LncRNA HOTAIRM1 And AP000487.1-Mediated PRKCB DNA Methylation Involved In Nano NiO-induced Pulmonary Fibrosis In Rats

Objective: A rat lung fibrosis model was established by tracheal drip injection of nickel oxide nanoparticles(Nano NiO),whole genome methylation sequencing(WGBS)and transcriptome sequencing(RNA-seq)were used to detect DNA methylation genes in rat fibrotic lung tissue.We aim to explore lncRNAs that bind to the promoter region of protein kinase C-beta(PRKCB)gene by performing chromatin RNA pulldown(ChRNA pulldown)experiments and then elucidate the role of lncRNA HOTAIRM1 and AP000487.1-mediated PRKCB DNA methylation in Nano NiO-induced pulmonary fibrosis.Methods: 1.In vivo experiment: Thirty-two adult healthy Wistar male rats were randomly divided into 0,0.015,0.06 and 0.24 mg/kg Nano NiO groups and intratracheally instilled with Nano NiO twice a week for 9 weeks.At the end of the experiment,the rats were executed and the lungs were removed and preserved for subsequent studies.2.In vitro experiment:(1)BEAS-2B cells were treated with 0,25,50,and 100 μg/m L Nano NiO suspension for 24 h.(2)BEAS-2B cells were pretreated with 10 μM SP600125 of JNK/c-JUN pathway inhibitor and TAK-242 of TLR4/MyD88/NF-κB pathway inhibitor for 1 h,followed by co-treatment with 50μg/m L Nano NiO for 24 h.(3)BEAS-2B cells overexpressing or low expressing PRKCB2 were treated with 50 μg/m L Nano NiO for 24 h.BEAS-2B cells were pretreated with 10 μM PRKCB inhibitor LY317615 for 1 h,followed by co-treatment with 50 μg/m L Nano NiO for 24 h.(4)BEAS-2B cells were transfected with the overexpressing lncRNA HOTAIRM1 and lncRNA AP000487.1 plasmids,and then treated with 50 μg/m L Nano NiO for 24 h.3.Research index detection:(1)WGBS and RNA-seq techniques were used to detect the genome-wide DNA methylation and gene expression levels in rat fibrotic lung tissue.(2)Ch-RNA pulldown was performed to explore lncRNAs that bind to the promoter region of the PRKCB gene.(3)Methylation-specific PCR(MSP)and bisulfite sequencing PCR(BSP)were used for qualitative and quantitative analysis of PRKCB DNA methylation in cells.(4)CCK-8and LDH leakage assays were used to detect cell cytotoxicity.(5)Western blot and cell immunofluorescence were used to detect the expressions of PRKCB2,signaling pathway-related proteins(JNK,p-JNK,c-Jun,p-c-Jun,TLR4,MyD88,NF-κB,and pNF-κB),and collagen-related proteins(Col-I,α-SMA,E-cadherin).(6)RT-q PCR was used to detect the expression levels of 13 methylation differential genes,as well as lncRNA HOTAIRM1,lncRNA AP000487.1,and PRKCB2 in lung tissues and BEAS-2B cells.Results: 1.LncRNA HOTAIRM1 mediated PRKCB DNA methylation and JNK/c-JUN pathway in Nano NiO-induced pulmonary fibrosis in ratsIn vitro and in vivo experiments showed that Nano NiO could downregulate the expression of lncRNA HOTAIRM1,promote PRKCB DNA methylation and increases the expression of PRKCB2,activates JNK/c-JUN pathway,and leads to collagen deposition(upregulation of Col-I and α-SMA).SP600125,a JNK/c-JUN pathway inhibitor,could alleviate excessive collagen formation induced by 50 μg/m L Nano NiO.Overexpression of PRKCB2 promoted JNK/c-JUN pathway activation and excessive collagen formation induced by Nano NiO in BEAS-2B cells,while DNA methylation inhibitor 5-Azacytidine could reduce the expression of PRKCB2,JNK/c-JUN pathway activation and excessive collagen formation induced by Nano NiO in BEAS-2B cells.Ch-RNA pull-down experiment revealed that lncRNA HOTAIRM1 could bind to the promoter region of the PRKCB gene,and the overexpressing lncRNA HOTAIRM1 could inhibit Nano NiO-induced the high expression of PRKCB2,hypomethylation of PRKCB,the activation of JNK/c-JUN pathway,and excessive collagen formation in BEAS-2B cells.2.LncRNA AP000487.1 mediated PRKCB DNA methylation and TLR4/MyD88/NF-κB involved in Nano NiO-induced collagen overload formation in BEAS-2B cellsAfter treating BEAS-2B cells with Nano NiO,it was found that Nano NiO could lead to the downregulated lncRNA AP000487.1,the upregulated PRKCB2,PRKCB DNA methylation,the activation of TLR4/MYD88/NF-κB pathway and excessive collagen deposition.However,TAK-242 of TLR4 inhibitor suppressed the activation of TLR4/MYD88/NF-κB pathway and excessive collagen deposition induced by 50μg/m L Nano NiO.After intervention of si PRKCB2 fragment and PRKCB2 protein inhibitor LY317615,it was found that the activity of NF-κB signaling pathway and collagen formation induced by Nano NiO were inhibited to varying degrees in BEAS-2B cells.Furthermore,DNA methylation inhibitor 5-Azacytidine could attenuated the expression of PRKCB2,activation of TLR4/MYD88/NF-κB pathway and excessive collagen formation induced by Nano NiO.Ch-RNA pull-down experiment revealed that lncRNA AP000487.1 could bind to the promoter region of PRKCB with the largest differential magnification,and the overexpressing lncRNA AP000487.1 inhibited Nano NiO-induced the high expression of PRKCB2 and low methylation of PRKCB,the activation of TLR4/MYD88/NF-κB pathway and excessive collagen deposition in BEAS-2B cells.Conclusion: 1.Nano NiO can induce pulmonary fibrosis in rats,and its mechanism is related to the down-regulated lncRNA HOTAIRM1 induced by Nano NiO,which prompts PRKCB DNA hypomethylation and the high expression of PRKCB2,and further activate the JNK/c-JUN signaling pathway.2.Nano NiO can lead to excessive collagen formation in BEAS-2B cells,and the mechanism is related to the downregulated lncRNA AP4000487.1 induced by Nano NiO,which trigger PRKCB DNA hypomethylation and further activated NF-κB signaling pathway in BEAS-2B cells.