MiR-344b-1-3p targets TLR2 and negatively regulates TLR2 signaling pathway

Figures & data

Table 1 Primer pairs of miRNA used for the RT-PCR

Gene	Top	Bottom
rno-miR-146a-5p	5′-GGGTGAGAACTGAATTCC-3′	5′-TGCGTGTCGTGGAGTC-3′
rno-miR-19b-3p	5′-GGGGGGTGTGCAAATCC-3′	5′-GTGCGTGTCGTGGAGTCG-3′
rno-miR-124-3p	5′-GGGTAAGGCACGCGGT-3′	5′-GTGCGTGTCGTGGAGTCG-3′
rno-miR-363-5p	5′-GCGGGTGGATCACGATG-3′	5′-GTGCGTGTCGTGGAGTCG-3′
rno-miR-9a-3p	5′-GGGGGGATAAAGCTAGATAACC-3′	5′-GTGCGTGTCGTGGAGTCG-3′
rno-miR-344b-1-3p	5′-GGGGTGATATAACCAAAGCC-3′	5′-GTGCGTGTCGTGGAGTCG-3′
rno-miR-125b-5p	5′-GCTCCCTGAGACCCTAAC-3′	5′-CAGTGCGTGTCGTGGAGT-3′

Abbreviations: miRNA, microRNA; RT-PCR, real-time polyerase chain reaction.

Table 2 The results of lung function tests analysis (n=5, mean ± SD)

Parameter	Control	COPD	T	P-value
MVV (mL)	111.04±2.92	108.8±2.86	0.960	0.392
Re (kPa s/L)	27.00±0.84	51.32±2.97*	−15.773	0.000
Ri (kPa s/L)	26.70±1.52	43.10±1.80*	−16.167	0.000
PEF (mL/s)	21.46±1.65	20.42±1.07	5.770	0.494
FEV0.3/FVC (%)	87.40±0.91	77.58±1.76*	9.541	0.001

Note:

* P<0.05 versus control group.

Abbreviations: FEV_0.3, forced expiratory volume in 0.3 second; MVV, minute ventilation volume; PEF, peak expiratory flow; Re, expiratory resistance; Ri, inspiratory resistance; SD, standard deviation.

Figure 1 CT analysis and histopathology examination of COPD rats.

Notes: The rats were treated with cigarette smoke and saline tracheal instillation for 90 days. Coronal CT images obtained in the control group (A) and in the COPD group (B). Histological examination of lungs in control-treated rats (C) and cigarette smoke-treated rats (D).

Figure 2 Expression profile and validation of selected miRNAs in AMs from COPD rats.

Notes: (A) miRNA arrays of AMs. The expression change indicated by the color bar was calculated by comparing the miRNA expression levels in AMs from COPD rats with those in control. Only differentially expressed miRNAs pass fold change filtering (≥2-fold change). The values presented are the mean of triplicate samples. (B) Comparison of expression levels of seven differentially expressed miRNAs in microarray and real-time PCR assay. Data are presented as mean ± SD of three independent tests with triplicate samples. The miRNA expression was normalized against an endogenous U6 RNA control.
Abbreviations: AMs, alveolar macrophages; miRNA, microRNA; qPCR, quantitative polymerase chain reaction; PCR, polymerase chain reaction; SD, standard deviation.

Figure 3 TLR2 is a direct target of miR-344b-1-3p.

Notes: psiCHECKTM-2 luciferase construct containing a WT or MUT TLR2-3′-UTR was cotransfected with NC, miR-344b-1-3p, NC inhibitor, or miR-344b-1-3p inhibitor. All experiments were repeated independently three times. Significant difference, **P<0.01.
Abbreviations: WT, wild type; MUT, mutated; miRNA, microRNA; hsa, homo sapien; NC, negative control.

Figure 4 Effect of CSE on NR8383 cell apoptosis.

Notes: (A) Dot plots and (B) histogram of NR8383 cells exposed to CSE at 5%, 10%, 15% and 0% for 24 hours before being harvested for apoptosis test. The detection was repeated independently three times. Significantly different from the control group: *P<0.05, **P<0.01, ***P<0.001.

Abbreviations: CSE, cigarette smoke extract; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 5 Effect of CSE on miR-344b-1-3p expression as detected by real-time PCR.

Notes: MiR-344b-1-3p level was detected after 24-hour exposure to CSE at the concentrations of 0%, 5%, 10%, and 15%. The detection was repeated independently three times. Significantly different from the control group, **P<0.05. ***P<0.01.
Abbreviations: CSE, cigarette smoke extract; miRNA, microRNA; PCR, polymerase chain reaction.

Figure 6 Effect of miR-344b-1-3p on TLR2 expression in NR8383 cells exposed to 10% CSE.

Notes: (A) Quantitative analysis results of RT-PCR validation of TLR2 mRNA levels. The expression of miR-334b-1-3p suppressed the activity of TLR2 even with the administration of TLR2 agonist. Once miR-334b-1-3p was suppressed, the expression of TLR2 was induced. Significantly different from the control group, *P<0.05. (B) Representative images of flow cytometry validation of TLR2 protein expression. A, 10% CSE-treated NR8383 cells administrated with miRNA-344b-1-3p inhibitor and 1 μg/mL of Pam3CSK4; B, 10% CSE-treated NR8383 cells administrated with control inhibitor and 1 μg/mL of Pam3CSK4; C, 10% CSE-treated NR8383 cells administrated with 1 μg/mL of Pam3CSK4.

Abbreviations: CSE, cigarette smoke extract; GAPDH, reduced glyceraldehyde-phosphate dehydrogenase; miRNA, microRNA; RT-PCR, real time polymerase chain reaction; FITC, fluorescein isothiocyanate.

Figure 7 Effect of miR-344b-1-3p inhibitor on the expressions or phosphorylation of downstream proteins of TLR2 in vitro.

Notes: The levels of (A) TNF-α, (B) IL-1β, and (C) MIP-2. The expression of miR-334b-1-3p suppressed the activity of all the indicators even with the administration of TLR2 agonist. Once miR-334b-1-3p was suppressed, the expression of those indicators was induced. (D) Representative images of Western blotting of p-IκBα, p-IRAK-1, and p-ERK. GAPDH functioned as a reference protein. (E) The quantitative analysis results of western blotting of p-IκBα, p-IRAK-1, and p-ERK. A, 10% CSE-treated NR8383 cells administrated with miRNA-344b-1-3p inhibitor and 1 μg/mL of Pam3CSK4; B, 10% CSE-treated NR8383 cells administrated with control inhibitor and 1 μg/mL of Pam3CSK4; C, 10% CSE-treated NR8383 cells administrated with 1 μg/mL of Pam3CSK4. **P<0.01, ***P<0.001.
Abbreviations: CSE, cigarette smoke extract; GAPDH, reduced glyceraldehyde-phosphate dehydrogenase; miRNA, microRNA.