The Role Of MiR-181b In Pulmonary Fibrosis Via Inhibiting TDFBR1Expression And Its Mechanism

Objective: To investigate the effect and mechanism of miR-181b in lungfibrosis.Method: We collecting biopsy lung specimens from7IPF patients and9healthy volunteers, and taking out lung tissue of normal mice and mice arediagnosed with IPF which induced by bleomycin (BLM), then extracting RNAto detect miR-181b level. Dividing the A549cells into control group, negativegroup, mimic group and inhibitor group. There is no intervention in controlgroup; Adding microRNA with no biological activities to negative group;Adding miR-181b mimic to the mimic group to overexpress microRNA; AddingmiR-181b inhibitor to inhibitor group to low express microRNA, detectingTGFBR1by western blot. Put TGFBR1mRNA3’UTR into luviferase reportingsystem (TGFBR1-3’UTR-wt), and also insert the mutated TGFBR1mRNA3’UTR into luviferase reporting system (TGFBR1-3’UTR-mut1,TGFBR1-3’UTR-mut2and TGFBR1-3’UTR-mut3). Then transfect miR-137and luciferase empty vector (or TGFBR1-3’UTR-wt vector or TGFBR1-3’UTR-mut1/2/3vector) together to HEK-293cells, detecting fluorescenceintensity of each group. Dividing the30mice into control group, miR-181bgroup and GFP group,10mice in each group. No intervention in control groupand injecting rAAV-miR-181b vector by caudal vein into mice of miR-181bgroup to over express miR-181b, and injecting rAAV-GFP vector by caudal veinof mice into GFP group. All mice are induced to IPF model by BLM, thenexecute these mice and extract lung tissues after IPF model making, detectpathological change and collagen deposition of lungs by HE staining and Masson staining, detect the expression of TGFBR1、phosphorylated SMAD2,phosphorylated SMAD3, α-SMA, E-cadherin, vimentin and Collgen I bywestern blot.Result: Expression of miR-181b in lung tissues of IPF patients is one thirdof normal patients, while comparing with normal mice, the expression ofmiR-181b in IPF mice is lower, which is a quarter. miR-181b mimic can reduceTGFBR1significantly in A549cells (p<0.05), however miR-181b inhibitor canimprove TGFBR1significantly in A549cells (p<0.05), there are no differencesof TGFBR1mRNA in cells among5groups. Results from luciferase assayshows fluorescence intensity of TGFBR1-3’UTR-wt group is lower greatly thancontrol group (miR-con), however there are no differences of fluorescenceintensity between TGFBR1-3’UTR-mut3group and control group or betweenempty vector group and control group. The pulmonary fibrosis of mice inmiR-181b group is significantly greater than BLM group and GFP group, andgreater than control group; No significantly differences of pulmonary fibrosis ofmice between BLM group and Vector group. Expression of TGFBR1, p-SMAD2,p-SMAD3, α-SMA、E-cadherin and Collgen I in lung tissue of IPF mice withAAV5-miR-181b transfected is significantly lower than control group and GFPgroup (p<0.05), while the expression of vimentin is greater than control groupand Vector group (p<0.05).Conclusion: The expression of miR-181b in IPF is reduced, the overexpression of miR-181b after transcription could inhibits the expression ofTGFBR1and blocks the conduction of TGF-β signal routing in lung tissues,then decrease the pulmonary fibrosis.