Figure 5
Figure 5. miR-142-3p regulates IL-6 expression in vivo. To analyze the effect of miR-142-3p, B6 mice were injected with either high-affinity LNA-anti–miR-142-3p with phosphorothioate modifications or scrambled control at various doses. The splenocytes were then harvested and used for the analyses of the expression of miR-142-3p as described in “Methods.” (A) Dose-dependent in vivo knockdown of miR-142-3p by anti–miR-142-3p in splenocytes measured by TaqMan quantitative RT-PCR. Data shown are from 1 of 2 experiments with 3-4 mice/group (mean ± SEM). (B) anti–miR-142-3p specifically knocked down miR-142-3p but did not affect off-target miRs. Expression levels of miR-142-3p and other miRs such as miR-142-5p or miR-155 were measured by TaqMan quantitative RT-PCR. Data shown are from 1 of 2 experiments with 3 mice/group (mean ± SEM). (C) In vivo knockdown of miR-142-3p by anti–miR-142-3p de-repressed LPS-induced IL-6 expression at both mRNA and protein levels: IL-6 mRNA levels in splenocytes were determined by quantitative RT-PCR and the protein levels was measured in the sera by ELISA. Data shown are from 1 of 2 experiments with 4-5 mice/group. (D) In vivo knockdown of miR-142-3p by anti–miR-142-3p does not affect LPS-induced TNFα expression at both mRNA and protein levels: TNFα mRNA levels in splenocytes were determined by quantitative RT-PCR and the protein levels was measured in the sera by ELISA of IL-6−/− and WT B6 mice following administration of LPS. Data shown are from 2 experiments with 6 mice/group. (E) Silencing of mir-142-3p mitigated endotoxin-induced mortality. Treatment of mice with LNA-modified oligonucleotide complementary to miR-142-3p (anti–miR-142-3p) followed by LPS injection (■) significantly reduced mortality compared with animals that were treated with scrambled anti-miR and LPS (●). P = .029. Data shown are combined from 2 different experiments with similar results. (F) Silencing of mir-142-3p does not alter endotoxin-induced mortality in IL-6−/− mice. Treatment of IL-6−/− mice with LNA-modified oligonucleotide complementary to miR-142-3p followed by LPS injection (dotted line, n = 6) did not affect the rate and overall mortality compared with IL-6−/− animals that were treated with scrambled anti-miR and LPS (solid line, n = 6). P = NS.

miR-142-3p regulates IL-6 expression in vivo. To analyze the effect of miR-142-3p, B6 mice were injected with either high-affinity LNA-anti–miR-142-3p with phosphorothioate modifications or scrambled control at various doses. The splenocytes were then harvested and used for the analyses of the expression of miR-142-3p as described in “Methods.” (A) Dose-dependent in vivo knockdown of miR-142-3p by anti–miR-142-3p in splenocytes measured by TaqMan quantitative RT-PCR. Data shown are from 1 of 2 experiments with 3-4 mice/group (mean ± SEM). (B) anti–miR-142-3p specifically knocked down miR-142-3p but did not affect off-target miRs. Expression levels of miR-142-3p and other miRs such as miR-142-5p or miR-155 were measured by TaqMan quantitative RT-PCR. Data shown are from 1 of 2 experiments with 3 mice/group (mean ± SEM). (C) In vivo knockdown of miR-142-3p by anti–miR-142-3p de-repressed LPS-induced IL-6 expression at both mRNA and protein levels: IL-6 mRNA levels in splenocytes were determined by quantitative RT-PCR and the protein levels was measured in the sera by ELISA. Data shown are from 1 of 2 experiments with 4-5 mice/group. (D) In vivo knockdown of miR-142-3p by anti–miR-142-3p does not affect LPS-induced TNFα expression at both mRNA and protein levels: TNFα mRNA levels in splenocytes were determined by quantitative RT-PCR and the protein levels was measured in the sera by ELISA of IL-6−/− and WT B6 mice following administration of LPS. Data shown are from 2 experiments with 6 mice/group. (E) Silencing of mir-142-3p mitigated endotoxin-induced mortality. Treatment of mice with LNA-modified oligonucleotide complementary to miR-142-3p (anti–miR-142-3p) followed by LPS injection (■) significantly reduced mortality compared with animals that were treated with scrambled anti-miR and LPS (●). P = .029. Data shown are combined from 2 different experiments with similar results. (F) Silencing of mir-142-3p does not alter endotoxin-induced mortality in IL-6−/− mice. Treatment of IL-6−/− mice with LNA-modified oligonucleotide complementary to miR-142-3p followed by LPS injection (dotted line, n = 6) did not affect the rate and overall mortality compared with IL-6−/− animals that were treated with scrambled anti-miR and LPS (solid line, n = 6). P = NS.

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