Hsa_circ_0049396 inhibited oral squamous cell carcinoma progression by regulating the miR-663b/ENDOU axis.

BACKGROUND: Circular RNA (circRNAs) play an important role in oral squamous cell carcinoma (OSCC) progression and has been widely reported. In this study, we aimed to investigate the role of a novel circRNA, circ_0049396, and its underlying mechanism in OSCC. METHODS: The expression levels of circ_0049396, miR-663b, and theuridylate-specific endoribonuclease (ENDOU) were assessed by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation and migration were evaluated using CCK-8 and Transwell assays, respectively. Western blotting was performed to measure the levels of the apoptosis-associated proteins (Bcl-2 and Bax). The functional role of circ _0049396 was further validated in a xenograft experiment in vivo. The interactions of miR-663b with circ_0049396/ENDOU were verified using the dual luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays. RESULTS: The expression of circ_0049396 and ENDOU was downregulated in OSCC tissues and cells, whereas miR-663b was upregulated. Circ_0049396 overexpression weakened OSCC cell proliferation and migration but enhanced their apoptosis. Circ_0049396 overexpression suppresses tumorigenesis in vivo. The circ_0049396/miR-663b/ENDOU regulatory network predicted through bioinformatic analysis was validated using RNA pull-down, luciferase reporter, and RIP experiments. MiR-663b mimic enhanced the migratory and proliferative abilities of OSCC cells, but suppressed apoptosis. Furthermore, circ_0049396 or ENDOU overexpression partially reversed the malignant behavior of miR-663b-overexpressing OSCC cells. CONCLUSIONS: Our study illustrated that circ_0049396 overexpression inhibited the malignant behavior of OSCC cells by regulating the miR-663b/ENDOU axis. Based on our findings, circ_0049396 can be used as a potential therapeutic agent for OSCC.

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways­interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)­activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung­derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Physiologic RNA targets and refined sequence specificity of coronavirus EndoU.

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U ↓ A and C ↓ A sequences (endoY ↓ A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2'-5' phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U ↓ A and C ↓ A sequences (endoY ↓ A) within viral (+) strand RNA to evade dsRNA-activated host responses.

EndoU is a novel regulator of AICD during peripheral B cell selection.

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22(-/-[B6])) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag-specific B cells in Ig(Tg) hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance.

A homologue of human placental protein, PP11, and mouse T cell-specific protein, Tcl-30, in exocrine pancreas of a teleost (Paralichthys olivaceus).

We cloned a 1401-bp cDNA encoding a novel pancreatic protein (PPSB) with two cysteine-rich somatomedin B-like domains from Japanese flounder (Paralichthys olivaceus). PPSB is predicted to be composed of 385 amino acids, including a signal sequence. The peptide sequence shares high homology with human placental protein 11 (PP11) and mouse T-cell specific protein (Tcl-30), which both contain a single somatomedin B-like domain. PPSB shares 47% and 44% identity with PP11 and Tcl-30, respectively. The unique point of PPSB is that an additional, somatomedin B-like domain is tandemly inserted. Unlike PP11 and Tcl-30, PPSB mRNA is specifically expressed by the exocrine pancreatic acinar cells, together with trypsinogen. Since PP11 has serine protease activity, it is predicted that its teleost homologue, PPSB, may function as a pancreatic digestive enzyme.