The Research Of The Regulation And Mechanism Of MiR-128-3p In Pulmonary Fibrosis Through SHH Pathway

Objective:To study the regulatory role of miR-128-3p in the occurrence and process of pulmonary fibrosis,and discuss its specific mechanism.Methods:MLE12 cells were divided into 4 groups,adding TGF-β1,SHH,TGF-β1+SHH,control group did not do any treatment,the cell fibrosis was observed under the microscope.TGF-β1stimulated MLE12 cells,and the protein expression of SHH pathway was detected by Western-blot,while the m RNA level of SHH pathway protein was detected by q RT-PCR.MLE12 cells were divided into the experimental group receiving TGF-β stimulation and the control group without intervention.The expression difference of miRNAs in each group was detected by Microarray technology,and then the level of miR-128-3p in vivo and in vitro pulmonary fibrosis models was detected by q RT-PCR technology.MLE12 cells were divided into Blank group,Control group,SMAD2 si RNA group,SMAD3 si RNA group,and Smad2/3si RNA group.By transfection of si RNA silenced the expression of related genes,the Blank group did not do any treatment,the Control group added TGF-β,SMAD2 si RNA group added SMAD2 si RNA+TGF-β,SMAD3 si RNA group added SMAD3 si RNA+TGF-β,SMAD2/3si RNA group was added with SMAD2/3 si RNA+TGF-β,and the miR-128-3p level was detected by real-time PCR.Meanwhile,SMAD2,SMAD3 and Smad2/3 plasmids were constructed to overexpress them,and MLE12 cells were divided into four groups: The Vector group consisted of no-load control group,SMAD2 group,SMAD3 group,and Smad2/3 group.After culture,the level of miR-128-3p was detected by q RT-PCR.miR-128-3p plasmid was constructed,and MLE12 cells were divided into three groups: miR-128-3p group was transfected with miR-128-3p plasmid,Control group was transfected without any treatment,Scramble group was transfected with empty plasmid,and fluorescence intensity was observed to predict intracellular Gli2 content.Then,the 3 ’UTR of SUFU m RNA was transferred into luciferase reporting system and represented by SUFU 3’ UTR.Similarly,the 3 ’UTR of SUFU m RNA after mutation was constructed into luciferase reporting system and represented by SUFU 3’ UTR-muts.MLE12 cells were divided into two groups: miR-128-3p group was separately added with miR-128-3p and the above plasmid,and empty vectors were constructed as controls;Scramble group was separately added with the above plasmid and empty vector,and fluorescence intensity of cells in each group was detected to reflect the protein expression level.MLE12 cells were further divided into three groups in vitro: The miR-128-3p group was transfected with miR-128-3p plasmid,the Scramble group was transfected with empty plasmid,and the Control group was not treated.After culture,the expression level of SUFU protein was detected by Western-blot,and the above experiment was repeated in p AECs cells.MLE12 cells were divided into two groups: the miR-128-3p group was transfected with miR-128-3p plasmid,and the Scramble group was transfected with empty plasmid.The expression of EMT-related proteins mediated by miR-128-3p was detected by Western-blot.Thirty mice were randomly divided into Control group,PBS group,Anti-NC group and Anti-miR-128-3p group.The Control group did not receive any treatment,the PBS group was injected with PBS via tail vein,and the Anti-NC group was injected with the control vector r AAV-Tu D via tail vein.Anti-miR-128-3p group was injected with r AAV-miR-128-3p-Tu D vector via caudal vein;After 3 days of the above operation,all mice except the Control group were injected with BLM through the trachea to establish a mouse model of pulmonary fibrosis.The animals were killed and lung tissues were taken,and the phenomena of pulmonary fibrosis and collagen deposition were observed by HE and Masson staining.Results:Under TGF-β/SHH stimulation,MLE12 cells can transform from normal form to fibroblast,and the two have synergistic effect.After TGF-β1 stimulation,SUFU protein level in SHH pathway changed significantly,in order to inhibit the expression,but the detection of m RNA content,TGF-β1 group and control group showed no significant difference.Compared with the control group,the expression of miR-128-3p was significantly increased after TGF-β stimulation,and the expression level was significantly increased with time(P <0.01),and the content of miR-128-3p in the BLM-induced in vitro model was also increased,about 3times as much as that in the control group(P <0.01).According to the database query,SMAD2/3 molecule in TGF-β pathway had a sequence matching the promoter of the gene encoding miR-128-3p.After silencing the expression of SMAD molecule,the level of miR-128-3p was significantly down-regulated compared with Control(P<0.05).After overexpression of SMAD,miR-128-3p levels were significantly up-regulated in the SMAD group compared with the Vector group(P <0.01).After overexpression of miR-128-3p,the fluorescence intensity of miR-128-3p group was higher than that of Scramble group and Control group,and there was no significant difference in fluorescence between Scramble group and Control group.Luciferase experiment indicated that after transfection of miR-128-3p,the fluorescence intensity of SUFU 3’UTR group was significantly increased compared with that of the negative control group(P <0.01).There was no significant difference in fluorescence intensity between SUFU 3 ’UT-Muts group and empty carrier group compared with negative control group.Compared with the control group and the empty plasmid group,the expression level of SUFU protein in miR-128-3p group was significantly decreased,and there was no significant difference between the control group and the empty plasmid group.After overexpression of miR-128-3p,the expressions of EMT-related proteins N-cadherin,vimentin,fibronectin and α-smooth muscle actin were higher than those in Scramble group,while the epithelial cell marker e-cadherin was lower than that in control group.The degree of pulmonary fibrosis in Anti-miR-128-3p group was significantly lower than that in PBS group and Anti-NC group,but still higher than that in control group.There was no significant difference in pulmonary fibrosis degree between PBS group and Anti-NC group.Conclusions:The expression of miR-128-3p was significantly increased in pulmonary fibrosis,and the down-regulation of the expression of the target protein-SUFU further enhanced the conduction of TGF-β and SHH signaling pathway in a post-transcriptional regulatory manner,which was a positive promoting effect on pulmonary fibrosis.Inhibition of the expression of miR-128-3p could reduce the degree of pulmonary fibrosis.