Fig 2: miR-185-5p directly targets RAGE 3'-UTR. (A) Predicted targeting site of miR-185-5p to the 3'-UTRs of RAGE. (B) RAGE protein expression was tested in the indicated cells by western blotting. (C) and (D) Expression levels of RAGE mRNA were detected by reverse transcription-quantitative polymerase chain reaction in the indicated cells. (E) Luciferase activity of pGL3-RAGE 3'-UTR WT or pGL3-RAGE 3'-UTR-MUT report in MDA231 and MCF-7 cells. (F) and (G) Expression of vimentin and E-cadherin was examined in (F) MDA231 and (G) MCF-7. For (B), (F) and (G), the results of western blotting were from a representative of at least three repeated experiments. *P<0.05, as indicated. miR, microRNA; RAGE, advanced glycosylation end-product specific receptor; WT, wild-type; MUT, mutant; NC, negative control; UTR, untranslated region; si-, small interfering RNA; Scr, scramble.

Fig 3: Reduced inflammatory response in lung tissue in ALI by administration of TLR2/4 and RAGE antagonists. Histopathological changes and injury scores in left lung are illustrated for different groups: (A) Control group, LPS group, LPS-RS group, and LPS + LPS-RS group (H&E staining, ×200). (B) Control group, LPS group, FPS-ZM1 group and D: LPS+FPS-ZM1 group (H&E staining, ×200). Effects of (C) LPS-RS and (D) FPS-ZM1 on the lung W/D ratio of LPS-induced ALI mice. Effects of LPS-RS and on (E) MPO activity in lung tissues and (F) infiltration of inflammatory cells in BALF of LPS-induced ALI mice. All experiments were repeated more than three times (n=4-6 mice per each group). Data presented is from a representative experiment. All data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. LPS group. Reduced inflammatory response in lung tissue in ALI by administration of TLR2/4 and RAGE antagonists. Effects of FPS-ZM1 on (G) MPO activity in lung tissues and (H) infiltration of inflammatory cells in BALF of LPS-induced ALI mice. ELISA results of secretion of inflammatory cytokines (TNF-a, IL-6, MCP-1, IL-1ß and IL-18) treated with (I) LPS-RS and (J) FPS-ZM1 in BALF. All experiments were repeated more than three times (n=4-6 mice per each group). Data presented is from a representative experiment. All data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. LPS group. LPS, lipopolysaccharide; IL, interleukin; MCP, myeloperoxidase; TNF, tumor necrosis factor; ALI, acute lung injury; rHMGB1, recombinant High mobility group box 1; RT-q, reverse transcription-quantitative; NF, nuclear factor; BMMs, bone-marrow derived macrophages; TLR, toll-like receptor; RAGE, receptor for advanced glycation end products; BALF, bronchoalveolar lavage fluid; H&E, hematoxylin and eosin; RS, Rhodobacter sphaeroides; Mac, macrophage; Neu, neutrophil; Lym, lymphocyte.

Fig 4: miR-185-5p is downregulated in metastatic patient tissues and cells and is correlated with the expression of RAGE. (A) The expression of miR-185-5p was detected in MCF-7, T47D, MDA231, MDA453, SK-BR-3 and MCF-10A cells. (B) Relative expression of miR-185-5p was tested in 30 pairs of fresh frozen tumors tissues (T group) and their adjacent normal tissues (ANT group). (C) Expression of RAGE compared between paired T tissues and ANTs tissues. The results of western blotting were from a representative of at least three repeated experiments. (D) Correlation of expression between miR-185-5p and RAGE. miR, microRNA; T, tumor tissues; ANT, adjacent normal tissues; P, patient number; RAGE, advanced glycosylation end-product specific receptor.

Fig 5: Overexpression of miR-185-5p and reduction of RAGE inhibits lung metastasis in SCID mice. (A) Lung metastasis and hematoxylin and eosin staining of the respective tissues from SCID mice. Scale bars, 50 µm. (B) The number of lung metastasis nodes were calculated (n=10). (C) RAGE expression was detected in SCID xenograft tumors. The results of western blotting were from a representative of at least three repeated experiments. miR, microRNA; NC, negative control; SCID, Severe Combined Immune-deficiency; RAGE, advanced glycosylation end-product specific receptor; Scr, scramble.