| Objective:Idiopathic pulmonary fibrosis(IPF)is a chronic and fatal interstitial lung disease with complex pathogenesis and unknown cause.Its main pathological feature is alveolar epithelial damage caused by various factors,followed by abnormal scar repair after the destruction of normal lung tissues,which eventually leads to respiratory failure of patients.IPF has a poor prognosis,and there is no effective treatment for the time being.In recent years,circular RNA(cric RNAs)have been a hot topic in the field of RNA research in recent years,and a large number of studies have demonstrated that circ RNAs can be therapeutic targets or biomarkers for certain diseases.However,the mechanism of circ RNAs in the pulmonary fibrosis needs to be further investigated.In this study,we used hsa-circ-0002490,which was screened in a previous study,to investigate the role of hsa-circ-0002490 on pulmonary fibrosis by regulating the Rho A/ROCK1 pathway.Methods:1.MRC-5 cell experiment: the experimental group was given medium with2ng/ml TGF-β1 added,and the control group was given the same volume of medium and treated for 2 weeks.The expression level of hsa-0002490 in TGF-β1-induced lung fibrogenic transformation model was verified by q RT-PCR.2.Construction of hsa-circ-0002490 overexpression cell model: The TGF-β1-induced lung fibrogenic transformation model was divided into experimental group(sh-RNA group)and control group(control group).The hsa-circ-0002490 overexpression lentiviral vector was constructed,and the experimental group was transfected,while the control group was left untreated.After 48 hours of transfection,hsa-circ-0002490 expression differences were verified by q RT-PCR.3.On sh-RNA group and control group,CCK-8 assay was performed to compare cell proliferation ability,Western Blot assay was performed to compare the effect of GTP-Rho A,ROCK1 and p-MYPT1 expression,and flow cytometry was performed to compare the degree of apoptosis.4.Dual-luciferase reporter was performed to assess the targeting relationship between hsa-circ-0002490 and miR-4446-3p,and Western Blot,CCK-8 and flow cytometry were performed on control group,sh-RNA group,control+mimic group and sh-RNA+mimic group to test To compare the expression effect of downstream proteins,cell proliferation ability and the degree of apoptosis.Results:1.The low expression of circ RNA-0002490 in the TGF-β1-induced lung fibrosis transformation model was verified by q RT-PCR,which is consistent with the sequencing results in the previous study.2.TGF-β1-induced lung fibroblast cell model was found to have a significant increase in hsa-circ-0002490 expression by q RT-PCR after transfection with hsa-circ-0002490 overexpression lentiviral vector for 48 hours,indicating that the circ RNA overexpression cell model was successfully constructed.3.CCK-8,Western Blot and flow cytometry results showed that hsa-circ-0002490 inhibited the proliferation of MRC-5 cells,promoted apoptosis and inhibited expression of GTP-Rho A,ROCK1 and p-MYPT1.4.Results of Dual-luciferase reporter verified the targeting relationship between hsa-circ-0002490 and miR-4446-3p.The results of Western Blot,CCK-8 and flow cytometry after transfection of miR-4446-3p mimic showed that compared with the control group,the control+mimic group had increased expression of GTP-Rho A and ROCK1,and cell proliferation ability was increased,while the degree of apoptosis was reduced,suggesting that miR-4446-3p may promote fibrosis progression through the Rho A/ROCK1 pathway;compared to the sh-RNA group,the sh-RNA+mimic group also showed the effect of reversing its biological behavior,further verifying the targeting relationship between hsa-circ-0002490 and miR-4446-3p.Conclusion:High expression of hsa-circ-0002490 has inhibitory effects on proliferation and apoptosis of lung fibroblasts,and can target miR-4446-3p to inhibit the Rho A/ROCK1 pathway thereby affecting lung fibrosis progression.The findings may provide a new target for IPF diagnosis and interventional therapy. |
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