A Study On MicroRNAs Associated With Lung Cancer And Lung Injury Induced By Radon And Its Progeny

BackgroudRadon is the second leading cause of lung cancer after tobacco smoke. Inhalation of radon and its progeny can promote lung cancer that account for 10% of human lung cancer. Radon and its progeny are environmental carcinogens of human lung cancer based on epidemiological data supported by experimental evidence from cells and animals' model. MicroRNAs (miRNAs) are small, noncoding RNAs with 18～24 nucleotides in length that regulate gene expression. They are involved in the regulation of a variety of biological processes, including cell cycle progression, cell differentiation and development as well as human diseases, such as cancer, diabetes and immunogic or neurodegenerative disorders. Currently,10581 miRNAs from over 50 organisms are registered in the miRBase database.721 human miRNAs are now known, but this number may increase, as bioinformatics studies predict that up to 1000 miRNAs exist. MiRNAs posttranscriptionally and translationally repress gene expression by recognizing complementary target sites in the 3 untranslated region of target messenger RNAs. Each miRNA has hundreds of different conserved or nonconserved targets. It has been estimated that approximately 30% of the genes are regulated by miRNAs. The pattern of miRNA expression can be correlated with cancer type, stage, diagnosis and prognosis. Let-7, miR-29 and miR-34 family can be associated with lung cancer.ObjectiveTo investigate the regulatory role and molecular mechanisms of miRNAs in lung cancer and lung injury induced by radon and its progeny using malignant transformed cells induced byα-particles, radon-exposure mice model and lung cancer tissues of high radon exposure residents, with the aim of laying a foundation for diagnosis and therapeutic strategies of lung cancer and lung injury induced by radon and its progeny.Methods1.The differentially expressed miRNAs in early transformed cells, late transformed cells and their parental BEP2D cells were identified by miRNA microarray assay.2.The results of miRNA microarray assay were confirmed by TaqMan qPCR to detect miR-200 family expression in malignant transformed cells induced byα-particles.3.The mRNA level of ZEB family and epithelial-mesenchymal transition marker in malignant transformed cells induced byα-particles was detected by qPCR.4.The protein expression of ZEB family and epithelial-mesenchymal transition marker in malignant transformed cells induced byα-particles was measured by Western Blot and immunofluorescence.5.The specific SiRNA for ZEB1 was transfected into RHT35 cells to repress ZEB1 expression and the specific miR-200 family mimics was transfected into RHT35 cells to increase miR-200 family expression.6.In vitro wound healing assay was used to investigate whether overexpression of miR-200b and miR-141 could block migration of malignant transformed cells induced by a particles.7.The concentration of TGFβ1 secreted by early transformed cells, late transformed cells and their parental BEP2D cells was determined by ELISA kit.8.The expression of miR-200, ZEB family and epithelial-mesenchymal transition marker in RH20 was measured by qPCR and immunofluorescence after TGFβ1 for 48h.9.A radon-exposure mice model was established.lO.The expression of let-7, miR-29 and miR-34 family in lung tissues of radon-exposure mice model was detected by TaqMan qPCR.11.The protein level of CDK6 and RAS in lung tissues of radon-exposure mice model was measured by Western Blot.12.The expression of let-7, miR-29, miR-34 and miR-200 family in lung cancer tissues from high radon exposure residents was detected by TaqMan qPCR.Results1.There were 38 differentially expressed miRNAs in RH20 cells versus BEP2D cells, with 18 upregulated and 20 downregulated miRNAs. RH20 and RHT35 cells shared 25 differentially expressed miRNAs compared with BEP2D cells, with 15 down-regulated and 10 up-regulated miRNAs.87 differentially expressed miRNAs in RHT35 cells versus BEP2D cells were identified, with 47 upregulated and 40 downregulated miRNAs.38 differentially expressed miRNAs in RHT35 cells versus RH20 cells were found, with 20 upregulated and 18 downregulated miRNAs.2.1n agreement with results of miRNA microarray assay, the downregulation of mature miR-200a, miR-200b, miR-200c and miR-141 expression in RHT35 cells was validated by TaqMan qPCR compared with RH20 and BEP2D cells.3.The mRNA level of ZEB 1 and ZEB2 in RHT35 cells upregulated 9.44 fold and 27.56 fold respectively, compared with RH20 and BEP2D cells, p<0.01. The mRNA level of E-cadherin in RHT35 cells downregulated 4.40 fold, with N-cadherin upregulated 4.30 fold, compared with RH20 and BEP2D cells,p<0.01. This suggested that an inverse expression pattern between downregulation of miR-200 and E-cadherin and overexpression of ZEB family in RHT35 cells.4.The protein level of ZEB1, ZEB2 and N-cadherin in RHT35 cells increased, with E-cadherin decreased, compared with RH20 and BEP2D cells.5.Downregulation of ZEB 1 mRNA and protein level in RHT35 cells was found after transfection of siZEB1, compared with negative control. Although miR-200a and miR-200b expression were not affected, we detected upregulation of miR-200c and miR-141 expression (1.5-2.0 fold) after ZEB1 knockdown. These data suggested that overexpression of ZEB1 in RHT35 cells could promote downregulation of miR-200c and miR-141 expression during malignant transformation induced byαparticles.6.Overexpression of miR-200b and miR-141 led to reduced expression of ZEB1, ZEB2 and TGFβ2 in RHT35 cells compared with mimic control. This indicated a double-negative feedback loop between miR-200 and ZEB family. Overexpression of miR-200b and miR-141 upregulated E-cad expression (about 2-fold) and downregulated N-cad expression in RHT35 cells. These results suggested that overexpression of miR-200 family could block epithelial-mesenchymal transition in RHT35 cells by targeting ZEB family.7.Overexpression of miR-200b and miR-141 strongly blocked migration of RHT35 cells using in vitro wound healing assays, compared with mimic control.8.The concentration of TGFβ1 secreted by RHT35 cells increased, compared with RH20 and BEP2D cells, p<0.05. There was a change from a cobblestone to a spindle-like morphology in RH20 cells after TGFβ1 for 48h compared with control. The expression of ZEB family, N-cadherin, Vimentin and Fibronectin in RH20 increased after TGFβ1 for 48h compared with control, p<0.01. The expression of miR-200a, miR-200c and miR-141 in RH20 decreased after TGFβ1 for 48h compared with control. These results suggested that TGFβ1 could induce epithelial-mesenchymal transition in RH20 cells.9.The expression of let-7a, let-7c and let-7f in lung tissues from mice with one and three months after exposure to 30 WLM and 60 WLM of radon significantly decreased compared with control, whereas no obvious difference in expression of let-7a, let-7c and let-7f in lung tissues from mice operated immediatedly after exposure to 30 WLM and 60 WLM was observed. No visible difference in expression of miR-29 and miR-34 family in lung tissues of radon-exposure mice was observed compared with control.10.The protein level of CDK6 in lung tissues from mice with one months after exposure to 60 WLM upregulated compared with control, whereas no obvious difference in protein level of RAS.11.The expression of let-7, miR-29, miR-34, miR-200a and miR-200b in lung cancer tissues from high radon exposure residents significantly reduced compared with control, p<0.05, whereas no obvious difference in level of miR-200c. No visible association of differentiation in lung cancer tissues from high radon exposure residents with miR-200a, miR-200b and miR-200c expression was observed compared with control through independent sample t test.Conclusion1.MiRNAs are differentially expressed in the stages of carcinogenesis of BEP2D cells induced by a particles, which suggests that miRNAs may play an important role during malignant transformation of BEP2D cells induced byαparticles. 2.RH20 and RHT35 cells share differentially expressed miRNAs compared with BEP2D cells, which suggests that miRNAs can act as biomarkers of early diagnosis of malignant transformation of cells induced by a particles.3.Overexpression of ZEB1 in RHT35 cells promote downregulation of miR-200c and miR-141 expression during malignant transformation induced by a particles.4.RHT35 cells exhibit features of epithelial-mesenchymal transition during malignant transformation induced by a particles. TGFP may play a role in epithelial-mesenchymal transition of RHT35 cells.5.Overexpression of miR-200 family blocks epithelial-mesenchymal transition and migration in RHT35 cells by targeting ZEB family.6.Downregulation of let-7 family expression is an early event during lung cancer and lung injury induced by radon and its progeny, which suggests that downregulation of let-7 family expression may play an important role in lung cancer and lung injury induced by radon and its progeny.7.Downregulation of miR-29, miR-34 and miR-200 family expression associates with lung cancer and lung injury induced by radon and its progeny.