Limits...
MicroRNA-19b-3p Modulates Japanese Encephalitis Virus-Mediated Inflammation via Targeting RNF11.

Ashraf U, Zhu B, Ye J, Wan S, Nie Y, Chen Z, Cui M, Wang C, Duan X, Zhang H, Chen H, Cao S - J. Virol. (2016)

Bottom Line: Japanese encephalitis virus (JEV) can invade the central nervous system and consequently induce neuroinflammation, which is characterized by profound neuronal cell damage accompanied by astrogliosis and microgliosis.The pathological features of JEV-induced encephalitis are inflammatory reactions and neurological diseases resulting from glia activation.The present study reveals that miR-19b-3p targets ring finger protein 11 in glia and promotes inflammatory cytokine production by enhancing nuclear factor kappa B activity in these cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China.

Show MeSH

Related in: MedlinePlus

Inhibition of miR-19b-3p suppresses JEV-mediated production of inflammatory cytokines. (A) U251 cells were transfected with miR-19b-3p inhibitors or control oligonucleotides (final concentration, 50 nM) for 24 h and then either left uninfected or infected with JEV at an MOI of 5 for 36 h. The level of miR-19b-3p was analyzed by quantitative real-time PCR and normalized to the U6 level. **, P < 0.01; ***, P < 0.001. (B) The protein levels of TNF-α, IL-6, and IL-1β were analyzed by ELISA. Data represent means ± SD from three independent experiments performed in duplicate. *, P < 0.05; **, P < 0.01. CCL5 mRNA levels were determined by quantitative real-time PCR and normalized to the expression of β-actin in each sample. Data represent means ± SD from three independent experiments. *, P < 0.05; ***, P < 0.001. (C) The transfected U251 cells were infected with JEV at an MOI of 5. Cells were collected at the indicated time points, and titers of infectious virus in the culture supernatants were determined by plaque assay. The data represent three independent experiments with identical results. (D) BV2 cells were transfected with miR-19b-3p inhibitors or control oligonucleotides (final concentration, 50 nM) for 24 h and then either left uninfected or infected with JEV at an MOI of 5 for 36 h. The level of miR-19b-3p was analyzed by quantitative real-time PCR and normalized to the U6 level. **, P < 0.01. (E) The protein levels of TNF-α, IL-6, and IL-1β were analyzed by ELISA. Data represent means ± SD from three independent experiments performed in duplicate. *, P < 0.05. CCL5 mRNA levels were determined by quantitative real-time PCR and normalized to the expression of β-actin in each sample. Data represent means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836334&req=5

Figure 3: Inhibition of miR-19b-3p suppresses JEV-mediated production of inflammatory cytokines. (A) U251 cells were transfected with miR-19b-3p inhibitors or control oligonucleotides (final concentration, 50 nM) for 24 h and then either left uninfected or infected with JEV at an MOI of 5 for 36 h. The level of miR-19b-3p was analyzed by quantitative real-time PCR and normalized to the U6 level. **, P < 0.01; ***, P < 0.001. (B) The protein levels of TNF-α, IL-6, and IL-1β were analyzed by ELISA. Data represent means ± SD from three independent experiments performed in duplicate. *, P < 0.05; **, P < 0.01. CCL5 mRNA levels were determined by quantitative real-time PCR and normalized to the expression of β-actin in each sample. Data represent means ± SD from three independent experiments. *, P < 0.05; ***, P < 0.001. (C) The transfected U251 cells were infected with JEV at an MOI of 5. Cells were collected at the indicated time points, and titers of infectious virus in the culture supernatants were determined by plaque assay. The data represent three independent experiments with identical results. (D) BV2 cells were transfected with miR-19b-3p inhibitors or control oligonucleotides (final concentration, 50 nM) for 24 h and then either left uninfected or infected with JEV at an MOI of 5 for 36 h. The level of miR-19b-3p was analyzed by quantitative real-time PCR and normalized to the U6 level. **, P < 0.01. (E) The protein levels of TNF-α, IL-6, and IL-1β were analyzed by ELISA. Data represent means ± SD from three independent experiments performed in duplicate. *, P < 0.05. CCL5 mRNA levels were determined by quantitative real-time PCR and normalized to the expression of β-actin in each sample. Data represent means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.

Mentions: JE is characterized by stimulation of resident glial cells and the excessive release of inflammatory cytokines, which leads to neuronal cell death (8, 9). To examine whether miR-19b-3p is involved in the JEV-mediated inflammatory process, the effect of miR-19b-3p on the regulation of inflammatory cytokine production after JEV infection was determined. First, we evaluated the consequences of synthetic miR-19b-3p mimics and inhibitors on the expression pattern of miR-19b-3p. miRNA mimics are double-stranded RNAs synthesized to simulate naturally occurring mature miRNAs, whereas inhibitors are chemically modified antisense single-stranded RNAs that curb the yielding of endogenous miRNAs by sequence complementarity. miRNA mimics and inhibitors can cause fluctuations in the levels of the physiological miRNAs. As expected, transfection of miR-19b-3p mimics increased miR-19b-3p levels significantly in mock- or JEV-infected glial cells at 36 h postinfection (Fig. 2A and D), whereas miR-19b-3p inhibitors diminished its levels (Fig. 3A and D). Interestingly, certain cytokines of JEV-induced neuroinflammation, such as TNF-α, IL-6, IL-1β, and CCL5 (9, 36), were found to be significantly upregulated upon transfection of miR-19b-3p mimics in both infected and uninfected U251 cells (Fig. 2B). In contrast, the inhibition of endogenous miR-19b-3p significantly repressed JEV-triggered cytokine production (Fig. 3B). Furthermore, we found that treatment of miR-19b-3p mimics or inhibitors did not produce any antiviral activity in JEV-infected U251 cells as viral titers were similar to those in control cells (Fig. 2C and 3C). The effects of miR-19b-3p mimics and inhibitors on inflammatory cytokine production were also examined in BV2 cells, and the results were analogous to those observed in U251 cells (Fig. 2E and 3E). Thus, these data indicate that miR-19b-3p participates in regulating JEV-mediated inflammation.


MicroRNA-19b-3p Modulates Japanese Encephalitis Virus-Mediated Inflammation via Targeting RNF11.

Ashraf U, Zhu B, Ye J, Wan S, Nie Y, Chen Z, Cui M, Wang C, Duan X, Zhang H, Chen H, Cao S - J. Virol. (2016)

Inhibition of miR-19b-3p suppresses JEV-mediated production of inflammatory cytokines. (A) U251 cells were transfected with miR-19b-3p inhibitors or control oligonucleotides (final concentration, 50 nM) for 24 h and then either left uninfected or infected with JEV at an MOI of 5 for 36 h. The level of miR-19b-3p was analyzed by quantitative real-time PCR and normalized to the U6 level. **, P < 0.01; ***, P < 0.001. (B) The protein levels of TNF-α, IL-6, and IL-1β were analyzed by ELISA. Data represent means ± SD from three independent experiments performed in duplicate. *, P < 0.05; **, P < 0.01. CCL5 mRNA levels were determined by quantitative real-time PCR and normalized to the expression of β-actin in each sample. Data represent means ± SD from three independent experiments. *, P < 0.05; ***, P < 0.001. (C) The transfected U251 cells were infected with JEV at an MOI of 5. Cells were collected at the indicated time points, and titers of infectious virus in the culture supernatants were determined by plaque assay. The data represent three independent experiments with identical results. (D) BV2 cells were transfected with miR-19b-3p inhibitors or control oligonucleotides (final concentration, 50 nM) for 24 h and then either left uninfected or infected with JEV at an MOI of 5 for 36 h. The level of miR-19b-3p was analyzed by quantitative real-time PCR and normalized to the U6 level. **, P < 0.01. (E) The protein levels of TNF-α, IL-6, and IL-1β were analyzed by ELISA. Data represent means ± SD from three independent experiments performed in duplicate. *, P < 0.05. CCL5 mRNA levels were determined by quantitative real-time PCR and normalized to the expression of β-actin in each sample. Data represent means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836334&req=5

Figure 3: Inhibition of miR-19b-3p suppresses JEV-mediated production of inflammatory cytokines. (A) U251 cells were transfected with miR-19b-3p inhibitors or control oligonucleotides (final concentration, 50 nM) for 24 h and then either left uninfected or infected with JEV at an MOI of 5 for 36 h. The level of miR-19b-3p was analyzed by quantitative real-time PCR and normalized to the U6 level. **, P < 0.01; ***, P < 0.001. (B) The protein levels of TNF-α, IL-6, and IL-1β were analyzed by ELISA. Data represent means ± SD from three independent experiments performed in duplicate. *, P < 0.05; **, P < 0.01. CCL5 mRNA levels were determined by quantitative real-time PCR and normalized to the expression of β-actin in each sample. Data represent means ± SD from three independent experiments. *, P < 0.05; ***, P < 0.001. (C) The transfected U251 cells were infected with JEV at an MOI of 5. Cells were collected at the indicated time points, and titers of infectious virus in the culture supernatants were determined by plaque assay. The data represent three independent experiments with identical results. (D) BV2 cells were transfected with miR-19b-3p inhibitors or control oligonucleotides (final concentration, 50 nM) for 24 h and then either left uninfected or infected with JEV at an MOI of 5 for 36 h. The level of miR-19b-3p was analyzed by quantitative real-time PCR and normalized to the U6 level. **, P < 0.01. (E) The protein levels of TNF-α, IL-6, and IL-1β were analyzed by ELISA. Data represent means ± SD from three independent experiments performed in duplicate. *, P < 0.05. CCL5 mRNA levels were determined by quantitative real-time PCR and normalized to the expression of β-actin in each sample. Data represent means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.
Mentions: JE is characterized by stimulation of resident glial cells and the excessive release of inflammatory cytokines, which leads to neuronal cell death (8, 9). To examine whether miR-19b-3p is involved in the JEV-mediated inflammatory process, the effect of miR-19b-3p on the regulation of inflammatory cytokine production after JEV infection was determined. First, we evaluated the consequences of synthetic miR-19b-3p mimics and inhibitors on the expression pattern of miR-19b-3p. miRNA mimics are double-stranded RNAs synthesized to simulate naturally occurring mature miRNAs, whereas inhibitors are chemically modified antisense single-stranded RNAs that curb the yielding of endogenous miRNAs by sequence complementarity. miRNA mimics and inhibitors can cause fluctuations in the levels of the physiological miRNAs. As expected, transfection of miR-19b-3p mimics increased miR-19b-3p levels significantly in mock- or JEV-infected glial cells at 36 h postinfection (Fig. 2A and D), whereas miR-19b-3p inhibitors diminished its levels (Fig. 3A and D). Interestingly, certain cytokines of JEV-induced neuroinflammation, such as TNF-α, IL-6, IL-1β, and CCL5 (9, 36), were found to be significantly upregulated upon transfection of miR-19b-3p mimics in both infected and uninfected U251 cells (Fig. 2B). In contrast, the inhibition of endogenous miR-19b-3p significantly repressed JEV-triggered cytokine production (Fig. 3B). Furthermore, we found that treatment of miR-19b-3p mimics or inhibitors did not produce any antiviral activity in JEV-infected U251 cells as viral titers were similar to those in control cells (Fig. 2C and 3C). The effects of miR-19b-3p mimics and inhibitors on inflammatory cytokine production were also examined in BV2 cells, and the results were analogous to those observed in U251 cells (Fig. 2E and 3E). Thus, these data indicate that miR-19b-3p participates in regulating JEV-mediated inflammation.

Bottom Line: Japanese encephalitis virus (JEV) can invade the central nervous system and consequently induce neuroinflammation, which is characterized by profound neuronal cell damage accompanied by astrogliosis and microgliosis.The pathological features of JEV-induced encephalitis are inflammatory reactions and neurological diseases resulting from glia activation.The present study reveals that miR-19b-3p targets ring finger protein 11 in glia and promotes inflammatory cytokine production by enhancing nuclear factor kappa B activity in these cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China.

Show MeSH
Related in: MedlinePlus