Host Non-Coding RNA Regulates Influenza A Virus Replication

Outbreaks of influenza, caused by the influenza A virus (IAV), occur almost every year in various regions worldwide, seriously endangering human health. Studies have shown that host non-coding RNA is an important regulator of host–virus interactions in the process of IAV infection. In this paper, we comprehensively analyzed the research progress on host non-coding RNAs with regard to the regulation of IAV replication. According to the regulation mode of host non-coding RNAs, the signal pathways involved, and the specific target genes, we found that a large number of host non-coding RNAs directly targeted the PB1 and PB2 proteins of IAV. Nonstructural protein 1 and other key genes regulate the replication of IAV and indirectly participate in the regulation of the retinoic acid-induced gene I-like receptor signaling pathway, toll-like receptor signaling pathway, Janus kinase signal transducer and activator of transcription signaling pathway, and other major intracellular viral response signaling pathways to regulate the replication of IAV. Based on the above findings, we mapped the regulatory network of host non-coding RNAs in the innate immune response to the influenza virus. These findings will provide a more comprehensive understanding of the function and mechanism of host non-coding RNAs in the cellular anti-virus response as well as clues to the mechanism of cell–virus interactions and the discovery of antiviral drug targets

TGM2 inhibits the proliferation, migration and tumorigenesis of MDCK cells.

Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2

A chelicerate-specific burst of nonclassical Dscam diversity

Abstract Background The immunoglobulin (Ig) superfamily receptor Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for both nervous and immune systems in insects. However, further information is required to develop a comprehensive view of Dscam diversification across the broad spectrum of Chelicerata clades, a basal branch of arthropods and the second largest group of terrestrial animals. Results In this study, a genome-wide comprehensive analysis of Dscam genes across Chelicerata species revealed a burst of nonclassical Dscams, categorised into four types—mDscam, sDscamα, sDscamβ, and sDscamγ—based on their size and structure. Although the mDscam gene class includes the highest number of Dscam genes, the sDscam genes utilise alternative promoters to expand protein diversity. Furthermore, we indicated that the 5′ cassette duplicate is inversely correlated with the sDscam gene duplicate. We showed differential and sDscam- biased expression of nonclassical Dscam isoforms. Thus, the Dscam isoform repertoire across Chelicerata is entirely dominated by the number and expression levels of nonclassical Dscams. Taken together, these data show that Chelicerata evolved a large conserved and lineage-specific repertoire of nonclassical Dscams. Conclusions This study showed that arthropods have a large diversified Chelicerata-specific repertoire of nonclassical Dscam isoforms, which are structurally and mechanistically distinct from those of insects. These findings provide a global framework for the evolution of Dscam diversity in arthropods and offer mechanistic insights into the diversification of the clade-specific Ig superfamily repertoire

Effect of TGM2 on MDCK tumorigenicity.

(A). Subcutaneous tumor display in nude mice. (B) Statistical results of subcutaneous tumor number in nude mice. (C). Effect of TGM2 overexpression on tumor volume. (D). Effect of TGM2 knockout on tumor volume. (E). Histological observation of liver, lung and subcutaneous tumors in nude mice 30 days after Con, TGM2-OE, WT, and TGM2-KO cells were injected subcutaneously (H&E staining). A large number of hepatic cells were seen in the tissues with mild watery degeneration, cell swelling, loose cytoplasm, and light staining (black arrow). Focal necrosis of the liver cells was observed with nuclear fragmentation or dissolution (yellow arrows), with a small amount of connective tissue hyperplasia (red arrows), and a small amount of lymphocyte and neutrophil infiltration (blue arrows). Positive control (HeLa cells), Negative control (MRC-5 cells), TGM2 overexpression (TGM2-OE), Carrier control (Con), TGM2 knockout (TGM2-KO), Wild type (WT).</p

Effects of TGM2 on proliferation of influenza A (H1N1) virus.

(A). mRNA levels of viral NP and NS1 genes overexpressed by TGM2 were determined by real-time PCR. (B, C). The effect of TGM2 overexpression on viral NP protein was determined by Western blotting. (D). Effects of TGM2 overexpression on titer of influenza A H1N1 virus determined by TCID50; (E). mRNA levels of NP and NS1 genes of TGM2 knockout virus were determined by real-time PCR. (F, G). Effect of TGM2 knockout on NP protein of the virus was determined by Western blotting. (H). Effect of TGM2 knockout on titer of influenza A (H1N1) virus measured by TCID50. TGM2-overexpressed cells (TGM2-OE); Vector control cells (Con); TGM2-knockout cells (TGM2-KO); Wild-type cell (WT). The experimental data are expressed as "mean ± standard deviation". *P < 0.05 and **P < 0.01 indicate significant differences, and ***P < 0.001 indicates extremely significant differences.</p

S3 File -

Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.</div

Raw data of the gels and blot.

Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.</div

Effects of TGM2 overexpression on proliferation, migration, and invasion of MDCK cells.

(A). mRNA levels of TGM2-overexpressed cells were determined with real-time PCR. (B). Protein levels of TGM2-overexpressed cells determined by Western blotting. (C). Effects of TGM2 overexpression on proliferation ability of MDCK cells determined by cell counting; (D,E) Effects of TGM2 overexpression on plate clone rate of MDCK cells determined by plate clone formation assays. (F,G) The effects of TGM2 overexpression on the invasion ability of MDCK cells were determined using Transwell assays. (H) The effects of TGM2 overexpression on the migration ability of MDCK cells were determined by scratch tests. TGM2-overexpressed cells (TGM2-OE); Vector control cell (Con). The experimental data are expressed as "mean ± standard deviation". *P < 0.05 and **P < 0.01 indicate significant differences, and ***P < 0.001 indicates extremely significant differences.</p