Role And Regulation Of MiR21-5p In Patient With Traumatic Tracheal Stenosis

Traumatic tracheal stenosis(TTS)is the most common complication of tracheal intubation and tracheotomy,also which is the main cause of complex and difficult airway.At present,there are no drugs with definite curative effect in clinic,mainly through thermal ablation,balloon dilation,cryotherapy,stent implantation and other respiratory interventional therapy,as well as surgical resection of the narrow trachea and end-to-end trachea anastomosis.Nevertheless,both medical interventional treatment and surgical procedure are secondary injuries and still lead to airway restenosis again.It dramatically impacts the physical and psychological wellbeing of patients and contributes to a huge financial burden on their family and society.Consequently,how to inhibit or reverse tracheal stenosis after injuries is a critical task in medical fields.Over recent years,as increasing numbers of functional non-coding RNAs has been discovered,a favored topic of the molecular mechanisms between coding and non-coding RNAs marks the arrival of a new paradigm of gene regulation.mi RNA is known as the "master regulator" of gene expression,and serves a critical function in cell growth,development,proliferation,differentiation,apoptosis and metabolism via negatively regulating the expression of target genes.Therefore,taking mi RNA as a key entry point for treatment and prevention of TTS,we used small-seq high-throughput technology to conduct gene sequencing on scar tissue of post-intubation and tracheotomy,mapped differential expression profile of mi RNAs,and constructed a mi RNA-m RNA network in TTS.The biological functions of mi RNAs on fibroblasts will be comprehensively demonstrated from multiple levels and dimensions,providing theoretical basis and accurate molecular targets for the intricate regulatory network in the real world.At the same time,whether pirfenidone inhibits inflammation and fibrosis by affecting mi RNA expression was explored,providing a strong experimental basis for clinical therapeutic drug of TTS.The research is made up of the following four sub-studies:Part Ⅰ Construction and Analysis of mi RNAs Expression Profiles in Patient with Traumatic Tracheal StenosisObjective To explore the biological function and signal transduction pathway of mi RNAs enrichment by drawing differential expression profile of mi RNAs and constructing a mi RNA-m RNA network in TTS.Methods Four subjects of stenosis tissues with post-tracheal intubation and tracheotomy as tracheal stenosis group(TS group),and the 4 paired adjacent normal tissues were collected from surgical resection of pulmonary disease as control group.Small-seq high-throughput gene sequencing was used to screen and identify mi RNAs differential expression profiles.GO and KEGG enrichment analysis of candidate target genes was performed by bioinformatics technology.Results Twenty-four differentially expressed mi RNAs were identified,of which 13 were up-regulated,such as mi R450a-5p,mi R450b-5p,mi R493-3p,mi R493-5p,mi R409-5p,mi R409-3p,mi R21-5p,mi R214-3p,mi R223-5p,mi R379-5p,mi R134-5p,mi R382-5p,mi R381-3p;and 11 were down-regulated,such as mi R29b-3p,mi R30a-3p,mi R195-5p,mi R200a-3p,mi R200a-5p,mi R141-3p,mi R3615,mi R34c-5p,mi R375-3p,mi R449c-5p,mi R335-5p.A total of 2496 target genes were predicted by bioinformatics software,and GO enrichment analysis was performed.The biological process(BP)mainly enriched in “single-organism metabolic process”,“single-organism cellular process”,“regulation of cell communication”,“regulation of signaling”;Cellular Component(CC)mainly include in “cytoplasm”,“intracellular”,“ Organelle ”;Molecular Function(MF)major enrichment in “ catalytic activity”,“protein binding”,“nucleoside binding”.KEGG enrichment analysis associated with 275 signaling pathways,such as ① Inflammatory-related signaling pathway: “ TNF signaling pathway ”,“ NF-kappa B signaling pathway”,“Toll-like receptor signaling pathway Toll”.② Fibrosis-related signaling pathway: “MAPK signaling pathway”,“Notch signaling pathway”,“Wnt signaling pathway”.③ “Cell cycle”,“Apoptosis”,“PI3K-Akt signaling pathway”,“m TOR signaling pathway”,“HIF-1 signaling pathway”.Conclusion Successfully,the differential expression profile of mi RNAs was mapped,and the mi RNA-m RNA network was constructed.These mi RNA-target genes are mainly enriched in cells and organelles which have catalytic activity and binding function,for example binding protein,small molecule,nucleotide,and finally process into TTS through a series of intercellular regulation and signal regulation of the related inflammatory signaling pathway and fibrosis signaling pathway.Part Ⅱ Verification of Differential mi RNAs in Scar Tissue of Patients with TTS and Establishment of Fibroblast ModelObjective To validate the expression of mi R21-5p,214-3p,141-3p and29b-3p in scar tissue of patients with TTS and build up LPS-induced fibroblast mode for further study on the biological function and molecular mechanism of mi RNA about TTS.Methods 10 subjects of stenosis tissues with post-tracheal intubation and tracheotomy as tracheal stenosis group(TS group),and the 10 paired adjacent normal tissues were collected from surgical resection of pulmonary disease as control group.The expression of mi R21-5p,214-3p,141-3p and 29b-3p in tissue specimens was verified by RT-q PCR.Respectively,at LPS concentration gradient of 0,1,10,100,1000μg/ m L and time gradient of 0,24,48,72 h,we test the highest mi R21-5p expression to find out the optimal LPS induction concentration and cell culture time.Results ① Compared with the control group,the relative expression of mi R21-5p,214-3p were 4.880±1.358 and 2.173±0.466,respectively,which were significantly up-regulated(p<0.01).The relative expression of mi R141-3p,29b-3p were 0.612±0.153 and 0.529±0.154,respectively,which were significantly down-regulated(p<0.01).② Cultured with 24 and 48 hours,compared with the other four concentration,the expression of mi R21-5p was the highest in LPS 1μg/ m L group(1.89±0.232,5.22±0.176)(p<0.01).③ Cultured with 1μg/ml LPS,mi R21-5p expression in the other three groups increased than0 h group,however the mi R21-5p expression was significant highest(6.15±0.333)at 48 h.Conclusion These results indicated that the expression of mi R21-5p and 214-3p were significantly up-regulated,while mi R141-3p and 29b-3p were significantly down-regulated,which was expected to be biomarkers for TTS.At the same time,fibroblast cells were cultured with 1μg/ m L lipopolysaccharide for 48 hours which would be the best cell model for subsequently research.Part Ⅲ mi R21-5p Promotes Proliferation and Inhibits Apoptosis of LPS-Induced Lung FibroblastObjective Insights into biological function and molecular mechanism of mi R21-5p in LPS-Induced Lung Fibroblast.Methods Fibroblasts were cultured with 1μg/ mL lipopolysaccharide for48 h in vitro and divided into control group,mimic NC group,mi R21-5p mimic group,inhibitor NC group,mi R21-5p inhibitor group.The cell proliferation was examined using a CCK-8 assay.The cell apoptosis was determined by flow cytometer analysis.The m RNA and protein relative expressions of TNF-α,Smad7,TGF-β1,COL1A1,VEGF-A,PDGF-B,BCL2,Bax and Caspase3 were detected by RT-q PCR and Western Blot.Results ①cck-8 assay was used to measure the cell growth curve from 0hours to 72 hours: compared with the control group,mimic NC group,and inhibitor NC group,mi R21-5p mimic group had the strongest fibrocyte viability,while mi R21-5p inhibitor group had the weakest cell activity(p<0.01).② The result of cell apoptosis showed that the apoptosis rate was 7.19% in control group,7.4% in mimic NC group,7.11% in inhibitor NC group,4.60% in mi R21-5p mimic group and 10.93% in mi R21-5p inhibitor group.③The result of RT-q PCR and Western Blot: compared with control group and mimic NC group,the m RNA and protein relative expressions of TNF-α,TGF-β1,COL1A1,VEGF-A,PDGF-B and BCL2 were significantly up-regulated,while the m RNA and protein relative expressions of Smad7,Bax and Caspase3 were down-regulated in mi R21-5p mimic group(p<0.01).In mi R21-5p inhibitor group,the results of cytokine expression above mentioned were reversed.Conclusion mi R21-5p may activate the effects of fibrosis and inflammation response,further promote the excessive proliferation and inhibit apoptosis of fibroblasts.Part Ⅳ Effect of mi R21-5p on LPS-Induced Fibroblast Proliferation Interfered with PirfenidoneObjective To explore the effect of mi R21-5p on LPS-induced fibroblast proliferation interfered with pirfenidone.Methods Fibroblasts were cultured with 1μg/ mL LPS for 48 h in vitro and divided into control group,pirfenidone group,mimic group,mimic+pirfenidone group.The cell proliferation was examined using a CCK-8 assay.The m RNA and protein relative expressions of TNF-α,Smad7,TGF-β1,COL1A1 were detected by RT-q PCR and Western Blot.Results ①cck-8 assay was used to measure the cell growth curve from 0hours to 72 hours: compared with the control group,mi R21-5p mimic group had the strongest fibrocyte viability,while pirfenidone group had the weakest cell activity(p<0.01);and the activity of fibroblasts increased in mimic +pirfenidone group increased,but still decreased than mimic group.② The result of RT-q PCR and Western Blot: compared with control group,the m RNA and protein relative expressions of TNF-α,TGF-β1,COL1A1 were significantly up-regulated,while Smad7 were down-regulated in mi R21-5p mimic group.However,the results of cytokine expression above mentioned were the opposite in pirfenidone group(p<0.01);and the m RNA and protein relative expressions of TNF-α,TGF-β1,COL1A1 increased,while the m RNA and protein relative expressions of Smad7 decreased in mimic +pirfenidone group,but compared with the mimics group,pirfenidone reversed the effects of mi R21-5p mimic.Conclusion Pirfenidone may inhibit fibrosis and inflammatory response by down-regulating mi R21-5p,up-regulating smad7,ultimately inhibit the fibrosis progression of fibroblasts.