparing to co-transfection of MAFG-AS1 expression vector and negative control (NC) miRNA. However, MAFG-AS1 and miR-146a failed to affect each other. Interestingly, MAFG-AS1 overexpression led to the upregulated TLR4. In addition, MAFG-AS1 overexpression also led to the inhibited proliferation of PDLSCs. Therefore, MAFG-AS1 may regulate the proliferation of PDLSCs and the expression of TLR4 to participate in periodontitis.

MiR-143-3p Inhibits Osteogenic Differentiation of Human Periodontal Ligament Cells by Targeting KLF5 and Inactivating the Wnt/ß-Catenin Pathway.

Human periodontal ligament cells (hPDLCs) play a vital role in cell regeneration and tissue repair with multi-directional differentiation potential. microRNAs (miRs) are implicated in the osteogenesis of hPDLCs. This study explored the mechanism of miR-143-3p in osteogenesis of hPDLCs. Osteogenic differentiation of isolated hPDLCs was induced. KLF5 expression during osteogenic differentiation of hPDLCs was detected and then silenced in hPDLCs. Binding relationship between KLF5 and miR-143-3p was predicted and verified. hPDLCs were treated with miR-143-3p mimic or overexpressing KLF5, and then osteogenic specific markers and mineralized nodules were measured. The key factors of the Wnt/ß-catenin pathway during osteogenesis of hPDLCs were measured. KLF5 expression was upregulated during osteogenesis of hPDLCs. KLF5 silencing or miR-143-3p mimic reduced osteogenic specific markers and mineralized nodules. Overexpression of KLF5 could reverse the inhibitory effect of miR-143-3p on osteogenic differentiation. miR-143-3p mimic and KLF5 silencing inactivated the Wnt/ß-catenin pathway. Activation of the Wnt/ß-catenin pathway reversed the repression effect of miR-143-3p mimic on osteogenesis of hPDLCs. In conclusion, miR-143-3p inhibited osteogenic differentiation of hPDLCs by targeting KLF5 and inactivating the Wnt/ß-catenin pathway.