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miR-370 mimic inhibits replication of Japanese encephalitis virus in glioblastoma cells

View Article: PubMed Central - PubMed

ABSTRACT

Japanese encephalitis (JE) is one of the most severe viral infections of the central nervous system. No effective treatment for JE currently exists, because its pathogenesis remains largely unknown. The present study was designed to screen the potential microRNAs (miRNAs) involved in JE. Glioblastoma cells were collected, after being infected with the Japanese encephalitis virus (JEV). Total miRNAs were extracted and analyzed using an miRNA chip. One of the most severely affected miRNAs was selected, and the role of miR-370 in JEV infection was investigated. Cell viability and apoptosis of the host cells were evaluated. JEV replication was detected via analysis of gene E expression. Real-time polymerase chain reaction was used to determine the levels of endogenous miR-370 and expression of innate immunity-related genes. Following JEV infection, 114 miRNAs were affected, as evidenced by the miRNA chip. Among them, 30 miRNAs were upregulated and 84 were downregulated. The changes observed in five miRNAs were confirmed by real-time polymerase chain reaction. One of the significantly downregulated miRNAs was miR-370. Therefore, miR-370 mimic was transfected into the cells, following which the levels of endogenous miR-370 were significantly elevated. Concurrently, JEV replication was significantly reduced 24 hours after transfection of miR-370 mimic. Functionally, miR-370 mimic mitigated both JEV-induced apoptosis and the inhibition of host cell proliferation. Following JEV infection, interferon-β and nuclear factor-kappa B were upregulated, whereas miR-370 mimic prevented the upregulation of the genes induced by JEV infection. The present study demonstrated that miR-370 expression in host cells is downregulated following JEV infection, which further mediates innate immunity-related gene expression. Taken together, miR-370 mimic might be useful to prevent viral replication and infection-induced host cell injury.

No MeSH data available.


Related in: MedlinePlus

miR-370 mimic inhibition of JEV replication (A), endogenous miR-370 expression (B), JEV-induced apoptosis (C), and proliferation-inhibition (D).Note: *P<0.05 and **P<0.01 compared with control inhibitor/mimic.Abbreviations: JEV, Japanese encephalitis virus; mRNA, messenger RNA; miRNA, microRNA; Ctrl, control; OD, optical density; CCK-8, Cell Counting Kit-8; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
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f2-ndt-12-2411: miR-370 mimic inhibition of JEV replication (A), endogenous miR-370 expression (B), JEV-induced apoptosis (C), and proliferation-inhibition (D).Note: *P<0.05 and **P<0.01 compared with control inhibitor/mimic.Abbreviations: JEV, Japanese encephalitis virus; mRNA, messenger RNA; miRNA, microRNA; Ctrl, control; OD, optical density; CCK-8, Cell Counting Kit-8; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Mentions: Endogenous miRNAs could be functionally mimicked or stimulated by exogenous miRNAs, whereas miRNA inhibitors specifically prevent the normal functioning of endogenous miRNAs. In the present study, miR-370 mimic and miR-370 inhibitor were selected to evaluate the role of miR-370 in JEV infection. As shown in Figure 2A, expression of gene E was significantly inhibited by miR-370 mimic at the 36-hour and 48-hour time points, whereas it was significantly enhanced by miR-370 inhibitor (versus control, P<0.05). These data suggest that viral replication was inhibited by miR-370 mimic and promoted by miR-370 inhibitor. In comparison with the control, miR-370 mimic significantly increased levels of endogenous miR-370 in U251 cells, 24 hours after transfection (Figure 2B). In successive experiments, cell proliferation and apoptosis were detected at 0, 12, 24, 36, and 48 hours following JEV infection. JEV infection significantly increased the number of apoptotic cells, 48 hours: F(3,15)=7.284, P<0.05 (Figure 2C) and inhibited cell proliferation, F(3,15)=4.21, P<0.05 (Figure 2D). In contrast, the effects of JEV on apoptosis were inhibited by miR-370 mimic, but not by miR-370 inhibitor (Figure 2C). JEV-induced inhibition of proliferation was slightly attenuated at the 48-hour time point by miR-370 mimic (P>0.05; Figure 2D).


miR-370 mimic inhibits replication of Japanese encephalitis virus in glioblastoma cells
miR-370 mimic inhibition of JEV replication (A), endogenous miR-370 expression (B), JEV-induced apoptosis (C), and proliferation-inhibition (D).Note: *P<0.05 and **P<0.01 compared with control inhibitor/mimic.Abbreviations: JEV, Japanese encephalitis virus; mRNA, messenger RNA; miRNA, microRNA; Ctrl, control; OD, optical density; CCK-8, Cell Counting Kit-8; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036624&req=5

f2-ndt-12-2411: miR-370 mimic inhibition of JEV replication (A), endogenous miR-370 expression (B), JEV-induced apoptosis (C), and proliferation-inhibition (D).Note: *P<0.05 and **P<0.01 compared with control inhibitor/mimic.Abbreviations: JEV, Japanese encephalitis virus; mRNA, messenger RNA; miRNA, microRNA; Ctrl, control; OD, optical density; CCK-8, Cell Counting Kit-8; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
Mentions: Endogenous miRNAs could be functionally mimicked or stimulated by exogenous miRNAs, whereas miRNA inhibitors specifically prevent the normal functioning of endogenous miRNAs. In the present study, miR-370 mimic and miR-370 inhibitor were selected to evaluate the role of miR-370 in JEV infection. As shown in Figure 2A, expression of gene E was significantly inhibited by miR-370 mimic at the 36-hour and 48-hour time points, whereas it was significantly enhanced by miR-370 inhibitor (versus control, P<0.05). These data suggest that viral replication was inhibited by miR-370 mimic and promoted by miR-370 inhibitor. In comparison with the control, miR-370 mimic significantly increased levels of endogenous miR-370 in U251 cells, 24 hours after transfection (Figure 2B). In successive experiments, cell proliferation and apoptosis were detected at 0, 12, 24, 36, and 48 hours following JEV infection. JEV infection significantly increased the number of apoptotic cells, 48 hours: F(3,15)=7.284, P<0.05 (Figure 2C) and inhibited cell proliferation, F(3,15)=4.21, P<0.05 (Figure 2D). In contrast, the effects of JEV on apoptosis were inhibited by miR-370 mimic, but not by miR-370 inhibitor (Figure 2C). JEV-induced inhibition of proliferation was slightly attenuated at the 48-hour time point by miR-370 mimic (P>0.05; Figure 2D).

View Article: PubMed Central - PubMed

ABSTRACT

Japanese encephalitis (JE) is one of the most severe viral infections of the central nervous system. No effective treatment for JE currently exists, because its pathogenesis remains largely unknown. The present study was designed to screen the potential microRNAs (miRNAs) involved in JE. Glioblastoma cells were collected, after being infected with the Japanese encephalitis virus (JEV). Total miRNAs were extracted and analyzed using an miRNA chip. One of the most severely affected miRNAs was selected, and the role of miR-370 in JEV infection was investigated. Cell viability and apoptosis of the host cells were evaluated. JEV replication was detected via analysis of gene E expression. Real-time polymerase chain reaction was used to determine the levels of endogenous miR-370 and expression of innate immunity-related genes. Following JEV infection, 114 miRNAs were affected, as evidenced by the miRNA chip. Among them, 30 miRNAs were upregulated and 84 were downregulated. The changes observed in five miRNAs were confirmed by real-time polymerase chain reaction. One of the significantly downregulated miRNAs was miR-370. Therefore, miR-370 mimic was transfected into the cells, following which the levels of endogenous miR-370 were significantly elevated. Concurrently, JEV replication was significantly reduced 24 hours after transfection of miR-370 mimic. Functionally, miR-370 mimic mitigated both JEV-induced apoptosis and the inhibition of host cell proliferation. Following JEV infection, interferon-&beta; and nuclear factor-kappa B were upregulated, whereas miR-370 mimic prevented the upregulation of the genes induced by JEV infection. The present study demonstrated that miR-370 expression in host cells is downregulated following JEV infection, which further mediates innate immunity-related gene expression. Taken together, miR-370 mimic might be useful to prevent viral replication and infection-induced host cell injury.

No MeSH data available.


Related in: MedlinePlus