Effects Of RNA Interference Silencing Smad3on Production Of Type â…  Collagen Of Human Lung Fibroblast

Asthma was a chronic airway inflammation with kinds of cells and cellularcomponents participated in, associated with lung function declining and airflow limitation.The main treatments were to eliminate airway inflammation and relieve airway spasm. Insome asthma patients, even with active anti-inflammatory and relaxation of bronchialtreatment, the airflow obstruction and airway hyperresponsiveness remained not prevented,and sharp declining lung function and hormone resistance are arising. Studies have shownthat the main reason was airway remodeling. Airway remodeling was mainly presentedwith the asthmatic airways pathological changes, including: epithelial cell shedding, submucosal deposition of collagen, hypertrophy of smooth muscle cell, proliferation ofmyofibroblast, excessive secretion of mucous cells and hypertrophy of mucus gland. Inrecent years, many studies indicated that the TGF-β1(transforming growth factor-β1) isthe most critical fibrosing cytokines in the asthmatic airway remodeling. Transforminggrowth factor β1plays an important role in the asthmatic airway remodeling，throughSmad3dependent signal transduction pathway to promote the synthesis and secretion oftype Ⅰcollagen. RAN interference technology has made great progress in gene therapy andfunctional studies in recent years. TGF-β1/Smad3signaling pathway played a veryimportant role in stimulation of extracellular matrix synthesis and inhibiting itsdegradation to promote the submucosal collagen deposition.Objective This study was to investigate the effects of RNA interference (RNAi)mothed silencing the expression of Smad3and inhibiting the type Ⅰcollagen production ofhuman lung fibroblast-1(HLF-1).Methods Short hairpin RNA (shRNA) was designed and synthesized, shRNA wastransferred into HLF-1using Lipofectamine. Reverse transfection PCR (RT-PCR) andwestern blot analysis were used to detect the expression of Smad3inhibited by shRNA.Enzyme-linked immunoadsordent assay (ELISA) was performed to detect the productionof type Ⅰcollagen.Results RT-PCR results indicted expression of Smad3mRNA in experimental groupsignificantly decreased68.2%and69.5%(P<0.05) than normal control group andnegative group, respectively; Western blot indicated expression of Smad3protein inexperimental group significantly decreased67.1%and64.7%(P<0.05) than normalcontrol group and negative group, respectively; ELISA results showed type Ⅰcollagensignificantly decreased59.3%and61.1%(P<0.05), compared with normal control groupand negative group, respectively.Conclusion The expression of Smad3mRNA and protein were efficiently silenced bySmad3shRNA, while the production of type Ⅰcollagen was also inhibited. RNAinterference has potential for clinical therapy of asthma in future.