While the incidence of pneumoconiosis is decreasing every year worldwide [16–19], the number of pneumoconiosis cases is still high in China [20–24]. Silicosis is the main type of pneumoconiosis, accounting for about 90% of all occupational diseases . Studies by Wang Dan and others show that the data reported on occupational diseases is less than 7% of the actual incidence . Silicosis is thought to emerge as one of the major occupational diseases that seriously affects the health of concerned populations.
The pathogenesis of silicosis is complex. The entry of silica dust into the body results in macrophage damage in the early stage, followed by the triggering of the immune system, formation of antigen-antibody immune complexes, and development of pathological changes related to silicosis. Lymphocytes in the human peripheral blood are important cellular components of immune response. The analysis and interpretation of genetic material in lymphocyte nucleus may reflect the genesis and development of silicosis. The occurrence of silicosis is affected by several targets. Therefore, it is essential to explore the mechanism underlying silicosis and identify the key reliable targets at the molecular level.
The recent development in biogenetic exploration technologies has facilitated the understanding of DEGs in several diseases to provide ideas for accurate treatment [24, 27, 28]. For instance, four molecular markers associated with the development of gliomas have been identified through biogenetic approaches . A study on hepatocellular carcinoma employed the microarray technology to search and analyze a biological database and finally identified 16 hub genes closely related to the development of hepatocellular carcinoma . Another study analyzed the expression levels of 322 immune genes and revealed the enhancement in the immune characteristics of patients with neuroblastoma; this study finally selected eight immune genes as the molecular markers of neuroblastoma . Biotechnology and its application in disease-related gene prediction may provide new cues and support for the selection of DEGs related to silicosis.
In the present study, we used bioinformatic tools to identify the DEGs and target molecular markers from peripheral blood lymphocytes. GEO2R for the analysis of 80813 whole gene transcriptome data sets may help us identify the DEGs between silicosis and non-silicosis samples. With enrichment analysis, we found that these DEGs were mainly associated with JAK-STAT, Wnt, and adhesion molecule signaling pathways with respect to EMT. Among the most important modules of the PPI network, three central genes (PI3K, ITGB1, and ILK) showed the highest score, suggestive of their important roles in the occurrence or development of EMT in silicosis.
ITGB1 is a member of the integrin family, while integrin is a member of the adhesion molecule group . Therefore, we detected the expression levels of SICAM in the serum samples from 89 silicosis patients and 94 dust-exposed workers, and found that the difference was statistically significant. After adjustment for age, length of service, and smoking index, the expression levels of SICAM in the serum were still statistically significant between the two groups. The results of ROC curve analysis also suggested that SICAM could be used as an index for the early screening of silicosis. Thus, PI3K, ITGB1, and ILK may play an important role in the occurrence and development of EMT in silicosis.
EMT refers to the loss of polarity of epithelial cells and the disappearance of tight junctions between cells under the effect of certain factors, thereby imparting the morphology and characteristics of interstitial cells . The process of pulmonary fibrosis may be regarded as EMT in tissue repair. The level of E-cadherin protein and the relative expression of E-cadherin gene in silicosis model group decreased, while the levels of alpha smooth muscle actin and vimentin proteins and the relative expression of their RNAs increased, suggestive of the occurrence of EMT during the development of silicosis [30–32].
PI3K is a very important member of the phospholipid kinase family. It has lipid kinase and protein kinase activities. P13K signaling pathway is an important pathway widely involved in cell proliferation, differentiation, cycle regulation, and apoptosis. Activated PI3K may produce the second messenger phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) and further phosphorylate protein kinase B (AKT) protein. Activated AKT enters the nucleus and induces or inhibits the downstream proteins for the regulation of cell survival and proliferation, apoptosis, angiogenesis, and pulmonary fibrosis [33–35]. Studies have confirmed that the inhibition of PI3K/AKT signaling pathway may significantly reduce hepatic stellate cell (HSC) proliferation, alpha-collagen type III mRNA expression, and collagen type III secretion . Over-activation of P13K/AKT/hypoxia-inducible factor-1a (HIF-1a) pathway may affect the normal repair of alveolar epithelial cells through the regulation of cell proliferation and apoptosis, leading to the production of collagen III and formation of pulmonary fibrosis . Some studies have shown that HIF-1a may be regulated by Snail and beta-catenin signaling pathways to promote the transformation of alveolar epithelial cells to interstitial cells and participate in the formation of pulmonary fibrosis . These results suggest that the activation of P13K signaling pathway is associated with the process of pulmonary fibrosis via EMT.
ITGB1 is a member of the integrin family of proteins that are mainly involved in the regulation of cell adhesion and recognition during embryogenesis, hemostasis, tissue repair, immune response and tumor cell metastasis .
As a member of the cell adhesion molecule family, integrins not only function as adhesion molecules but also are also involved in transmembrane connection. These molecules can connect the extracellular matrix-integrin molecule-cytoskeleton protein and participate in the activation of multiple signal transduction pathways. Integrins play an important role in several physiological and pathological processes. The connection of the extracellular matrix with integrin may result in the formation of focal adhesion (FAP). Many cytoskeletal proteins are expressed in FAP, including fibronectin (FN), laminin (LN), collagen, and wavin (VN). In addition, many important signal transduction molecules such as FAK, ILK, and PI3K could be activated by the extracellular matrix and integrin, subsequently leading to the activation of a variety of downstream signal transduction pathways regulating cell proliferation, survival, and differentiation.
Integrin is thought to be closely related to the occurrence of EMT. The abnormal secretion of integrin in response to trauma-induced tissue stimulation may promote EMT and development of fibrosis . Galliher et al. found that integrin αvβ3 promotes EMT by regulating the expression of transforming growth factor-beta (TGF-β) receptor. In the absence of integrin β3, TGF-β may not induce EMT . Thus, integrin is an upstream molecule that promotes EMT.
ILK is a serine/threonine protein kinase that binds to the cytoplasmic domain of integrin β1 and β3 subunits and participates in integrin, growth factor, Wnt, and TGF-β/Smad signal transduction pathways. ILK also regulates cell growth, survival, cell cycle, EMT, apoptosis, invasion, migration, cancer, and angiogenesis.
Studies have shown that ILK is closely related to EMT of cells and promotes the occurrence and development of EMT. Serrano et al. found that EMT in mammary epithelial cells results in a significant increase in the expression of ILK; this transformation process may be effectively reversed by blocking ILK expression . Mao Zhongyi et al. found that the expression of ILK and TGF-β1 increased with the development of liver fibrosis and positively correlated with the degree of liver fibrosis . Li Xiaoqin and others confirmed that ILK was closely related to the occurrence and development of renal interstitial fibrosis, and that its expression may serve as an important reference index to judge the progression of renal diseases and prognosis . Cui Hongshuai's research shows that the level of ILK increases with the severity of pulmonary fibrosis in mice, suggestive of the close relationship between ILK and occurrence of pulmonary fibrosis. ILK may reduce the degradation of β-catenin and its transfer to the nucleus. It binds to T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) in the nucleus and promotes the transcription of target genes, thereby promoting EMT . Thus, the activation of ILK is closely related to EMT.
Our study has some limitations. First, the screening of the three key genes related to the development and progression of silicosis is based on bioinformatic technology, which needs further verification with in vitro and in vivo experiments. Collection of samples from silicosis and non-silicosis subjects and establishment of a suitable animal model of silicosis are warranted for the verification of the mechanism of action of the three genes in lymphocytes. Second, the data set of the transcription group is based on the lymphocytes from three cases of silicosis and three control subjects. The data were collected thrice and analyzed without mixing to obtain maximum and minimum values; the minimum values were used. In the second phase, we plan to re-derive the transcription group data set according to the conventional method to verify the similarities and differences between the two detection methods.