Alteration In LncRNA-ErbB Signal Pathway In Lung And Bronchial Epithelial Cells Damage Induced By Radon

Objective:Based on the experimental model of radon exposure both in vivo and in vitro, the aim of the present study was to investigate the adverse effect of radon on mice lung tissue and human bronchial epithelial cells, and to explore the potential role of non-coding RNA（lnc RNA） in regulation of the ErbB signaling pathway in radon to provide an epigenetic mechanism in the process.Methods:（1） Setup of animal model with Radon exposure:Thirty BALB/c mice were randomly divided into two experimental groups and one control group with each of 10 mice. The mice were exposed to radon in a multi-function ecological radon room at a constant concentration to cumulative doses of 60 and 120WLM （Working Level Month） respectively. Pathological changes of lung tissue in mice after radon exposure were observed by HE stain, and the proteins related to ErbB signal pathway were measured by Western Blot. （2） Setup of cell model with radon exposure in vitro:HBE cells were exposed to radon at 20000Bq/m3 for 20min and repeated in 3 days to generate first generation （Rn1）, and continued to obtain Rn5 and Rn10 cells. The ability of cell proliferation and colony formation, cell cycle and levels of ROS, superoxide dismutase （SOD）, catalase （CAT）, and the protein level related to ErbB signal pathway were measured after radon exposure. （3）Measurement of ErbB signal pathway proteins:The proteins level related to ErbB signal pathway were detected by Western Blot and immunohistochemical （IHC） stain, including ErbB2, K-ras, C-abl and Mig6. （4） Screening of differential lncRNA expression:Differentially expressed lncRN A between radon exposure mice and control were screened by microarray, and the target genes and the gene interaction network were analyzed by GO and KEGG software. The most significant differential expression of lncRNA were selected according to the Function Prediction, FISH fluorescent stain was used to locate the intracellular site. The base sequence of IncRNA and the target protein was predicted by using NCBI databases. （5） The RNAi of lncRNA was transfected into V79 cell, and the target protein expression was measured by Western Blot. The vectors of IncRNA and the dual UTR of its target gene by splicing of luciferase were constructed, and the target sites were verified by dual luciferase reporter gene assay.Results:（1） Pathological observation revealed congestion and edema of the lung interstitial, thickening of alveolar septum with infiltration of inflammatory cells and collagen fiber after long-term radon exposure. The expression of ErbB2 and K-ras increased and those of C-abl, Mig6 and p73 decreased as detected by IHC. The phosphorylation levels of ErbB2 also increased with the dose accumulation of radon, and the proteins expression of C-abl, Mig6 and K-ras measured by Western Blot were identical to those by IHC method. （2） Long-term exposure of HBE cells to radon resulted in a malignant phenotype as evidenced by high proliferation rate and the formation of multiple colonies in soft agar. In addition, the levels of ROS and CAT increased and the activity of SOD was down-regulated. The S-phase of cell cycle in HBE cells showed a higher proportion after radon exposure. The protein levels of ErbB2 and K-ras and phosphorylation levels of ErbB2 were up-regulated, but the expression of C-abl and Mig6 were down-regulated in HBE cells after long-term exposure to radon. （3） The microarray screening revealed 1256 differentially expressed lncRNAs, among which 825 were up-regulated and 431 were down-regulated. The GO and KEGG analysis found that most of these lncRNAs were involved in the ErbB signal pathway. Nine of the differentially expression lncRNAs were chosen to be verified by real-time PCR, and the expressions of ELK1, FR146301, FR052959, n417375 and XR₁68810 were confirmed to be identical with the microarray result, and only the FR052959 was up-regulated. （4） The intracellular location of the up-regulated lncRNA_FR052959 was showen in the nuclear and cytoplasmic by FISH. The target gene of lncRNA_FR052959 was predicted to be C-abl according to the NCBI database. （5） The RNAi and dualluciferase reporter gene assay revealed that lncRNA_FR052959 could inhibit the activation of 5’-UTR initiator of the C-abl gene to regulate the expression in the ErbB pathway. In cells exposed to radon, this inhibitive effect was reduced significantly.Conclusions:1. Mice and HBE cell models with radon exposure were established successfully. Long-term exposure to radon could induce malignant cell phenotype in vitro, as shown by accelerated cell growth, cell cycle alteration, increased oxidative stress and enhanced cell invasion.2. Radon exposure induced alterations in expression of genes and proteins in the ErbB signaling pathway. Among which ErbB and K-ras were up-regulaed and Mig6 and C-abl were down-regulated, indicating an involvement of the pathway in the process of cell malignant transformation induced by radon exposure.3. A differentially expressed lncRNA-FR052959 was screened and up-regulated by radon exposure. The RNAi and dual luciferase reporter gene assay revealed that lncRNA_FR052959 could inhibit the activation of 5’-UTR initiator of the C-abl gene to regulate the C-abl expression in the ErbB pathway. In cells exposed to radon, this inhibitive effect was reduced significantly, indicating that lncRNA_FR052959 was involved in regulation of radon-induced cell damage via the ErbB signaling pathway. The findings may provide a new direction of study on epigenetic mechanism of radon carcinogenesis.