MicroRNA-7 negatively regulates Toll-like receptor 4 signaling pathway through FAM177A

Abstract

Toll-like receptor (TLR) 4 signalling is critical for innate immunoinflammatory response and widely triggers the development of various types of clinical diseases. MicroRNA-7 (miR-7) is well documented to play an important regulatory role in various biological events. However, the exact role of miR-7 in TLR4 signalling pathway remains to be fully elucidated. In the present study, we found that miR-7 expression in TLR4 signalling-activated bone marrow-derived macrophages (BMDMs) stimulated by LPS was dramatically increased. Importantly, miR-7 deficiency significantly enhanced the production of related inflammatory cytokines including IL-1β, IL-6 and IL-12, as well as TNF-α, on LPS-activated BMDMs, accompanied by elevated transduction of TLR4 signalling including Myd88-dependent and Myd88-independent pathways, whereas miR-7 overexpression significantly decreased the transduction of TLR4 signalling and the production of related inflammatory cytokines. Mechanistically, we identified family with sequence similarity 177, member A (FAM177A) as a novel target molecule of miR-7. Furthermore, down-regulation of FAM177A using RNAi could impair the transduction of TLR4 signalling. Finally, down-regulation of FAM177A also reversed the effect of miR-7 deficiency on TLR4 signalling transduction and production of related inflammatory cytokines on BMDMs. Therefore, we provide the new evidence that miR-7 acts as a novel negative fine-tuner in regulating TLR4 signalling pathways by targeting FAM177A, which might throw light on the basal understanding on the regulatory mechanism of TLR4 signalling and benefit the development of therapeutic strategies against related clinical diseases.

Keywords: FAM177A; LPS; TLR4; macrophages; microRNA-7.

Figure 1

The expression of miR‐7 is up‐regulated during TLR4 signalling activation. Bone marrow cells were extracted from C57BL/6 WT mice (aged 8–10 weeks, n = 8) and then cultured in the presence of 20 ng/ml GM‐CSF in vitro for 7 days to induce BMDMs. (A) Schematic diagram of experiment. (B) BMDMs were treated with LPS (100 ng/ml, Escherichia coli 0111: B4; Sigma) for 12 h, and then, the morphology change was observed (magnification: 100×, 200× and 400×). (C) BMDMs were treated with different concentrations of LPS (0, 50, 100 or 200 ng/ml) for 12 h. The relative expression levels of cytokines IL‐1β, IL‐6, IL‐12 and TNF‐α, as well as INOS, were detected by real‐time PCR assay and calculated, respectively. (D) The relative expression level of miR‐7 in BMDMs, which were treated with different concentrations of LPS (0, 50, 100 or 200 ng/ml), was determined by real‐time PCR assay and calculated, respectively. (E) The relative expression level of miR‐7 in BMDMs, which were treated with 100 ng/ml of LPS for 0, 6, 12 or 24 h, was determined by real‐time PCR assay and calculated, respectively. (F) RAW 264.7 cells were treated with 100 ng/ml of LPS for 12 h. Then, the relative expression level of miR‐7 was determined by real‐time PCR assay and calculated, respectively. Representative data of three independent experiments are shown. *p < 0.05, **p < 0.01.

Figure 2

miR‐7 deficiency promotes the transduction of TLR4 signalling including Myd88‐dependent and Myd88‐independent pathways. Bone marrow cells were extracted from C57BL/6 miR‐7^def and WT mice (aged 8–10 weeks, n = 8) and then cultured in the presence of 20 ng/ml GM‐CSF in vitro for 7 days to induce BMDMs. (A) BMDMs from miR‐7^def and WT mice were treated with 100 ng/ml LPS for 12 h, and then, the morphology change was observed (magnification: 100×, 200× and 400×). (B) The relative expression level of miR‐7 in BMDMs from miR‐7^def and WT mice was detected by real‐time PCR assay and calculated, respectively. The mRNA levels of cytokines including IL‐1β (C), IL‐6 (D) and IL‐12 (E), as well as TNF‐α (F), in BMDMs were detected by real‐time PCR assays and calculated, respectively. The protein levels of cytokines including IL‐1β (G), IL‐6 (H) and IL‐12 (I), as well as TNF‐α (J), in cultural supernatants of BMDMs were detected by ELISAs and calculated, respectively. The protein levels of signalling molecules including TLR4, Myd88 and TRIF, as well as TRAF6, in cells were detected by Western blot assay (K) and calculated (L), respectively. The protein levels of signalling molecules including NF‐κB p65, phos‐NF‐κB p65, Akt, phos‐Akt, Erk1/2, phos‐Erk1/2 and JNK, as well as phos‐JNK, in cells were detected by Western blot assay (M) and calculated (N), respectively. phosphorylation = phos‐ = p‐. Representative data of three independent experiments are shown. *p < 0.05, **p < 0.01.

Figure 3

Overexpression of miR‐7 represses the transduction of TLR4 signalling including Myd88‐dependent and Myd88‐independent pathways. RAW 264.7 cells were infected with lentiviruses encoding miR‐7 (LV‐miR‐7^OE) or negative control lentiviruses (LV‐NC) and then cultured in the presence of Puromycin (6 μg/ml) for 15 days. (A) Schematic diagram of experiment. (B) The expression level of miR‐7 in RAW 264.7 cells was detected by real‐time PCR assays and calculated, respectively. Cells were stimulated by 100 ng/ml LPS for 12 h. Then, the mRNA levels of cytokines IL‐1β (C), IL‐6 (D) and IL‐12 (E), as well as TNF‐α (F), in RAW 264.7 cells were detected by real‐time PCR assays and calculated, respectively. The protein levels of cytokines IL‐1β (G), IL‐6 (H) and IL‐12 (I), as well as TNF‐α (J), in cultural supernatants of cells were detected by ELISAs and calculated, respectively. The protein levels of signalling molecules, including TLR4, Myd88 and TRIF, as well as TRAF6, were detected by Western blot assay (K) and calculated (L), respectively. The protein levels of signalling molecules, such as NF‐κB p65, phos‐NF‐κB p65, Akt, phos‐Akt, Erk1/2, phos‐Erk1/2 and JNK, as well as phos‐JNK, were detected by Western blot assay (M) and calculated (N), respectively. phosphorylation = phos‐ = p. Representative data of three independent experiments are shown. **p < 0.01.

Figure 4

FAM177A is a novel target of miR‐7. BMDMs from C57BL/6 miR‐7^def and WT mice (aged 8–10 weeks, n = 8) were treated with 100 ng/ml LPS for 12 h. Then, the global gene expression in BMDMs was analysed by RNA sequencing technology. (A) Heatmap. (B) The candidate target genes of miR‐7 in BMDMs were screened by bioinformatic prediction software (TargetScan, MiBase, etc.) and Wayne analysis. (C) Heatmap of above‐mentioned candidate target genes of miR‐7. (D) The expression change of the above candidate target genes in BMDMs before and after TLR4 signalling activation was determined by real‐time PCR assays and calculated, respectively. (E) Prediction of the binding site of FAM177A mRNA 3′‐UTR and miR‐7. (F) Luciferase assay. Representative data of three independent experiments are shown. *p < 0.05, **p < 0.01.

Figure 5

Down‐regulation of FAM177A impairs the transduction of TLR4 signalling. (A) RAW 264.7 cells were stimulated by 100 ng/ml LPS (Escherichia coli 0111: B4; Sigma) for 12 h. Then, the expression of FAM177A (green, arrow indication) was analysed by immunofluorescence; DAPI (blue). (B) Schematic diagram of experiment. RAW 264.7 cells were infected with FAM177A shRNA lentiviruses (LV‐FAM177A^shRNA) or negative control shRNA lentiviruses (LV‐NC^shRNA) and then were cultured in the presence of Puromycin (6 μg/ml) for 15 days. (C) The expression level of FAM177A in cells was detected by real‐time PCR assays and calculated, respectively. (D) The protein level of FAM177A in cells was detected by Western blot assay and calculated, respectively. The mRNA levels of cytokines including IL‐1β (E), IL‐6 (F) and IL‐12 (G), as well as TNF‐α (H), in cells were detected by real‐time PCR assays and calculated, respectively. The protein levels of cytokines including IL‐1β (I), IL‐6 (J) and IL‐12 (K), as well as TNF‐α (L), in cultural supernatants of cells were detected by ELISAs and calculated, respectively. The protein levels of signalling molecules including TLR4, Myd88 and TRIF, as well as TRAF6, were detected by Western blot assay (M) and calculated (N), respectively (bands were cropped to show 1 population). The protein levels of signalling molecules including NF‐κB p65, phos‐NF‐κB p65, Akt, phos‐Akt, Erk1/2, phos‐Erk1/2 and JNK, as well as phos‐JNK, were detected by Western blot assay (O) and calculated (P), respectively (bands were cropped to show 1 population). phosphorylation = phos‐ = p‐. Representative data of three independent experiments are shown. *p < 0.05, **p < 0.01.

Figure 6

Down‐regulation of FAM177A reverses the effect of miR‐7 deficiency on TLR4 signalling transduction. BMDMs from miR‐7^def mice were infected with LV‐FAM177A^shRNA or LV‐NC^shRNA. (A) Schematic diagram of experiment. (B) The expression level of FAM177A in cells was detected by real‐time PCR assays and calculated, respectively. (C) The protein level of FAM177A in cells was detected by Western blot assay and calculated, respectively. BMDMs from miR‐7^def mice were infected with LV‐FAM177A^shRNA or LV‐NC^shRNA. Then, cells were stimulated by 100 ng/ml LPS for 12 h. The protein levels of signalling molecules including TLR4, Myd88 and TRIF, as well as TRAF6, were detected by Western blot assay (D) and calculated (E), respectively. The protein levels of signalling molecules including NF‐κB p65, phos‐NF‐κB p65, Akt, phos‐Akt, Erk1/2, phos‐Erk1/2 and JNK, as well as phos‐JNK, were detected by Western blot assay (F) and calculated (G), respectively. The mRNA levels of cytokines including IL‐1β (H), IL‐6 (I) and IL‐12 (J), as well as TNF‐α (K), were detected by real‐time PCR assays and calculated, respectively. The protein levels of cytokines including IL‐1β (L), IL‐6 (M) and IL‐12 (N), as well as TNF‐α (O), in cultural supernatants of cells were detected by ELISAs and calculated, respectively. phosphorylation = phos‐ = p‐. Representative data of three independent experiments are shown. **p < 0.01.