Effect Of MiR-29c On Silicosis In Mice By Regulating Wnt/?-catenin Pathway

ObjectiveTo construct a miR-29c overexpression lentiviral vector and study the effect of increased expression of miR-29c on the expression of Wnt/?-catenin pathway-associated genes and on blocking fibrosis in the silicosis model induced by SiO₂ in mice,finding a new target for the prevention and treatment of silicosis.MethodsThe mouse pre-miR-29c gene was amplified by PCR,ligated into the pLenti-CMV-EGFP vector and packaged into a miR-29c over-expression lentiviral vector to infect the MLE-12 cells to determine whether they were successfully constructed.72 BALB/c mice were randomly divided into 4 groups,with 18 rats in each group.Dust exposed by the dust exposed endotracheal injection method.Saline group:inject 0.1ml sterile saline;SiO₂ model group:inject dust 0.1ml containing SiO₂ 3mg suspension;SiO₂+Lv-miR-29c group:inject 0.1ml of a dust suspension of3 mg of silica dust and 5×10⁷ IU of Lv-miR-29c;SiO₂+Lv-NC group:inject 0.1ml of a dust suspension of containing 3 mg of SiO₂ and 5×10⁷ IU miRNAs lentivirus.The animals were sacrificed on days 7,14,and 28,respectively.Real-time PCR was used to detect the expression of miR-29c in lung tissue;Real-time PCR and Western Blot were used to detect the expression of?-catenin and MMP2 and MMP9 in lung tissue;The expression of TGF-?1 and IL-6 in BALF was detected by ELISA.The content of hydroxyproline in lung tissue of mice was measured.The coefficient of lung organ was calculated according to the ratio of lung wet weight and weight in mice.The pathological changes of lung tissue were observed by HE staining and Masson staining.ResultsThe miR-29c lentiviral vector was successfully constructed in this experiment.The expression of miR-29c was significantly increased in MLE-12 cells transfected with Lv-miR-29c?P<0.001?;In animal experiments,real-time PCR results showed that the miR-29c expression levels in the SiO₂+Lv-miR-29c group were significantly higher than those in the other three groups at 7,14,and 28 days?P<0.001?;Compared with the SiO₂ model group,the mRNA and protein expression levels of?-catenin,MMP2,and MMP9 in the SiO₂+Lv-miR-29c group were significantly lower,and the differences were statistically significant?P<0.001?.The results of ELISA showed that the levels of TGF-?1 and IL-6 in the SiO₂+Lv-miR-29c group were significantly lower than those in the SiO₂ model group?P<0.001?.The results of hydroxyproline assay showed that the content of hydroxyproline in four groups had not statistically significant differences at 7 days.At 14 days and 28 days,the content of hydroxyproline in SiO₂+Lv-miR-29c group was significantly lower than that in SiO₂ model group?P<0.001?.The calculation results of the mouse lung coefficient showed that there was no significant difference in the four groups at 7 days?P=0.66?,and the lung organ coefficient of the SiO₂+Lv-miR-29c group was lower than that of the SiO₂ model group at 14 days and28 days,the difference was statistically significant?P<0.001?;Pathological examination showed that compared with the SiO₂ model group,the degree of pulmonary fibrosis in the SiO₂+Lv-miR-29c group was significantly reduced.ConclusionIn this experiment,the miR-29c lentiviral expression vector was successfully constructed and the mouse silicosis model was established.Increasing the expression of miR-29c in the mouse silicosis model,the expression of?-catenin,MMP2 and MMP9 were inhibited,so miR-29c inhibits the Wnt/?-catenin signaling pathway related genes;In mouse silicosis model the expression of miR-29c increasing,reduced the content of hydroxyproline in lung tissue,the coefficient of lung organ and the degree of pulmonary fibrosis,so miR-29c has an inhibitory effect on the fibrosis of the lung in mice.It can be seen that miR-29c inhibits the development of silicosis induced by SiO₂ in mice by modulating Wnt/?-catenin signaling pathway,which provides new ideas for the molecular mechanism of silicosis.