MiR-140 increased the sensitivity of AECs to PTX.

<p>A: Morphological characteristics. Scale bars: 150 µm. TGF-β1 was added into miR-140- or ASO-140-transfected A549 cells for 24 h prior to PTX (50 nM) treatment. A549 cells treated with TGF-β1 or ASO-140 presented a fibroblast-like morphology, and the fibroblast-like morphology was reversed to epithelial-like characteristics in PTX- or PTX+miR-140-treated cultures. B: Western blot analysis. The levels of vimentin, Smad3 and p-Smad3 levels were increased and E-cadherin was downregulated in TGF-β1-treated A549 cells, which were reversed in PTX-, or PTX+miR-140-treated cells, especially in both PTX and miR-140-treated cultures. n = 3 replicates.</p

The sequences of chemically synthesized oligos.

a<p>The selected miRNAs were chemically synthesized in the form of small interfering RNA (siRNA) duplexes.</p>*<p>the bold and underlined letters were the mutation sites of miRNA.</p

PTX ameliorates pulmonary fibrosis andu pregulates miR-140 in fibrotic lungs.

<p>A: Upper, Haematoxylin and eosin (HE). The lungs initiated alveolar disruption on 7 d, and more severe on 28 d, while pulmonary fibrosis was ameliorated by PTX (0.6 mg/kg) treatment. Lower, Masson's trichrome for collagen and elastin evaluation. The lungs underwent a slight fibrotic invasion on 7 d, and more aggressive on 28 d, which was alleviated by PTX. Scale bars: 150 µm. B: Type-I collagen ELISA analysis. The type-I collagen level showed basally low in lung tissues of rats instilled with saline (sham control rats), similar to those in the untreated lungs (0 d). The collagen I levels in BLM-treated lung tissues on 28 d were increased seven folds than those in untreated or sham control lung tissues. Collagen I levels were reduced obviously in PTX-treated lungs compared to BLM-treated 28 d lung tissues. *<i>P</i><0.05 versus 28 d. C: Hydroxyproline analysis. BLM-treated lungs had much higher levels of hydroxyproline, but PTX treatment significantly decreased hydroxyproline levels compared to BLM-treated 28 d lungs. *<i>P</i><0.05 versus 28 d. D: Quantitative RT-PCR analysis of miR-140 expression. U6 was used as a control. n = 3 replicates. The miR-140 level in BLM-instilled rat lungs reached its nadir on 7 d, approximately two folds lower than that in untreated or sham control lung tissues, while it was dramatically upregulated in PTX-treated lung tissues. *<i>P</i><0.05 versus 0 d. **<i>P</i><0.05 versus PTX. E: miR-140 expression in human pulmonary fibrotic lungs. a–e: healthy control lungs, f–j: human pulmonary fibrosis lungs. The miR-140 levels are obvious lower in humanulmonary fibrotic lung tissues compared with those in healthy control lungs.</p

MiR-140 suppresses the TGF-β1/Smad3 pathway in A549 cells.

<p>A: Fluorescence microscope detection. Scale bars: 150 µm. B: Flow cytometry analysis. n = 3 replicates. Control, cells untreated with miRNA or plasmid; GFP-Smad3, cells treated with pcDNA-GFP-UTR; D-NC, cells treated with both D-NC (miR-140 mutation control) and pcDNA-GFP-UTR plasmid; S-NC, cells treated with both S-NC (the single-stranded RNA control) and pcDNA-GFP-UTR; miR-140, cells treated with both miRNA-140 and pcDNA-GFP-UTR; ASO-140, cells treated with both ASO-140 and pcDNA-GFP-UTR. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070725#pone-0070725-g004" target="_blank">Fig. 4 A, B</a>, GFP expression and the number of GFP-positive cells in miR-140-treated cultures were much lower than those in D-NC- or ASO-140-treated controls. C: Western blot analysis. The gene expression/α-tubulin ratios were shown on the right of gel. The miR-140 treatment led to an obvious reduction in the expression of TGF-β1, vimentin, Smad3 and p-Smad3 compared to the D-NC control groups. α-tubulin was used as loading controls. *<i>P</i><0.05 versus miR-140 treatment, **<i>P</i><0.05 versus ASO-140 treatment, n = 3 replicates.</p

MiR-140 affected EMT and Smad3/p-Smad3 expression.

<p>A: Morphological characteristics. Scale bars: 150 µm. A549 cells treated with TGF-β1 or TGF-β1+ D-NC underwent a fibroblast-like morphology, while their morphologies were reversed by miR-140 or miR-140 plus PTX treatment. B: Western blot analysis. α-tubulin was used as loading controls. The Smad3 and p-Smad3 levels were decreased obviously in miR-140- or PTX+miR-140-treated A549 cells, especially in PTX+miR-140-treated cells, compared to those in TGF-β1- or TGF-β1+D-NC-treated cultures. n = 3 replicates.</p

PTX suppresses the TGF-β1/Smad3 signaling pathway.

<p>A: Smad3 and p-Smad3 expression in A549 cells by western blot analysis. α-tubulin was used as loading controls. B: Quantitative RT-PCR analysis of Smad3 expression in A549 cells. GAPDH was used a control gene. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070725#pone-0070725-g003" target="_blank">In Fig. 3A,B</a>, TGF-β1-treatment increased the expression of p-Smad3 and Smad3 in A549 cells, which could be reduced by PTX treatment. *<i>P</i><0.05 versus TGF-β1 treatment group, n = 3 replicates. C: Immunohistochemical staining of Smad3, p-Smad3 and α-SMA in rat lung tissues. Black arrows, Smad3, p-Smad3 or α-SMA positive staining AECs. Stars, vasculature. Scale bars: 150 µm. A majority of Smad3, p-Smad3 and α-SMA staining was found to be localized in the AECs of fibrotic lungs. The BLM-untreated (0 d) or sham control lung tissues showed almost a total absence of α-SMA and p-Smad3. TGF-β1-treatment increased the expression of p-Smad3 and α-SMA in fibrotic lungs (7 d, 28 d), which was restored by PTX treatment to some extent. D: Western blot analysis. E: The ratios of Smad3 (p-Smad3, or α-SMA)/α-tubulin of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070725#pone-0070725-g003" target="_blank">Fig. 3D</a>. α-tubulin was used as the internal standard. n = 3 replicates. F: Quantitative RT-PCR analysis of Smad3 and α-SMA expressions in rat lung tissues (n = 3 replicates). GAPDH was used as a control gene. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070725#pone-0070725-g003" target="_blank">Fig. 3D–F</a>: the quantity of p-Smad3 in lung tissues reached peaked on day 7 and the quantity of α-SMA in lung tissues reached peaked on day 28 after BLM treatment. PTX could reduce the expression of p-Smad3 and α-SMA obviously, but had no effect on Smad3. *<i>P</i><0.05 versus 0 d. **<i>P</i><0.05 versus PTX.</p

Lung-to-body weight ratio, the pulmonary inflammation and fibrosis scores.

*<p><i>P</i><0.05 versus saline-treated control,</p>**<p><i>P</i><0.05 versus BLM-treated 28 d rats, n = 12.</p

Calcium Carbonate/Polydopamine Composite Nanoplatform Based on TGF‑β Blockade for Comfortable Cancer Immunotherapy

Cancer pain seriously reduces the quality of life of cancer patients. However, most research about cancer focuses solely on inhibiting tumor growth, neglecting the issue of cancer pain. Therefore, the development of therapeutic agents with both tumor suppression and cancer pain relief is crucial to achieve human-centered treatment. Here, the work reports curcumin (CUR) and ropivacaine (Ropi) coincorporating CaCO3/PDA nanoparticles (CaPNMCUR+Ropi) that realized efficient tumor immunotherapy and cancer pain suppression. The therapeutic efficiency and mechanism are revealed in vitro and in vivo. The results indicate that CaPNMCUR+Ropi underwent tumor microenvironment-responsive degradation and realized rapid release of calcium ions, Ropi, and CUR. The excessive intracellular calcium triggered the apoptosis of tumor cells, and the transient pain caused by the tumor injection was relieved by Ropi. Simultaneously, CUR reduced the levels of immunosuppressive factor (TGF-β) and inflammatory factor (IL-6, IL-1β, and TNF-α) in the tumor microenvironment, thereby continuously augmenting the immune response and alleviating inflammatory pain of cancer animals. Meanwhile, the decrease of TGF-β leads to the reduction of transient receptor potential vanilloid 1 (TRPV1) expression, thereby alleviating hyperalgesia and achieving long-lasting analgesic effects. The design of the nanosystem provides a novel idea for human-centered tumor treatment in the future