TP53 Targets Mitochondria In Mediation Of The Radon-induced Malignant Transformation Of Lung Cells

Background:Lung cancer is a multi-causal disease.Among the many pathogenic factors,radioactive radon and its progeny are considered to be the second most important risk factor for lung cancer after smoking,and the most important risk factor among never-smokers.Radon(Rn)is a colorless and odorless radioactive gas,which has been designated as a human carcinogen by the International Agency for Research on Cancer(IARC)and the U.S.Environmental Protection Agency.Long-term radon exposure can increase the incidence of lung cancer in non-smokers,which is usually related to mutations in the TP53 gene.The genetic variation of the tumor suppressor gene TP53 can promote the occurrence and development of human cancer in different ways,and it occurs in almost all types of cancer.Mitochondria are organelles involved in multiple catabolic and anabolic pathways necessary for the maintenance and proliferation of cells.It is now generally believed that they are closely related to the occurrence and development of tumors.The TP53 gene directly participates in the intrinsic apoptotic pathway by interacting with the multi-domain members of the BCL2 family,thereby inducing changes in mitochondrial outer membrane permeabilization(MOMP),resulting in the release of mitochondrial membrane interstitial proteins(such as Cyt-c),and leading to cell apoptosis.Although the exact mechanism of MOMP is unclear,it is known to be regulated by the interaction between pro-apoptotic proteins and anti-apoptotic Bcl2 proteins.Objective:In previous experimental studies on radon-induced lung cancers,the main observed biological effects were the mutational damage to nuclear DNA.However,the damage to extranuclear organelles,especially mitochondria,was not considered.This research uses TP53 wild-type human bronchial epithelial cells(BEAS-2B cells)to construct a long-term radon model to explore the mechanism of p53-mediated radon-induced lung cancer through mitochondria.Through a series of in vivo and in vitro experiments,we observed the effects of radon exposure and TP53 knockout on cells and mitochondria,and then screened for and verified the mitochondrial-related genes targeted by TP53 through high-throughput sequencing and bio-informatics analysis,so as to provide new experimental evidence for clarifying the mechanism of radon-induced malignant transformation of lung cells.Methods:(1)In vivo and in vitro experiments were performed to observe the effect of radon and/or TP53 knockout.In the in-vivo experiments,the radon inhalation mouse model established in our research group was used.The radon inhalation-exposed mice were divided into four groups:control group(Control),low-dose group(30WLM),medium-dose group(60WLM)and high-dose group(120WLM).After the exposure,the lung tissues of each group of mice were taken for HE staining to examine the pathological changes in the lungs.The mitochondrial DNA copy number and p53 protein expression were also determined from the samples.In the in-vitro experiments,the TP53 gene knockout and long-term radon-induced cell malignant transformation models were constructed and were divided into four groups,namely the normal passage control BEAS-2B cells(BEAS-2B),TP53 knockout cells(TP53-/-),and radon exposure cells(Rn),and TP53 knockout cells with radon exposure(TP53-/-+Rn).After processing,the clone formation ability of each group of cells was analyzed by a soft agar cloning experiment.EdU and CCK-8 cell proliferation experiments were used to observe the proliferation ability of each group of cells.Scratch experiment and transwell experiment were performed to test the migration ability of each group of cells.The changes in mitochondrial membrane potential in cells of each group were analyzed by flow cytometry,and the cytoplasm and mitochondrial proteins were separated to determine the expressional differences of pro-apoptotic proteins BAX,Cyt-c,and anti-apoptotic protein Bcl2 by western blot analysis.Mitochondrial DNA copies counts were determined in each group of cells.(2)High-throughput sequencing and bioinformatics analysis were conducted on TP53 knockout and/or radon-stained cells.The total RNA of the above four groups of cells was extracted and transcriptome high-throughput sequencing was performed.Through pairwise difference analysis,compared with normal passage BEAS-2B cells,the differentially expressed genes in the TP53-/-,Rn,and TP53-/-+Rn groups of cells were identified.DAVID online tool was used to perform GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis for differentially expressed genes.The total RNA of each group of cells was extracted with TRIZol kit,and the mRNA expression levels of the top five up-regulated and down-regulated genes identified by the above-mentioned high-throughput analysis were verified with qPCR measurement.Using the TCGA(The Cancer Genome Atlas,Cancer Genome Atlas)database of lung adenocarcinoma(LUAD,Lung adenocarcinoma)and lung squamous cell carcinoma(LUSC,Lung squamous cell carcinoma)mRNA expression data,the differential expression of the top 5 up-regulated and down-regulated genes identified by the above-mentioned high throughput analysis in lung cancer tissues and adjacent normal tissues were analyzed.The R language "Mfuzz" package was further used to perform cluster analysis of transcriptome expression patterns on the expression data of each group of cells to screen for affected genes that have a synergistic effect between TP53 gene knockout and radon treatment.Combining literature and bioinformatics analysis based on the TCGA database,the key genes related to the differential expression of mitochondria after TP53 gene knockout and radon exposure were further screened.(3)To verify the mechanism of radon exposure and TP53 knockout through gene knockdown and overexpression and pathway analysis.Western blot analysis was used to determine the protein expression of BTG2 in TP53 knockout and/or radon-exposed cells,and the mRNA expression levels of BTG2 and BCL2 in the lung tissue of the radon-exposed mouse model were verified using qPCR techniques and immunohistochemical observation.The knockdown shRNA plasmids and overexpression plasmids of BTG2 and BCL2 were constructed using pGreen plasmid and pCDH plasmid as the backbone respectively,and then the constructed plasmids were transfected into normal BEAS-2B cells and TP53 knockout cells,respectively.The MOMP-related molecular markers,the BAX and Cyt-c,in the transfected cells were detected with western blot to determine their distribution inside and outside the mitochondria,and the changes in mitochondrial membrane potential and apoptosis levels of transfected cells were detected by flow cytometry.The Cancer Genome Atlas TCGA database and Gene Expression Omnibus database(Gene Expression Omnibus database,GEO)were used to analyze the difference in the expression of BTG2 in normal tissues adjacent to cancer and lung cancer tissues,and survival analysis of BTG2 was performed based on online tool KMplot.Results:(1)The lung tissue was altered in mice exposed to radon.The results of HE staining of the lung tissues of mice exposed to radon showed that there were varying degrees of pathological changes in the lung tissues of mice exposed to different doses of radon,manifested as lung tissue consolidation,alveolar fusion,alveolar shrinkage or disappearance,and pulmonary interstitium thickening.The copy number of mitochondrial DNA in the lung tissue of mice exposed to radon at each dose increased significantly,the expression of p53 protein decreased,and a dose-effect relationship was observed.The mitochondrial DNA copy number in the 120WLM high-dose radon-exposed group increased the most,significantly higher than that in 30WLM group.The p53 expression level in the high-dose group decreased the most,which was significantly lower than that of the low-dose and medium-dose groups.In the in-vitro experiments,the function of BEAS-2B cells altered after TP53 knockout and/or radon exposure.After long-term radon exposure of BEAS-2B cells,the TP53-/-and Rn groups showed consistent changes,namely,decreased apoptosis rate,increased soft agar clone formation rate,and enhanced cell proliferation and migration capabilities.Corresponding to the changes in the growth characteristics of these cells,the morphological and functional regulation of mitochondria has also undergone corresponding changes,manifested as an increase in Bcl2 protein expression,a decrease in the apoptotic protein BAX on the outer mitochondrial membrane and an increase in Cyt-c in the mitochondrial membrane space,an increase in mitochondrial membrane potentialand mitochondrial DNA copy number.Compared with the Rn and TP53-/-groups,the mitochondrial changes in the combined group(TP53-/-+Rn)were significantly higher than those in the TP53 knockout alone and radon alone groups,indicating that the two mitochondria-influencing factors have a synergistic effect on malignant transformation of cells.(2)The high-throughput sequencing data bioinformatics analysis results in the TP53 knockout and/or radon-exposured cells uncovered a series of differentially expressed genes in TP53-/-and radon-exposured cells,and they were significantly enriched in multiple tumor-related biological functions and signaling pathways,including the regulation of Cyt-c release from mitochondria and functional biological processes such as cell proliferation,migration,and apoptosis,as well as PI3K-Akt,TNF and NF-κB signaling pathways.Analysis based on the TCGA database showed that the top 5 up-or down-regulated genes in most lung cancer tissues showed consistent changes,and the differences were significant.Through cluster analysis,all genes were grouped into 6 categories,and the genes in cluster 2 whose expression was continuously down-regulated,and cluster 3 whose expression was continuously up-regulated were analyzed by protein association network,and 76 genes related to TP53 were found.Enrichment analysis revealed that these genes were related to apoptotic mitochondrial changes and mitochondrial DNA damage checkpoints and tumor-related signaling pathways.Further analysis of the expression of these 76 genes in TCGA lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)showed that 8 genes significantly up-regulated in both LUAD and LUSC were in the Cluster3,and 6 down-regulated genes were in the Cluster2,which expressions showed a consistent decrease in the radon-exposed group and the TP53 knock-out combined radon group.Through a literature search,combined with the bioinformatics analysis in the TCGA database,two genes with the strongest correlation with mitochondrial changes and abnormal TP53 were found,namely the anti-proliferative tumor suppressor gene BTG2 and the anti-apoptotic gene BCL2.(3)Verification of the functions of TP53 targeting mitochondrial-related genes BCL2 and BTG2.Western blot was used to verify the protein expression levels of the selected BTG2 gene.The results were consistent with the results of the high-throughput mRNA sequencing.In the four groups of cells,the expression level of BCL2 from high to low was BEAS-2B>Rn>TP53-/->TP53-/-+Rn.The immunohistochemical results of mouse lung tissue showed that radon exposure can significantly increase the expression of BCL2 protein in lung tissue and reduce the expression of BTG2 protein.In TP53 wild-type cells,overexpression of BCL2 or knockdown of BTG2 significantly reduced BAX expression in mitochondria and increased Cyt-c,decreased cell apoptosis,and increased mitochondrial membrane potential.Knockdown of BCL2 or overexpression of BTG2 can significantly rescue the changes in MOMP,the decrease of apoptosis level,and the increase of mitochondrial membrane potential caused by TP53 knockout.Bioinformatic analysis showed that BTG2 expression level is significantly lower in lung cancer tissues,and its expression changes were related to tumor stage,depth of invasion,and patient prognosis.Conclusions:(1)Long-term radon exposure can cause lung tissue damage in mice,accompanied by down-regulation of p53 expressions and an increase in mitochondrial copy number.In human bronchial epithelial cells,both TP53 knockout and/or radon exposure can reduce the apoptosis rate,increase the proliferation ability and migration ability,and the mitochondrial morphology and functions also change correspondingly,as manifested by the increased permeability of the mitochondrial outer membrane,increased mitochondrial membrane potential and number of mitochondrial DNA copies.These changes are more prominent in TP53 knockout cells,suggesting that the TP53 gene is involved in the mitochondrial-mediated malignant transformation of the cells.(2)The biological functions of the differentially expressed genes after TP53 knockout and radon exposure are mostly enriched in signal pathways related to tumors and are closely related to the apoptotic function of mitochondria.Through bioinformatics analysis of gene expression patterns,two genes BCL2 and BTG2 related to TP53 targeting mitochondria were found.(3)At the animal and cellular level,it was confirmed that BCL2 and BTG2 played an important role in the malignant transformation of BEAS-2B cells and changes in mitochondrial function caused by TP53 knockout and radon exposure.