CircHECTD1 Mediates Silica-induced Pulmonary Fibrosis Via HECTD1

Objective: Silicosis is a type of pulmonary fibrosis caused by long-term inhalation of silica(SiO2)particles.Multiple inflammatory reactions occur in the early stage,and interstitial transformation occurs accompanied by excessive proliferation of fibroblasts.Developed as an irreversible pulmonary fibrosis at the advanced stage.The specific pathophysiological mechanism of silicosis fibrosis is unclear.Although the role of chemokines and cytokines released by SiO2 in alveolar macrophages have received significant attention,the direct effects of SiO2 on protein production and functional changes in pulmonary fibroblasts have been less extensively studied.Circular RNA(circRNA),a new class of non-coding RNA,has been shown its importance in silicosis for unique role as transcription regulators or sponges of small RNA regulators.Previous data from our lab using circRNA microarray assay suggested that circHECTD1 level in mouse lung exposure to SiO2 was increased,which may target an E3 ubiquitin ligase-HECTD1.Here,the mechanisms underlying circHECTD1 and HECTD1 in fibroblast activation and subsequent fibrosis induced by SiO2 were investigated.Methods:1)The role of HECTD1 in the activation of fibroblast proliferation induced by SiO2.a)Human pulmonary fibroblasts(HPF-a)treated with SiO2 particles,Western blotting combined with cellular immunofluorescence to detect the xpression of HECTD1 and fibroblasts’ markers;b)The silicosis mouse models were established by instillation of SiO2.The lung tissue of the mice were obtained,and the expression levels of fibroblast markers and HECTD1 were detected by immunofluorescence;c)Immunofluorescence was performed to check fibrosis markers and HECTD1 in lung tissue of silicosis patients.d)After CRISPR/Cas9 knockout HECTD1,explore the cell proliferation and viability changes were measured by BrdU,MTT and gel contraction;e)CRISPR/Cas9 knockout HECTD1,cell scratch assay,3D nested migration assay to assess changes in HPF-a cell migration.2)The specific mechanism of circHECTD1/HECTD1 affects fibroblast activation.a)WB detects the expression level of autophagy markers in fibroblasts exposed to SiO2;b)WB and LC3B-LV-RFP lentivirus were applied to evaluate autophagic flow levels after specific knockout of HECTD1;c)Treatment of rapamycin investigate whether HECTD1 affects fibroblast function through autophagy;d)qRT-PCR and in situ hybridization to observe the expression and situation of circHECTD1 in HPF-a cells;e)Overexpress circHECTD1,detecting the changes of HECTD1 expression changes and cellular function.Results: 1)Compared with the control group,SiO2 induced the expression of HECTD1 in HPF-a up to 1.6 times,and the expression of HECTD1 in fibroblasts of silicotic mice and silicosis patients was also increased.The amount of newborn cells was increased,the vitality enhanced by 30% and promoted migration capacity;CRISPR/Cas9 knocked out HECTD1,inhibited the proliferation and activation of HPF-a and inhibited the capacity of migration.2)After SiO2 treatment,the expression level of autophagy markers were increased,and the level of autophagy flux are increased else.After treatment with rapamycin,the cell function changes caused by knockout of HECTD1 could be significantly reversed;SiO2 resulted in circHECTD1 decline reduced by 0.5 times,circHECTD1 were mainly distributed in the cytoplasm;Overexpress circHECTD1 can cause a decrease in the expression of HECTD1,thereby inhibiting the viability and migration of fibroblasts.Conclusion: Our data suggested a link between circHECTD1/HECTD1 and fibroblast activation with subsequent fibrosis induced by SiO2,providing a novel insight into the potential of circHECTD1/HECTD1 in terms of opening up novel therapeutic avenues for silicosis.