A transcriptomics-based investigation of the mechanism of pulmonary fibrosis induced by nickel oxide nanoparticles.

Nickel oxide nanoparticles (NiONPs) are an emerging nanomaterial, which poses a huge threat to the health of workplace population. Nanoparticles induce pulmonary fibrosis, and its mechanisms are associated with noncoding RNAs (ncRNAs). However, ncRNAs and competing endogenous RNA (ceRNA) networks which involved in NiONP-induced pulmonary fibrosis are still unclear. This study aimed to identify ncRNA-related ceRNA networks and investigate the role of the Wnt/ß-catenin pathway in pulmonary fibrosis. Male Wistar rats were intratracheally instilled with 0.015, 0.06, and 0.24 mg/kg NiONPs twice a week for 9 weeks. First, we found there were 93 circularRNAs (circRNAs), 74 microRNAs (miRNAs), 124 long non-coding RNAs (lncRNAs), and 1675 messenger RNAs (mRNAs) differentially expressed through microarray analysis. Second, we constructed ceRNA networks among lncRNAs/circRNAs, miRNAs and mRNAs and identified two ceRNA networks (lncMelttl16/miR-382-5p/Hsd17b7 and circIqch/miR-181d-5p/Stat1) after real time-quantitative polymerase chain reaction (RT-qPCR) validation. Furthermore, based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, ncRNAs were found to be involved in biological processes and signaling pathways related to pulmonary fibrosis. KEGG analysis showed that NiONPs activated the Wnt/ß-catenin pathway in rats. In vitro, HFL1 cells were treated with 0, 50, 100, and 200 µg/mL NiONPs for 24 h. We found that NiONPs induced collagen deposition and Wnt/ß-catenin pathway activation. Moreover, a blockade of Wnt/ß-catenin pathway alleviated NiONP-induced collagen deposition. In conclusion, these observations suggested that ncRNAs were crucial in pulmonary fibrosis development and that the Wnt/ß-catenin pathway mediated the deposition of collagen.

LncRNA AP000487.1 regulates PRKCB DNA methylation-mediated TLR4/MyD88/NF-κB pathway in Nano NiO-induced collagen formation in BEAS-2B cells.

Nickel oxide nanoparticles (Nano NiO) have been shown to cause pulmonary fibrosis; But, the underlying epigenetic mechanisms remain poorly understood. In this study, we aimed to investigate the role of lncRNA AP000487.1 in regulating PRKCB DNA methylation and the Toll-like receptor 4 (TLR4)/ Myeloid differentiation primary response 88 (MyD88)/ Nuclear factor kappa-B (NF-κB) pathway in Nano NiO-induced collagen formation. We found that lncRNA AP000487.1 was able to bind to the promoter region of the PRKCB gene by Chromosomal RNA pull-down experiments (Ch-RNA pull-down). Moreover, Nano NiO exposure led to down-regulation of lncRNA AP000487.1 expression and PRKCB DNA methylation, resulting in up-regulation of PRKCB expression, activation of the TLR4/MyD88/NF-κB pathway, and increased collagen formation in BEAS-2B cells. Conversely, overexpression of lncRNA AP000487.1 restored PRKCB expression, reduced its hypomethylation and attenuated TLR4/MyD88/NF-κB pathway activation and collagen formation. Furthermore, treatment with the DNA methylation inhibitor, decitabine, alleviated Nano NiO-induced PRKCB2 expression, TLR4/MyD88/NF-κB pathway activation, and collagen formation. Additionally, using PRKCB2 overexpression plasmid, PRKCB2 siRNA, and PRKCB2 protein inhibitor LY317615 influenced NF-κB pathway activity and collagen formation. Finally, TLR4 inhibitor (TAK-242) restrained Nano NiO-induced MyD88/NF-κB pathway activation and excessive collagen formation. In summary, we demonstrated that the down-regulated lncRNA AP000487.1 could cause PRKCB hypomethylation and increased expression, resulting in NF-κB pathway activation and collagen formation in Nano NiO-induced BEAS-2B cells. This is the first study to reveal the role of lncRNA AP000487.1 in regulating collagen formation in Nano NiO-exposed BEAS-2B cells. Our study identified that lncRNA AP000487.1/PRKCB hypomethylation/NF-κB pathway was a regulatory axis of BEAS-2B cells collagen excessive formation. Our findings indicate that lncRNA AP000487.1 and PRKCB DNA methylation may function as biomarkers or potential targets in response to Nano NiO exposure.

Whole-transcriptome sequencing revealed differentially expressed mRNAs and non-coding RNAs played crucial roles in NiONPs-induced liver fibrosis.

Nickel oxide nanoparticles (NiONPs) induced liver fibrosis, while its mechanisms associated with transcriptome remained unclear. This study aimed to investigate the roles of differentially expressed (DE) messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) in NiONPs-induced liver fibrosis, and further confirm whether JNK/c-Jun pathway enriched by the DE RNAs was involved in the regulation of the disease. A liver fibrosis rat model was established by intratracheal perfusion of NiONPs twice a week for 9 weeks. Whole-transcriptome sequencing was applied to obtain expression profiles of mRNAs, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) in the model rat and control liver tissues. Comparing the RNA expression profiles of the model and control liver tissues, we identified 324 DE mRNAs, 129 DE lncRNAs, 24 DE miRNAs and 33 DE circRNAs, and the potential interactions among them were revealed by constructing two co-expression networks, including lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA networks. Using RT-qPCR, we verified the sequencing results of some RNAs in the networks and obtained similar expression profiles, indicating our sequencing results were reliable and referable. Through Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we predicted the biological functions and signaling pathways potentially related to NiONPs-induced liver fibrosis, such as "positive regulation of JNK cascade", "inflammatory response", "transcription factor binding", and MAPK, Wnt, PI3K-Akt signaling pathways. JNK/c-Jun pathway, a subclass of MAPK signal, was selected for further investigation because it was significantly enriched by fibrosis-related DE genes and activated in animal models. In vitro, we detected the cytotoxicity of NiONPs on LX-2 cells and treated the cells with 5 µg/ml NiONPs for 12 h. The results showed NiONPs induced the up-regulated protein expression of fibrotic factors collagen-1a1 (Col-1a1) and matrix metalloproteinas2 (MMP2) and JNK/c-Jun pathway activation. While these effects were reversed after JNK/c-Jun pathway was blocked by SP600125 (JNK pathway inhibitor), indicating the pathway was involved in NiONPs-induced excessive collagen formation. In conclusion, our results revealed the DE mRNAs and ncRNAs played crucial roles in NiONPs-induced liver fibrosis, and JNK/c-Jun pathway mediated the development of the disease.

LncRNA HOTAIRM1 Involved in Nano NiO-Induced Pulmonary Fibrosis via Regulating PRKCB DNA Methylation-Mediated JNK/c-Jun Pathway.

Nickel oxide nanoparticles (Nano NiO) lead to pulmonary fibrosis, and the mechanisms are associated with epigenetics. This study aimed to clarify the regulatory relationship among long noncoding RNA HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1), DNA methylation and expression of protein kinase C beta (PRKCB), and JNK/c-Jun pathway in Nano NiO-induced pulmonary fibrosis. Therefore, we constructed the rat pulmonary fibrosis model by intratracheal instillation of Nano NiO twice a week for 9 weeks and established the collagen deposition model by treating BEAS-2B cells with Nano NiO for 24 h. Here, the DNA methylation pattern was analyzed by whole-genome bisulfite sequencing in rat fibrotic lung tissues. Then, we integrated mRNA transcriptome data and found 93 DNA methylation genes with transcriptional significance. Meanwhile, the data showed that Nano NiO caused the down-regulation of lncRNA HOTAIRM1, the hypomethylation, and up-regulation of PRKCB2, JNK/c-Jun pathway activation, and collagen deposition (the up-regulated Col-I and α-SMA) both in vivo and in vitro. DNMTs inhibitor 5-AZDC attenuated Nano NiO-induced PRKCB2 expression, JNK/c-Jun pathway activation, and collagen deposition, but overexpression of PRKCB2 aggravated the changes mentioned indicators in Nano NiO-induced BEAS-2B cells. Furthermore, JNK/c-Jun pathway inhibitor (SP600125) alleviated Nano NiO-induced excessive collagen formation. Additionally, overexpression of HOTAIRM1 restrained the PRKCB hypomethylation, the activation of JNK/c-Jun pathway, and collagen formation induced by Nano NiO in BEAS-2B cells. In conclusion, these findings demonstrated that HOTAIRM1 could arrest Nano NiO-induced pulmonary fibrosis by suppressing the PRKCB DNA methylation-mediated JNK/c-Jun pathway.

LncRNA MEG3 ameliorates NiO nanoparticles-induced pulmonary inflammatory damage via suppressing the p38 mitogen activated protein kinases pathway.

The lung inflammatory damage could result from the nickel oxide nanoparticles (NiO NPs), in which the underlying mechanism is still unclear. This article explored the roles of long noncoding RNA maternally expressed gene 3 (lncRNA MEG3) and p38 mitogen activated protein kinases (p38 MAPK) pathway in pulmonary inflammatory injury induced by NiO NPs. Wistar rats were treated with NiO NPs suspensions (0.015, 0.06, and 0.24 mg/kg) by intratracheal instillation twice-weekly for 9 weeks. Meanwhile, A549 cells were treated with NiO NPs suspensions (25, 50, and 100 µg/ml) for 24 h. It can be concluded that the NiO NPs did trigger pulmonary inflammatory damage, which was confirmed by the histopathological examination, abnormal changes of inflammatory cells and inflammatory cytokines (IL-1ß, IL-6, TGF-ß1, TNF-α, IFN-γ, IL-10, CXCL-1 and CXCL-2) in bronchoalveolar lavage fluid (BALF), pulmonary tissue and cell culture supernatant. Furthermore, NiO NPs activated the p38 MAPK pathway and downregulated MEG3 in vivo and in vitro. However, p38 MAPK pathway inhibitor (10 µM SB203580) reversed the alterations in the expression levels of inflammatory cytokines induced by NiO NPs. Meanwhile, over-expressed MEG3 significantly suppressed NiO NPs-induced p38 MAPK pathway activation and inflammatory cytokines changes. Overall, the above results proved that over-expression of lncRNA MEG3 reduced NiO NPs-induced inflammatory damage by preventing the activation of p38 MAPK pathway.

LncRNA MEG3 restrained pulmonary fibrosis induced by NiO NPs via regulating hedgehog signaling pathway-mediated autophagy.

Long noncoding RNA maternally expressed gene 3 (lncRNA MEG3) was down-regulated in pulmonary fibrosis of rats induced by Nickel oxide nanoparticles (NiO NPs), while the downstream regulatory mechanisms of MEG3 remain unclear. This study aimed to investigate the relationship among MEG3, Hedgehog (Hh) signaling pathway and autophagy in pulmonary fibrosis caused by NiO NPs. The pulmonary fibrosis model in rats was constructed by intratracheal instillation of 0.015, 0.06, and 0.24 mg/kg NiO NPs twice a week for 9 weeks. Collagen deposition model was established by treating A549 cells with 25, 50, and 100 µg/mL NiO NPs for 24 h. Our results indicated that NiO NPs activated Hh pathway, down-regulated the expression of MEG3, and reduced autophagy activity in vivo and in vitro. Meanwhile, the autophagy process was promoted by Hh pathway inhibitor (CDG-0449), while the collagen formation in A549 cells was reduced by autophagy activator (Rapamycin). Furthermore, the overexpressed MEG3 inhibited the activation of Hh pathway, resulting in autophagy activity enhancement along with collagen formation reduction. In summary, lncRNA MEG3 can restrain pulmonary fibrosis induced by NiO NPs via regulating hedgehog signaling pathway-mediated autophagy, which may serve as a potential therapeutic strategy for pulmonary fibrosis.

LncRNA MEG3 Involved in NiO NPs-Induced Pulmonary Fibrosis via Regulating TGF-ß1-Mediated PI3K/AKT Pathway.

Long noncoding RNA maternally expressed gene 3 (MEG3) involves in fibrotic diseases, but its role in nickel oxide nanoparticles (NiO NPs)-induced pulmonary fibrosis remains unclear. The present study aimed to explore the relationships among MEG3, transforming growth factor-ß1 (TGF-ß1) and phosphoinositide 3-kinase (PI3K)/AKT pathway in NiO NPs-induced pulmonary fibrosis. Wistar rats were intratracheally instilled with NiO NPs twice a week for 9 weeks, and human lung adenocarcinoma epithelial cells (A549 cells) were exposed to NiO NPs for 24 h. The pathological alterations and increased hydroxyproline indicated that NiO NPs caused pulmonary fibrosis in rats. The up-regulated type I collagen (Col-I) suggested that NiO NPs-induced collagen deposition in A549 cells. Meanwhile, NiO NPs could significantly down-regulate MEG3, up-regulate TGF-ß1 and activate PI3K/AKT signaling pathway both in vivo and in vitro. However, we found that the PI3K/AKT pathway activated by NiO NPs could be suppressed by 10 µM TGF-ß1 inhibitor (SB431542) in A549 cells. The protein markers (Col-I, Fibronectin, and alpha-smooth muscle actin) of collagen deposition up-regulated by NiO NPs were reduced by 10 µM PI3K inhibitor (LY294002). Furthermore, we further found that overexpressed MEG3 inhibited the expression of TGF-ß1, resulting in the inactivation of PI3K/AKT pathway and the reduction of collagen formation. In summary, our results validated that MEG3 could arrest NiO NPs-induced pulmonary fibrosis via inhibiting TGF-ß1-mediated PI3K/AKT pathway.

Evidence that ghrelin may be associated with the food intake of gibel carp (Carassius auratus gibelio).

Ghrelin, a non-amidated peptide hormone, is a potent anorectic neuropeptide implicated in feeding regulation in mammals and non-mammalian vertebrates. However, the involvement of ghrelin in the feeding behavior of teleosts has not been well understood. To better understand the role of ghrelin in the regulation of appetite in fish, in this study, we cloned the cDNAs encoding ghrelin and investigated their mRNA distributions in gibel carp tissues. We also assessed the effects of different nutritional status on ghrelin mRNA abundance. Ghrelin mRNAs were ubiquitously expressed in ten tissues (intestine, liver, brain, mesonephron, head kidney, spleen, skin, heart, muscle, gill and pituitary gland), and relatively high expression levels were detected in the gut. Postprandial studies analysis revealed a significant postprandial decrease in ghrelin mRNA expression in the gut (1 and 3 h after the regular feeding time). In addition, ghrelin mRNA expression in the gut significantly increased at day 7 after fasting and declined sharply after refeeding, which suggested that ghrelin might be involved in the regulation of appetite in gibel carp. Overall, our result provides basis for further investigation into the regulation of feeding in gibel carp.