A Transcriptomics-based Study Of The Role Of JNK/c-jun Pathway In Liver Fibrosis Induced By Nickel Oxide Nanoparticles

Objective:This study aims to establish a liver fibrosis rat model induced by nickel oxide nano particles（NiONPs）,and to obtain expression profiles of messenger RNAs（mRNAs）and non-coding RNAs（ncRNAs）in liver tissues.We screen differentially expressed（DE）RNAs and explore the biological functions of DE RNAs and related signaling pathways in the NiONPs-induced liver fibrosis.The study also aim to establish a NiONPs-induced collagen deposition model of LX-2 cells,and to clarify the regulatory role of JNK/c-Jun pathway in NiONPs-induced liver fibrosis.Methods:1.In vivo experiment:（1）experimental design:40 male adult Wistar rats are randomly divided into saline group and NiONPs groups（0.015,0.06 and 0.24mg/kg）,who are exposed to NiONPs suspension by tracheal instillation twice a week for 9 weeks.At the end of the exposure,the rats are sacrificed and their liver tissues are harvested for subsequent experiments.（2）index detection:（1）Three rats are randomly selected from saline group and 0.24 mg/kg NiONPs group respectively（n=3）,whose liver tissues（100mg）are taken for whole transcriptome sequencing.And DE RNAs are screened by differential expression analysis.（2）We apply miRanda 3.3a and Target Scan to predict the binding sites in lncRNAs,circRNAs and mRNAs to miRNAs,and construct two RNA co-expression networks.（3）The sequencing results of DE mRNAs,DE lncRNAs,DE miRNAs and DE circRNAs are verified by using RT-qPCR.（4）Gene Ontology（GO）annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis are used to perform functional annotation and pathway analysis of the DE mRNA and the target genes of DE ncRNAs.（5）Western blot is used to detect the protein expression levels of JNK,p-JNK,c-Jun and p-c-Jun in rat liver tissues.2.In vitro experiments:（1）experimental design:（1）LX-2 cells are treated with different concentrations of NiONPs suspension（0,1.25,2.5,5 and 10μg/mL）for 6,12 and 24h,and the cell survival rate is detected.Based on the cytotoxicity assays,three doses and one time of subsequent NiONPs treatment are determined.（2）The above doses and time of NiONPs treatment are used to establish the collagen deposition model of LX-2cells,and the protein content changes of JNK/c-Jun signaling pathway are detected to determine a subsequent dose of NiONPs treatment.（3）LX-2 cells are pretreated with JNK pathway inhibitor SP600125 for 1h,and then culture with NiONPs for 12h.（2）index detection:（1）CCK-8 kit is used to detect cell survival rate.（2）Western blot is used to detect the protein expression levels of JNK,p-JNK,c-Jun,p-c-Jun,collagen type 1 alpha1（Col-1a1）and matrix metalloproteinase 2（MMP2）.Results:1.Whole transcriptome sequencing analysis of NiONPs-induced liver fibrosis rat model:（1）Analyzing the expression profiles of RNAs,26691 mRNAs,6082 lncRNAs,912 miRNAs and 9852 circRNAs are identified totally.And 324mRNAs（including 167 up-regulated and 157 down-regulated）129 lncRNAs（including46 up-regulated and 83 down-regulated）,24 miRNAs（including 14 up-regulated and10 down-regulated）and 33 circRNAs（including 18 up-regulated and 15 down-regulated）are statistically differentially expressed compared with the control group with|log₂foldchange|≥1 and P<0.05.（2）We predict the possible targeted interactions of 32 DE lncRNAs,12 DE miRNAs and 15 DE mRNAs for construction of the lncRNA-miRNA-mRNA network.The targeted interactions of 5 DE circRNAs,8 DE miRNAs and 11 DE mRNAs are also predicted and the circRNA-miRNA-mRNA network is constructed.（3）By RT-qPCR verification,we find that the expression profiles of 19 randomly selected DE RNAs in the RNA co-expression networks are similar to the sequencing results.（4）Through GO and KEGG functional enrichment analysis,we find biological processes and signaling pathways related to liver fibrosis are significantly enriched by the DE mRNAs and the target genes of DE ncRNAs,among which JNK and MAPK pathways are significantly enriched in GO and KEGG analysis.2.The role of JNK/c-Jun pathway in NiONPs-induced liver fibrosis:（1）Compared with the control group,the protein contents of p-JNK and c-Jun in 0.015,0.06 and 0.24 mg/kg NiONPs groups are significantly up-regulated,while JNK and p-c-Jun are significantly up-regulated in 0.24 mg/kg NiONPs group,indicating that the JNK/c-Jun pathway is activated by NiONPs in vivo.（2）Cell viability assay show that,compared with the control group,1.25,2.5,5 and 10μg/mL NiONPs reduce LX-2 cell viability from 88%to 47%within 6 hours,from 80%to 41%within 12 hours,and from70%to 35%within 24 hours,respectively.According to the above results,we choose1.25,2.5 and 5μg/mL NiONPs to treat LX-2 cells for 12h for construction of excessive collagen formation cell model.Western blot results show that the protein contents of Col-1a1 and MMP2 are both significantly upregulated in 2.5 and 5μg/mL NiONPs groups,indicating the cell model is successfully established.（3）After LX-2 cells are treated with the above doses and time,the protein contents of JNK and p-JNK are upregulated in the 2.5 and 5μg/mL NiONPs groups,while c-Jun and p-c-Jun are significantly upregulated in the 1.25,2.5 and 5μg/mL NiONPs groups,suggesting NiONPs active JNK/c-Jun pathway in LX-2 cells.（4）After LX-2 cells are treated with JNK pathway inhibitor SP600125 combined with 5μg/mL NiONPs for 12h,the protein contents of Col-1a1,MMP2,JNK,p-JNK,c-Jun and p-c-Jun are all decreased in 5μg/mL NiONPs+SP600125 group compared with 5μg/mL NiONPs group,indicating that SP600125 restrain NiONPs-induced JNK/c-Jun signaling pathway activation and collagen overformation,and JNK/c-Jun pathway participates in NiONPs-induced collagen deposition in LX-2 cells.Conclusions:（1）NiONPs induce transcriptome changes of rat liver tissues,and DE mRNAs and DE ncRNAs interact and participate in the process of NiONPs-induced liver fibrosis by regulating the corresponding biological processes and signaling pathways.（2）NiONPs promote excessive collagen deposition by activating JNK/c-Jun signaling pathway in LX-2 cells.