Rare earth (RE) recognized as “industry gold” is an indispensable strategic mineral resource in many fields, such as the national defense industry, new technology, and so on. China has unique rare earth resources. With the massive mining and processing of rare earths, the prevalence of rare-earth pneumoconiosis (REP) among workers exposed to rare earths is increasing year by year (Ji et al., 2017). REP is the main occupational disease of rare earth exposed workers and there is no effective treatment methods. Therefor it is necessary to further explore the unknown molecular mechanism and discover potential biomarkers.

With the development of biological sequencing technology, it has been gradually found that more than 70% of the genes in the human genome can be transcribed, and the transcripts contain non-coding RNA (ncRNA) (Jalali et al., 2016). Studies have shown that ncRNAs have regulatory significance in tumors (Evangelista et al., 2021; Viviana and Massimo, 2021). Experts have conducted extensive in-depth studies on pneumoconiosis, focusing on symptoms from oxidative stress to epithelial-mesenchymal transformation (EMT) and found ncRNAs are involved in the regulation of fibrosis process (Perdas et al., 2016; Sun et al., 2016). As one of ncRNA, previous studies have shown that miRNA is dysfunctional in cancer and is involved in a variety of cancer-related biological processes, including apoptosis, proliferation and EMT (Bartel, 2009; Wang et al., 2016). Because the expression of miRNA has obvious characteristics in different cancers, it has a great prospect in the clinical diagnosis of different diseases (Thum et al., 2008; Yamada et al., 2013; Mirzaei et al., 2016). Existing evidence shows that lncRNA can participate in multiple biological processes, including cell proliferation, cell cycle, cell differentiation and apoptosis (Zhang et al., 2019). Abnormal expression of lncRNAs has been associated with the occurrence and development of inflammation, cancer and immune diseases (Roux and Lindsay, 2015). But there are few researches on pathogenesis of REP at the genetic level.

In 2011, the competitive endogenous RNA (ceRNA) hypothesis was proposed (Leonardo et al., 2011). The ceRNA hypothesis details that all kinds of RNA molecules in the human body are closely connected and form a huge network of interactions. Under normal conditions, various types of molecules in the human body are in dynamic equilibrium. When the expression of one or more molecules is up-regulated or down-regulated, there will be a chain reaction that disrupts the homeostasis, and eventually leads to the occurrence of disease. A large number of experimental studies have confirmed lncRNA-miRNA-mRNA ceRNA regulatory networks in liver cancer, lung cancer, and breast cancer (He et al., 2020; Xiao et al., 2020; Luo et al., 2021). Study Yi et al. (2018) has found that lncRNA-ATB could adsorb miRNA200c and lead to the high expression of ZEB1 protein, which promoted the occurrence of pulmonary fibrosis induced by cellular EMT.

However, at present, the related research at gene level on REP has been rarely reported. Therefore, in this study, we analyses the expression profiles of ncRNA by RNA sequencing, and predicted related functions through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), then established the ceRNA network and Survival Analysis to discover the relationships between ncRNAs and mRNA and do the lung cancer survival analysis. Our finding might discover new pathogenesis of SAP and provide new treatment ideas.

At present, more and more studies have shown that ncRNAs play an important role in the occurrence and development of various diseases. Our research studied the lncRNA miRNA and mRNA expression profile REP plasma to explore the underlying function of lncRNA and miRNA. In total, we identified 125 differentially expressed lncRNA, 49 of which were up-regulated and 76 of which were down-regulated, 5 differentially expressed miRNAs and 82 differentially expressed mRNA, 38 were up-regulated and 44 were down-regulated. Among them, the first two up-regulated and down-regulated RNA were verified by qRT-PCR, and the results were consistent with the sequencing results.

In the screening of miRNA expression profile of REP patients, there were 5 differentially expressed miRNA, and all of them were down-regulated, namely hsa-let-7d-3p, hsa-miR-16-2-3p, hsa-miR-1296-5p, hsa-miR-7106-5p, and hsa-miR-4515. Let-7d is one of the earliest and most deeply studied miRNA. Researches have shown that the expression of let-7 is decreased in a variety of tumor cells (Perdas et al., 2016; Sun et al., 2016), while restoring the expression of let-7 in tumor cells can inhibit the migration and invasion of tumor cells, so the let-7 family often plays the role of tumor suppressor genes. Recent studies have shown that let-7 plays an important role in fibrotic diseases (Pandit et al., 2010; Wang et al., 2016; Chen et al., 2017; Matsuura et al., 2018), as an important member of the let-7 family, let-7d has the same function. For example in the process of pulmonary fibrosis, transforming growth factor β1 (TGF-β1) can bind to the let-7d promoter through Smad3, resulting in down-regulation of let-7d, which further up-regulates the high mobility group protein A2 (HMGA2) of its target gene, promotes EMT, and leads to fibrosis (Pandit et al., 2010). There is also a low expression of let-7d in the EMT process of renal fibrosis, while the high expression of let-7d can inhibit the EMT process of adrenocortical cells induced by TGF-β1 (Wang et al., 2016). Our results are consistent with the above researches, let-7d expression of REP shows down-regulated.

Beside let-7d, the other four miRNA are rarely reported in pneumoconiosis. But for miR-16-2-3p, its role in other diseases has been reported such as in ovarian cancer. Study has found that miR-16-2-3p might be involved in the progression of Ig A nephropathy (Yan et al., 2015) and non-syndromic cleft lip (Zou et al., 2016). In a study by Liang et al. (2015), it was found that miR-16-2-3p negatively regulates the protein expression level of IL-6 by binding to the 3′UTR of IL-6 for post-transcriptional regulation. As we all know, IL-6, as a pro-inflammatory factor, plays an important role in the occurrence and development of pneumoconiosis. Our previous study have shown that inflammatory factors in the blood of workers exposed to rare earths are increased.

Studies have found that lncRNAs which have miRNA response elements have the ability of sponge miRNA (Arun et al., 2018). So LncRNAs can affect the abundance of target gene mRNA and thus its protein level through sponge miRNA. LncRNAs are involved in the occurrence and development of many diseases by acting as a competitive endogenous RNA (ceRNA), such as tumors (Chao et al., 2020), non-small cell lung cancer (Wang et al., 2020), nervous system development (Yang et al., 2018), metabolic diseases (Zhu et al., 2019), cardiovascular diseases (Cai et al., 2020). In order to better understand the role and interactions of differentially expressed lncRNA, miRNA, and mRNA in REP, we constructed a lncRNA-miRNA-mRNA-related ceRNA regulatory network. And the ceRNA network showed that the expression of lncRNAs and mRNAs are consistent in trend, and the main trend is up-regulated. Some researchers also found that the expression trends of lncRNAs and mRNAs are consistent when they analyzing co-expression of lncRNAs-mRNAs in other disease (Feng et al., 2018; Wang et al., 2021). This result proves that some lncRNAs act as miRNA sponge to affect one or more downstream gene expression and participate in the occurrence and development of REP. Then, we focused on miR-16-2-3p and its upstream and downstream RNA and constructed miR-16-2-3p ceRNA regulatory network. We found that miR-16-2-3p can affect the NM_006889 which is downstream gene of CD86. CD86 is a prominent surface molecule of dendritic cells and plays an important role in the immune system. Another study found that the increase of IL-6 can promote the expression of CD86 and enhance its ability to stimulate the proliferation of allogeneic T cells. Meanwhile, miR-16-2-3p also connected with NM_006889, which is related to lncRNA small nucleolar RNA host gene 5 (SNHG5). SNHG5 has been shown to be involved in the development and tumorigenesis of a variety of cancers (liver cancer, breast cancer, osteosarcoma, colorectal cancer; Ju et al., 2018; Li et al., 2018; Chi et al., 2019; Zhang et al., 2019; Li et al., 2020). Its disorder is closely related to metastasis, pathological staging, and prognosis (Li et al., 2020; Shen et al., 2020). For example, SNHG5 is also involved in the p38/MAPK signal pathway, Wnt/CTNNB1, and other pulmonary fibrosis-related signaling pathways (Chen et al., 2019; Hu et al., 2019). So it may be used as a key RNA for further study of. This result proves that miR-16-2-3p may be involved in the key process of immune response and pulmonary fibrosis-related signaling pathways. MiR-16-2-3p and SNHG5may be as a potential specific biomarker of the pathogenesis of REP.

Measnwhile the GO enrichment analysis results indicated that these differentially expressed miRNA were participated in the response of cells to extracellular stimulation, cell response to DNA damage stimulation, cell adhesion. KEGG analysis showed that the differential miRNA was mainly concentrated in the Hedgehog signaling pathway, pantothenic acid and CoA biosynthesis. Among these, Hedgehog signaling pathway is activated in different tumors, which may promote the occurrence of multiple types of tumors by promoting the process of tumorigenesis and metastasis (Bailey et al., 2007; Aval et al., 2017). These results suggesting differentially expressed miRNA may related to the process of EMT, inflammatory reaction and Hedgehog signaling pathway.

In addition, in order to better understand the relationship between the differentially expressed mRNAs, we used the STRING database to analyze the differentially expressed mRNAs and construct a PPI network graph, which consists of 18 nodes and 14 edges. Among them, SNW1 had the highest score of PPI relationship, which can fully activate the TGF-β/Smad3 signaling pathway by interacting with Smad3. At the same time, it was also found to be involved in NF-κB and Notch signaling pathways, which are closely related to the occurrence and development of pneumoconiosis (Verma et al., 2019). ATG4C is a molecule related to autophagy. Other study Gao et al. (2016) found that ATG4c is targeted by miR-142-3p and inhibits its expression in mouse macrophages, resulting in a significant down-regulation of LC3 II protein, thus regulating autophagy. The above two molecules can be used as key targets for further study of the mechanism of REP.

At last, we explore the survival analysis of the differentially expressed lncRNA, miRNA, and mRNA with the top five scores obtained by PPI network analysis in patients with REP. We found that four RNAs were significant in the survival analysis of lung cancer, in which the expression levels of miR-16-2-3p, lnc CEP83-AS1, and KRR1 were positively correlated with the overall survival rate, while the expression levels of SNW1 were negatively correlated with the overall patient survival rate. Study Maximov et al. (2019) found that miR-16-2-3p plays an important role as a tumor suppressor gene in osteosarcoma. In this analysis, miR-16-2-3p also showed its anti-cancer effect, which was positively correlated with the overall survival rate of patients with lung cancer, but it was down-regulated in patients with REP. Whether the down-regulation of miR-16-2-3p will increase the risk of lung cancer in patients with REP remains to be further studied.

In conclusion, we found 125 lncRNA, 5 miRNA, and 82 mRNA differentially expressed in the plasma of patients with REP and constructed a REP-related ceRNA network. In particular, MiR-16-2-3p and SNHG5 is closely related to the occurrence and prognosis of REP through inflammatory reaction, which could be as biomarker for REP.

The datasets generated for this study can be provided if requested. Requests to access these datasets should be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Baotou Medical College. The patients/participants provided their written informed consent to participate in this study.

S-hW, X-mS, and Y-hZ designed the research. Y-cB, Y-rG, and L-hH carried out the data collection and data analysis. NB and H-yS collected clinical samples. X-mS and Y-cB wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.