Mechanism Study On Effect Of MiR-702-3p Regulating TGF-?1 In Pulmonary Epithelial-mesenchymal Transition Induced By Nanosized SiO₂

Nanosized silicon dioxide?SiO₂?,named white carbon,were extensively used fields such as food,paint,rubber,cosmetics and medicine because of its special physical and chemical properties,having the reputation of ndustrial monosodium glutamate.China is one of the world's largest producer of white carbon,and with the increasing application of nano-SiO₂,more and more people come into contact with it,and its potential harm can't be ignored.Animal studies have shown that,Nanosized SiO₂ can cause rat/mice lung inflammatory reaction and lung fibrosis after entering the body by respiratory tract.The mechanism of the effect of nanosized SiO₂ on pulmonary fibrosis has not yet been fully understood.Epithelial-mesenchymal transition?EMT?is an important link in the process of pulmonary fibrosis.It has been reported that the abnormal expression of miRNAs is related to the development of EMT,Let-7d,miR-192,miR-124,miR-26a,miR-10b and miR-487a may play important regulatory roles in the development of EMT through TGF-(31 signaling pathway.TGF-?1 can induce EMT by Smads dependent manner,stimulate the transformation of pulmonary epithelial cells into interstitial cells,and promote the occurrence of pulmonary fibrosis.This study was based on the present study that the primary pulmonary fibrosis appeared after rats exposed to nanosized SiO₂ at 60 and 90 days by tracheal instillation,and premised high throughput sequencing showed that miR-702-3p was a miRNA with differential expression in rats lung fibrosis induced by nanosized SiO₂.It was found that miR-702-3p may be related to transforming growth factor beta 1?TGF-?1?pathway through biological function analysis.MiR-702-3p may participate in the development of EMT through the predicted target genes of bone morphogenetic protein-7?BMP-7?to regulate TGF-?1 signaling pathway in pulmonary fibrosis induced by nanosized SiO₂.In order to elucidate the biological regulatory role of miR-702-3p in rats' lung fibrosis induced by nanosized SiO₂ and the relationship of miR-702-3p,TGF-(31 signaling pathway and EMT.Our study investigated from two levels of the whole animal and cell,and the experimental results are as follows:1.There were 136 target genes of miR-702-3p predicted by The TargetScan.GO analysis and KEGG pathway analysis showed that miR-702-3p had a significant role in the regulation of TGF-P signaling pathway and Smad signal transduction.BMP-7,one of the members of the TGF-P superfamily,was chosen to be the important predictor gene of miR-702-3p to explore the mechanisms of miR-702-3p regulating the process of EMT induced by nanosized SiO₂.2.qRT-PCR and Western Blot were respectively applied to detect miRNA,mRNA and protein level.The expression of miR-702-3p in lung tissue of rats exposed to 25mg/mL SiO₂ nanoparticles at 60d was 2.04 times more than control group?P<0.05?.The expression of BMP-7,TGF-?1,Smad3,E-cadherin?E-cad?,a-smooth muscle acti?a-SMA?and Collagen type 3?COL3?mRNA were individually 0.40,1.28,1.71,0.57,2.70,1.53 times than control group and the differences were statistically significant?P<0.05?.In the group of lung tissue of rats exposed to SiO₂ nanoparticles,the expression of BMP-7,TGF-?1,Smad3,E-cad,a-SMA and COL3 protein were individually 0.60,1.48,1.24,0.65,1.43,1.23 times than control group and the differences were statistically significant?P<0.05?.Results of double luciferase reporter gene detected that the reported fluorescence values of miR-702-3p mimic group and miR-702-3p NC group were 0.73±0.010 and 1±0.005 after transfecting the 3 UTR luciferase reporter plasmid vector of wild type BMP-7 and miR-702-3p mimic/NC into human lung adenocarcinoma epithelial cells?A549 cells?respectively and the differences were statistically significant?P<0.05?.The reported fluorescence values of miR-702-3p mimic group and miR-702-3p NC group were 0.98±0.060 and 1.00±0.016 after transfecting the 3 UTR luciferase reporter plasmid vector of mutational type BMP-7 and miR-702-3p mimic/NC into A549 cells respectively and the differences had no statistically significant?P>0.05?.These data demonstrated that EMT occurred during the process of rat's lung interstitial fibrosis induced by SiO₂ nanoparticles.MiR-702-3p can inhibit the expression of target gene BMP-7 through combining with the binding sites of CCACCCG-GGUGGGC,activate TGF-?1 signaling pathway,induce lung epithelial cells translate into pulmonary interstitial cells,increase the secretion of collagen and promotes the occurrence of fibrosis.3.IC₅₀ of rat alveolar macrophages?NR8383 cells?treated 24 hours with SiO₂ nanoparticles was 0.761 mg/mL with a confidence interval of 95%?0.001?3.844 mg/mL?,which detected with 3-?4,5-dimethyl-2-thiazolyl?-2,5-diphenyl-2-H-tetrazolium bromide?MTT?assay.The survival rate of NR8383 cells was above 85%after exposed to 50?g/mL SiO₂ nanoparticles suspension 24h later.The content of TGF-?1 examined by enzyme-linked immunosorbent assay?ELISA?in 24h supernatant of NR8383 cells exposed to SiO₂ nanoparticles was 1612.67±15.32 pg/mL while the content of TGF-?1 in control group was 1072.67±60.10 pg/mL and the differences were statistically significant?P<0.05?.The expression level of miR-702-3p in NR8383 cells of SiO₂ nanoparticles group was 3.11 times higher than that in the control group?P<0.05?.The expression level of mRNA and protein of BMP-7 were 0.39 and 0.30 times than control group and the differences were statistically significant?P<0.05?.The proliferation rates of A549 cells in blank control group and NR8383 supernatant group were?100±6?%and?101±5?%respectively and the differences had no statistically significant?P>0.05?when A549 cells co-culturing 24h with the supernatant of NR8383 cells exposed to 50g/mL SiO₂ nanoparticles.The expression of miR-702-3p in A549 cells of SiO₂ nanoparticles group was 1.22 times than the control group.The expression of BMP-7,TGF-?1,Smad3,E-cad,?-SMA and COL3 mRNA were individually 0.25,1.33,1.53,0.99,6.75,2.68 times than control group and the differences were statistically significant?P<0.05?except E-cad.The expression of BMP-7,TGF-(31,Smad3,E-cad,?-SMA and COL3 protein were individually 0.48,1.88,2.20,0.40,1.91,3.1 times than the control group and the differences were statistically significant?P<0.05?.The proliferation rate of NR8383 cells in miR-702-3p mimic,mimic NC,miR-702-3p inhibitor,inhibitor NC and control group were?101.40±3.45?%,?100.03±2.91?%,?100.76±0.96?%,?92.37±3.26?%,?100±3.89?%after transfected 48 hours respectively and the differences had no statistically significant?P>0.05?and no obvious changes were observed in the NR8383 cells under the microscope.The expression of miR-702-3p in NR8383 cells of miR-702-3p mimic group was 27.78 times than the control group after transfected 48 hours?P<0.05?,indicating that miR-702-3p mimic transfection was successful.The expression of miR-702-3p in NR8383 cells of miR-702-3p inhibitor group was 0.19 times than the control group?P<0.05?.The expression of mRNA and protein of BMP-7 in NR8383 cells of miR-702-3p mimic group were 1.07 and 0.43 times than the control group?P<0.05?,while the expression of mRNA and protein of BMP-7 in NR8383 cells of miR-702-3p inhibitor group were 2.02 and 1.32 times than the control group?P<0.05?.The expression of mRNA and protein of TGF-?1 in NR8383 cells of miR-702-3p mimic group were 1.23 and 1.58 times than the control group?P<0.05?,while the expression of mRNA and protein of TGF-?1 in NR8383 cells of miR-702-3p inhibitor group were 0.52 and 0.63 times than the control group?P<0.05?.These data demonstrated that transfection of miR-702-3p could increase the production of TGF-?1 by inhibiting the expression of BMP-7.The cell proliferation rate of control group,TGF-?1+RB group and TGF-?1 group were?100±5.04?%,?106.28±1.38?%and?108.99±6.51?%after A549 cells induced 48 hours with TGF-?1 and TGF-?1+TGF-?1 receptor blocker and the differences had no statistically significant?P>0.05?.A549 cells of TGF-?1 group translated from epithelial-like cells into spindle-shaped fibroblast cells after stimulating 48h with 5ng/ml TGF-?1.The expression of miR-702-3p,BMP-7,Smad3,E-cad,?-SMA and COL3 mRNA were individually 1.37,0.39,0.57,0.18,3.14 and 2.64 times than control group and the differences were statistically significant?P<0.05?.The protein expression level of BMP-7,Smad3,E-cad,a-SMA and COL3 were individually 0.41,1.82,0.72,1.61 and 14.51 times than the control group?P<0.05?.There have no obvious changes were observed in the A549 cells under the microscope after cells induced 5 hours with TGF-?1 receptor blocker following with TGF-(31 culturing 48 hours.Results of qRT-PCR and Western Blot showed that the expression of miR-702-3p,Smad3,E-cad,a-SMA and COL3 did not change evidently except the expression of BMP-7 protein increased than the control group.These data demonstrated that TGF-?1 has an up regulation effect on miR-702-3p and can promote the occurrence of EMT.In summary,our study suggests that EMT occurs in the process of rat's pulmonary fibrosis induced by SiO₂ and BMP-7 is the target gene of miR-702-3p.MiR-702-3p,TGF-?1 signaling pathways are closely related to the EMT process,on the one hand,miR-702-3p inhibits the expression of target gene BMP-7 through combining with the binding sites of CCACCCG-GGUGGGC,activates TGF-?1 signaling pathway,induces lung epithelial cells translate into pulmonary interstitial cells,increases the secretion of collagen and promotes the occurrence of fibrosis.On the other hand,TGF-?1 has an up regulation effect on miR-702-3p and can promote the occurrence of EMT.