| Nano-sized silicon dioxide (SiO2) were used extensively in variety fields such as painting, cosmetics, catalysts, building materials and biomedicine. Human exposure to nano-sized SiO2 is inevitably increasing, but it is still vague about its biosecurity. Severe pneumonia or death of the animal was caused by nano-sized SiO2 after short-term exposure and pulmonary fibrosis after chronic or subchronic instillation. The actual exposure for workers may be long-term exposure in low dose, whether it can cause pulmonary fibrosis has not been definite based on insufficient evidence. We firmly believe that revealing the molecular mechanism of pulmonary fibrosis may provide a scientific basis to chronic or subchronic toxicity assessment of nano-sized SiO2 and early sensitive biomarkers and specific therapeutic options.Pulmonary fibrosis is a kind of irreversible diseases characterized by lung injury, alveolar structure destruction, fibroblasts concentration, uncontrolled extracellular matrix production and abnormal lung tissue structure remodeling induced by multi-pathogenesis. Lung fibroblasts and myofibroblasts may be derived from alveolar epithelial cells by Epithelial-Mesenchymal Transition (EMT), which is an important event in and development of pulmonary fibrosis. EMT were verified to associate with idiopathic pulmonary fibrosis (IPF) or pulmonary fibrosis induced by silicious dust, bleomycin (BLM), paraquat (PQ) and radioactive substances. A variety of cell growth factors, such as transforming growth factor beta (TGF-?), fibroblast growth factor (FGF), insulin-like growth factor (IGF), connective tissue growth factor (CTGF) are closely related to EMT, and TGF-? is generally recognized as a central mediator. TGF-? receptor type ? (TGF-? R ?) is one of important receptors in TGF-? pathway. Studies have shown that TGF-?? is very similar to TGF-? R ? in structure, which is considered as a coreceptor to increase the affinity of ligand binding to TGF-? R ? and TGF-? R ? and then enhance the role of TGF-? signaling pathway. Fibroblast growth factor 7 (FGF7) is a member of the FGF family, it has a biological effect on proliferation and differentiation of alveolar epithelial cell in acute lung injury. Over-expression of TGF-? ? and FGF7 is related to EMT in pulmonary fibrosis. MiRNAs (microRNAs, miRNAs) as a kind of post-transcriptional regulatory factors has been confirmed to be closely related to pulmonary fibrotic diseases on in recent years, they may play an important role in development of EMT. Our previous studies have confirmed that rats' pulmonary injury was induced by nano-sized SiO2 after endotracheal instillation administration, which mainly performed as pulmonary inflammation at 7d,15d and 30d and interstitial pulmonary fibrosis at 60d and 90d. Results of Illumina Hiseq 2000 high throughput sequencing showed that miR-541 was a down-regulated miRNA in nano-sized SiO2-treated lungs at 60d and 90d, miR-541 may play a significant role in development of pulmonary fibrosis induced by nano-sized SiO2 through regulating EMT. To elucidate the biological effects of over-expression of miR-541 and confirm the relationship among miR-541, TGF-? signaling pathway and TGF-?-mediated EMT, experiments in vivo and vitro cell were performed to explore its biological effects and molecular mechanisms. Results were showed as follows:1. Target genes prediction of miR-541 was performed by the Target-Scan and results showed that 83 predicted target genes may be regulated by miR-541. Gene enrichment and signal pathway analysis of 83 predicted target genes were performed by GO and KEGG database respectively. Results of functional classification by GO and pathway analysis by KEGG showed that miR-541 is obvious in regulating TGF-P receptor signaling pathway and cell differentiation. Combining with pathological results and bioinformatics analysis above, the predicted gene TGFBR3 related to TGF-P signal pathway and FGF7 that may be regulated by miR-541 were confirmed to further study. MiR-541 may have an important role in the differentiation of alveolar epithelial cells by regulating the TGF-P receptor signaling pathway and the FGF pathway.2. Results of RT-qPCR showed that the expression levels of miR-541 and E-cad mRNA in lungs of nano-sized SiO2 treated rats were significantly down-regulated at 7d,15d,30d,60d and 90d when compared with the control group (fold change?-2). Both of them have a diminishing tendency as administration time went on (P <0.05), and were significantly down-regulated at 60d and 90d when compared with 7d,15d and 30d (P<0.05). The expression levels of FGF7 mRNA were significantly up-regulated at all the administration time, TGF-?1? TGF-? R ? and COL ? mRNA were significantly up-regulated at 60d and 90d and a-SMA mRNA was significantly up-regulated at 30d,60d and 90d when compared with the control group(P<0.05). All of them have an increasing tendency as administration time went on (P<0.05), and were significantly down-regulated at 60d and 90d when compared with 7d,15d and 30d (P<0.05). Results of Western Blot showed that the expression level of E-cad protein in lungs of nano-sized SiO2 treated rats was significantly down-regulated at all the administration time when compared with the control group (P<0.05), which had a diminishing tendency as administration time went on (P<0.05), and were significantly down-regulated at 60d and 90d when compared with 7d,15d and 30d (P<0.05). The protein expression levels of TGF-? R?? FGF7? TGF-?1??-SMA?COL ? were all were significantly up-regulated at 30d,60d and 90d when compared with the control group (P<0.05). And all of them were significantly down-regulated at 60d and 90d when compared with 7d,15d and 30d (P< 0.05).The result of Dual Luciferase Reporter Assay revealed that respective co-transfection of miR-541 mimic and mimic NC with the luciferase reporter carrying a portion of the human TGF-? R ? and FGF7 3'-UTR of wild type to A549 cells can significantly reduce the luciferase activity level at (65±9)% and (35±4)%, respectively. But respective co-transfection of miR-541 and mimic NC with the luciferase reporter carrying the mutated TGF-P R ? and FGF7 3'-UTR failed to diminish the luciferase activity level when compared with its control (P>0.05). These data demonstrated that TGF-? R ? and FGF7 were target genes of miR-541, they were directly regulated by miR-541 in a post-transcriptional manner.3. A549 cells changed from epithelial-like cells into spindle-shaped fibroblast cells after 5ng/ml TGF-?1induced for 48h. Results of RT-qPCR and Western Blot showed that the expression of miR-541 and E-cad in mRNA and protein were significantly down-regulated (fold change?-2), whereas ?-SMA?COL ??TGF-? R ??FGF7 in mRNA and protein were significantly up-regulated when compared with the control group (P<0.05). But there was no observation on morphological change of A549 cells for 5ng/ml TGF-?1 treatment 48h after TGF-?1 receptor blocking 3h. And the expression of miR-541 and E-cad, ?-SMA, COL ?, TGF-?R ?, FGF7 in mRNA and protein were not statistically significant when compared with the control group (P>0.05). After respective transient transfection of miR-541 mimic, mimic NC, miR-541 inhibitors and inhibitor NC for 24 hours and TGF-?1 treatment for 48 hours, there was no observation on morphological change in all the transfected and TGF-?1-induced A549 cells. Results of MTT assay showed that cell proliferation rate was (115±7)%, (106±3)%, (97±8)% and (103±3)%, respectively, there is no statistically significant differences between transfected groups and control group (P>0.05). Results of RT-qPCR and Western Blot showed that the expression of miR-541 and E-cad in mRNA and protein in miR-541 inhibitor-transfected group were significantly down-regulated (fold change?-2), whereas ?-SMA?COL ??TGF-? R ??FGF7 in mRNA and protein were significantly up-regulated (P<0.05). The expression of miR-541was significantly up-regulated (fold change?-2), TGF-?R ? mRNA, FGF7 mRNA and protein were significantly down-regulated (P<0.05) and E-cad??-SMA and COL ? in mRNA and protein were not statistically significant in miR-541 mimic-transfected group (P>0.05). All the expression of miR-541 and TGF-? R ?, FGF7, E-cad??-SMA and COL ? in mRNA and protein were not statistically significant in mimic NC-transfected group and inhibitor NC-transfected group (P>0.05). Those data indicated thatTGF-?1 promote the development of EMT by suppressing the expression of miR-541 and over-expression of miR-541 attenuated TGF-? pathway and FGF pathway to exhibit its effect of EMT.In summary, our study showed that EMT was implicated in the process of pulmonary fibrosis induced by nano-sized SiO2, TGF-? R ? and FGF7 were target genes of miR-541. The relationship between miR-541 and TGF-? pathway was closely related, TGF-?1 promoted the development of EMT by suppressing the expression of miR-541 and over-expression of miR-541 attenuated TGF-? pathway and FGF pathway to exhibit its effect of inhibiting pulmonary fibrosis by suppressing the expression of TGF-?R ? and FGF7. |
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