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. 2016 Nov 22;7(47):77978-77986.
doi: 10.18632/oncotarget.12866.

miR-135b-5p Inhibits LPS-induced TNFα Production via Silencing AMPK Phosphatase Ppm1e

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Free PMC article

miR-135b-5p Inhibits LPS-induced TNFα Production via Silencing AMPK Phosphatase Ppm1e

Ping Li et al. Oncotarget. .
Free PMC article

Abstract

AMPK activation in monocytes could suppress lipopolysaccharide (LPS)-induced tissue-damaging TNFa production. We are set to provoke AMPK activation via microRNA ("miRNA") downregulating its phosphatase Ppm1e. In human U937 and THP-1 monocytes, forced expression of microRNA-135b-5p ("miR-135b-5p") downregulated Ppm1e and activated AMPK signaling. Further, LPS-induced TNFα production in above cells was dramatically attenuated. Ppm1e shRNA knockdown in U937 cells also activated AMPK and inhibited TNFα production by LPS. AMPK activation is required for miR-135b-induced actions in monocytes, AMPKα shRNA knockdown or T172A dominant negative mutation almost abolished miR-135b-5p's suppression on LPS-induced TNFα production. Significantly, miR-135b-5p inhibited LPS-induced reactive oxygen species (ROS) production, NFκB activation and TNFα mRNA expression in human macrophages. AMPKα knockdown or mutation again abolished above actions by miR-135b-5p. We conclude that miR-135b-5p expression downregulates Ppm1e to activate AMPK signaling, which inhibits LPS-induced TNFα production via suppressing ROS production and NFκB activation.

Keywords: AMPK; LPS; Ppm1e; TNFα; miR-135b-5p.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Expression of miR-135b-5p downregulates Ppm1e in human macrophages
A. miR-135b-5p complements Ppm1e's 3′ untranslated regions (UTRs, position 517-524) (A). Human U937 or THP-1 macrophages were transfected with miR-135b-5p construct or non-sense control microRNA (“miR-C”), and stable cells were established via neomycin selection. Expression of miR-135b-5p B and D. and Ppm1e mRNA (C and E., left panels) were tested by quantitative real-time PCR (“RT-qPCR”) assay; Ppm1e protein expression was examined by Western blot assay (C, right panels). Experiments in this figure were repeated three times, and similar results were obtained. Ppm1e protein expression (vs. β-actin) was quantified (C). “Ctrl” stands for non-transfected control cells (B-E). *p<0.05 vs. “miR-C” group (B-E).
Figure 2
Figure 2. miR-135b-5p activates AMPK signaling and inhibits LPS-induced TNFα production in human macrophages
Stably U937 macrophages expressing miR-135b-5p (two lines, “Line-1/-2”) or non-sense control microRNA (“miR-C”) were subjected to Western blot assay of phosphorylated- (“p”) and regular AMPKα and ACC A. Above cells were treated with LPS (100 ng/mL) or medium control (“C”) for 24 hours, TNFα content in conditional medium was tested by ELISA assay B. Experiments in this figure were repeated for three times, and similar results were obtained. AMPKα and ACC phosphorylations were quantified (A). “Ctrl” stands for un-transfected control cells. * p<0.05 vs. group “C” (B). # p< 0.05 vs. LPS treatment of “miR-C” group (B).
Figure 3
Figure 3. Ppm1e shRNA knockdown activates AMPK and inhibits LPS-induced TNFα production in human macrophages
Stably U937 cells expressing Ppm1e shRNA (“shPpm1e-1” or “shPpm1e-2”, with non-overlapping sequences) or scramble control shRNA (“sh-C”) were subjected to Western blot assay of listed proteins A. or RT-qPCR assay of miR-135b-5p and Ppm1e mRNA B. Above cells were treated with LPS (100 ng/mL) or medium control (“C”) for 24 hours, TNFα production was tested by ELISA assay C. U937 cells with shPpm1e-1 were also transfected with miR-135b-5p construct, and stably cells were again established; miR-135b-5p D. and Ppm1e mRNA E. expressions were tested by RT-qPCR assay. Above cells were treated with LPS (100 ng/mL) for 24 hours, TNFα production was measured F. Ppm1e expression (vs. Erk1/2) and AMPKα phosphorylation were quantified (A). “Ctrl” stands for un-transfected control cells. Experiments in this figure were repeated for three times, and similar results were obtained. # p < 0.05 vs. “sh-C” group (B-F). * p < 0.05 vs. “C” group (C and F).
Figure 4
Figure 4. AMPK activation is required for miR-135b-5p's inhibition on LPS-induced TNFα production
miR-135b-5p expressing U937 cells were constructed with AMPKα shRNA (“shAMPKα-No.1”/“shAMPKα-No.2”) or scramble control shRNA (“sh-C”), and stably cells were established; Expressions of listed proteins in these cells were tested by Western blot assay A.; Cells were treated with LPS (100 ng/mL) for 24 hours, TNFα production was tested B. miR-135b-5p expressing U937 cells were constructed with dominant negative AMPKα (T172A, “dnAMPKα”, GFP-tagged) or empty vector (“pSuper-puro”), stably cells were established; Expressions of listed proteins in these cells were tested by Western blot assay C.; LPS-induced TNFα production was also examined D. Experiments in this figure were repeated for three times, and similar results were obtained. AMPKα and ACC phosphorylations were quantified (vs. regular ACC, A and C). “Ctrl” stands for un-transfected control cells (B and D). # p < 0.05 vs. “sh-C” (B). # p < 0.05 vs. “Vector” (D).
Figure 5
Figure 5. miR-135b-5p inhibits LPS-induced ROS production, NFκB activation and TNFα mRNA expression
miR-135b-5p expressing U937 cells were constructed with scramble control shRNA (“sh-C”), AMPKα shRNA (“shAMPKα”, No.1) or dominant negative AMPKα (T172A, “dnAMPKα”), these cells or the control U937 cells were treated with LPS (100 ng/mL) or medium control (“C”) for applied time, relative ROS intensity A., NFκB activation B and C. and TNFα mRNA expression D. were tested by listed assays. Experiments in this figure were repeated for three times, and similar results were obtained. IKKα/β phosphorylation was quantified (B). *p < 0.05 vs. “C” group (A, C and D). # p < 0.05 vs. LPS only group (A, C and D). ** p < 0.05 (A, C and D).

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