Analysis of the effect of siRNA silencing α1 antitrypsin on rheumatoid arthritis fibroblasts / 中华风湿病学杂志

ABSTRACT

Objective To investigate the effect of siRNA silencing α1 antitrypsin (α1-AT) gene on the biological behavior of rheumatoid arthritis fibroblast-like synoviocytes (RA-SFs).Methods Primary culture of knee synovial tissue from 5 patients with rheumatoid arthritis (RA) was performed.The artificially synthesized silencing α1-AT siRNA specifically inhibits the expression of α1-AT in RA-SFs.After 24 and 36 hours of transient transfection,the inhibition efficiency was detected by Quantitative reverse transcription polymerase chain reaction (RT-qPCR),and the expression of related genes after α1-AT gene silencing was detected.Furthermore,ethyl thiazolyl tetrazolium (MTT) assay,Trans-well chamber,cell scratch and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of interfering α1-AT expression on cell proliferation,invasion and migration,and secretion of interleukin (IL)-17,Tumor necrosis factor (TNF)-α,IL-1α,IL-1β and other related inflammatory factors.At the same time,when the pathway inhibitor (ERK inhibitor,signal transducer and activator of transcription 3 (STAT3) inhibitor,NF-κB inhibitor) stimulated cells,the effect on α1-AT was changed.One-way analysis of variance was used for comparison between the two groups;further pairwise comparison using LSD-t test;The count data was checked by x2 test.Results Compared with the negative control group,the siRNA α1-AT group was transfected for 24 hours,the α1-AT mRNA level was significantly inhibited (P＜0.05),and the proliferation rate of RA-SFs was not affected (P＞0.05),and the invasion and migration ability of cells decreased significantly (P＜0.05).The secretion of IL-17 and IL-1β was slightly decreased (P＞0.05),while TNF-α and IL-1α were not affected (P＞0.05).In addition,the expression of α1-AT downstream genes Matrix metallop roteinases (MMP)-2 169,Hu-Bax1,MMP-9,Hu Bax and Hu Bcl-xl were significantly decreased (P＜0.05).Moreover,the signaling pathway inhibitors ERK inhibitor (PD98059) and NF-κB inhibitor (PDTC) had significant effects on α1-AT (P＜0.05).Conclusion The α1-AT gene silencing has potential effect on the biological behavior of RA SFs.Further study of this mechanism is helpful to provide evidence for the treatment of RA.