m 6 A demethylase ALKBH5 regulates TP53 to promote malignant transformation of lung cells induced by radon exposure via BTG2-mediated mitochondrial pathway

doi:10.21203/rs.3.rs-3864065/v1

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m 6 A demethylase ALKBH5 regulates TP53 to promote malignant transformation of lung cells induced by radon exposure via BTG2-mediated mitochondrial pathway

https://doi.org/10.21203/rs.3.rs-3864065/v1

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Lung cancer is a multi-etiological disease and radon is known as the second most common risk factor for the disease. The tumor suppressor gene TP53 is commonly mutated in human lung cancer, thus contributing to the alteration of mitochondrial outer membrane permeability (MOMP). Thus, this study aimed to investigate the role of the P53-mediated mitochondrial pathway in radon-induced lung cancer. Long-term radon exposure downregulated P53, enhanced mitochondrial membrane potential (MMP), and increased mitochondrial DNA copy number in BEAS-2B cells, as well as in the lung tissues of mice exposed to radon. The above effects significantly contributed to the radon-induced malignant transformation of BEAS-2B cells. In addition, high-throughput sequencing analysis revealed that BTG2 downregulation was involved in the P53 mitochondrial pathway-mediated radon-induced malignant transformation. BTG2 overexpression significantly restored the TP53-mediated alterations in MOMP, apoptosis, and MMP. Mechanically, TP53 downregulation was regulated by m⁶A demethylase ALKBH5, which was validated by treatment of ALKBH5 inhibitor IOX1 and mRNA stability assay in BEAS-2B and H1299 cells. Our research shows that the ALKBH5-mediated downregulation of TP53 affects the carcinogenesis of radon, and the BTG2 suppression in the P53-mediated mitochondrial pathway plays an important role in radon-induced lung cancer.

Radon

malignant transformation

mitochondria

P53

BTG2

m6A methylation

The etiology of lung cancer is likely multifactorial. Among numerous environmental carcinogens, radioactive radon and its progenies are the second leading cause of lung cancer, after smoking, and the first among nonsmokers [1, 2]. According to the International Agency for Research on Cancer (IARC) and United States Environmental Protection Agency (USEPA), radon is considered a human carcinogen [3–7]. Previous epidemiological studies showed that the risk of lung cancer was positively associated with radon exposure with no threshold [8–10]. Long-term and high-concentration exposure to radon may induce oxidative stress and inflammatory reactions in the lung tissue, thus leading to chronic lung diseases, including pulmonary fibrosis and lung cancer. After radon and its progenies enter the respiratory tract, ionizing radiation is emitted, which in turn damages DNA, proteins, and lipids in lung cells, ultimately resulting in their malignant transformation [11].

Gene encoding tumor protein p53 (TP53) is mutated in more than 90% of small cell lung cancers and more than 50% of non-small cell lung cancers [12]. Mutations in the TP53, mainly occurring in exons 5–8 and with G:C → T:A transversion being the most common mutation [13], have been associated with smoking [14, 15]. The oncogenic phenotypes resulting from mutations in TP53 commonly include enhanced cell proliferation, chemoresistance, and anti-apoptotic functions as well as increased cell migration and invasion abilities. Unusual patterns of mutations in the TP53 gene have also been reported in lung tumor cells from underground miners [16]. Previous studies demonstrated that radon exposure can induce TP53 mutations, which further contribute to lung carcinogenesis [17–20]. In addition, P53-deficient cell lines were reported as more susceptible to irradiation injury compared with P53 wild-type (WT) cell lines [21]. Moreover, a previous study has shown that both radon exposure and P53 are associated with apoptosis [22]. Our previous study revealed that p53-associated metabolic pathways may be altered in radon-mediated malignant transformation [23]. However, the more specific mechanisms involved are not well defined.

Mitochondria are organelles, where several catabolic and anabolic pathways necessary for the maintenance and proliferation of cellular functions take place [24]. In addition to their involvement in metabolism, mitochondria are also involved in other biochemical processes, such as signal transduction via the production of reactive oxygen species (ROS) [25]. The original Warburg hypothesis suggested that cancer cells could have dysfunctional mitochondria. However, it is currently accepted that cancer cells depend on both aerobic glycolysis and mitochondrial metabolism [26, 27]. P53 binds to the pro-apoptotic factors, including the BCL2 homologous antagonist killer (BAK) and BCL2 associated X Protein (BAX), at the outer mitochondrial membrane (OMM) to change its permeability and to promote apoptosis via inducing the release of cytochrome C (Cyt-C) [28]. Another study showed that mutations in TP53 or P53 downregulation could not only inhibit the respiratory activity of mitochondria, but they could also affect OMM permeability and cell apoptosis [29].

Due to the prevalence of TP53 gene mutations in lung cancer and the inevitability of daily radon exposure, the present study aimed to investigate the role of the P53-mediated mitochondrial pathway in radon-induced lung cancer. Therefore, TP53-deficient human bronchial epithelial cells (BEAS-2B) were used to establish an in vitro malignant transformation model induced by long-term exposure of cells to radon.

2.1. Long-term radon exposure promotes lung tissue damage in mice

HE staining in lung tissues derived from radon-exposed mice showed that radon exposure could induce the development of several adverse pathological changes in lung tissues, such as lung tissue consolidation, alveolar fusion and pulmonary interstitial thickening, which were further aggravated by exposure to increasing doses of radon (Fig. 1A-B). Compared with the control group, MDA content was significantly increased (P < 0.05) and SOD activity was significantly decreased (P < 0.05) in a dose-dependent manner (Fig. 1C-D). Compared with sham-exposed control mice, P53 was downregulated (P < 0.05) in the lung tissues of mice in the radon-exposed groups in a dose-dependent manner (Fig. 1E). The results of the current study showed that the mitochondrial DNA (mtDNA) copy number was significantly increased (P < 0.05) in the lung tissues of mice exposed to radon (Fig. 1F).

2.2. Radon exposure and TP53 KO induces malignant transformation of BEAS-2B cell

To explore the relationship between radon exposure and P53, we used TP53 knockout cells to observe the effects of radon exposure. The dynamic radon exposure model in vitro is shown in Fig. 2A. In the cell model of long-term radon-exposed TP53 knockout cells (TP53^-/- + Rn), cell proliferation was notably increased compared with control BEAS-2B cells, radon-exposed cells (Rn) and TP53 knockout cells (TP53^-/-) (Fig. 2B-C). On the other hand, the cell apoptosis rate was decreased in all groups (Fig. 2D). The colony formation (Fig. 2E) and migration (Fig. 2F-I) abilities of BEAS-2B cells were affected by the synergistic effect of TP53 KO and radon exposure. At the molecular level, radon exposure and TP53 KO enhanced the protein expression levels of the anti-apoptosis protein BCL2, cell proliferation-related marker Ki67 and the EMT-related marker N-cadherin (CDH2), and reduced those of the epithelial marker E-cadherin (CDH1) (Fig. 2J-K).

2.3. Radon exposure and TP53 KO induces MOMP in BEAS-2B cell

Enhanced MMP was found in the Rn, TP53^-/- and TP53^-/- + Rn experimental groups, with the highest observed in the TP53^-/- + Rn group (Fig. 3A-B). The mtDNA copy number was also markedly increased in the Rn, TP53^-/-, and TP53^-/- + Rn groups (Fig. 3C). An increased accumulation and release of BAX and Cyt-C, respectively, were also observed in the mitochondria of the above groups, while the opposite changes were observed in the cytoplasm (Fig. 3D-F). In particular, these changes were most prominent in the TP53^-/- + Rn group.

2.4. Radon exposure and TP53 KO can alter the gene expression profile in BEAS-2B cells

High-throughput sequencing was used to identify differentially expressed genes (DEGs) in the Rn, TP53^-/-, and TP53^-/- + Rn groups compared with the control BEAS-2B group and in the TP53^-/- + Rn group compared with the TP53^-/- group. The results revealed several clusters of DEGs between cells of different treatment groups (Fig. 4A). Therefore, in radon-exposed cells (Rn), a total of 2,175 DEGs were identified, including 1,377 upregulated and 798 downregulated ones (Figure S1A). A total of 2,005 upregulated and 1,310 downregulated genes were detected in TP53^-/- cells compared with WT BEAS-2B cells (Figure S1C). Additionally, a total of 1,820 upregulated and 1,270 downregulated genes were identified in radon-exposed TP53 KO cells (TP53^-/- + Rn) (Figure S1E). Furthermore, the mRNA expression levels of the top 5 upregulated and downregulated DEGs were validated by qPCR analysis (Figure S1B, D, F). Correspondingly, the mRNA expression levels of the top 10 DEGs in lung cancer datasets, including those from lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), were further analyzed based on the TCGA database (Figure S2). Therefore, DEGs in radon-exposed and TP53 KO cells were significantly enriched in several cancer-related biological processes, including "regulation of cell proliferation", "cell adhesion" and "angiogenesis" (Fig. 4B-D). These findings suggested that TP53 KO and/or cell exposure to radon could promote tumorigenesis by affecting different signaling transduction pathways and biological processes. Furthermore, a total of 807 and 300 intersected upregulated and downregulated DEGs, respectively, were identified among the Rn, TP53^-/-, and TP53^-/- + Rn groups (Fig. 4E-F). Enrichment analysis revealed that the 807 intersected upregulated DEGs were mainly enriched in the terms "cell adhesion", "extracellular matrix organization", "angiogenesis" and several other significant biological processes (Fig. 4G). In addition, the 300 intersected downregulated DEGs were enriched in "cell adhesion", "endothelial cell migration", "endocytosis" and "lung development" (Fig. 4H). The above intersected DEGs were unexceptionally enriched in several cancer-related pathways (Figure S1G-H), thus highlighting their roles in TP53 KO- and radon exposure-induced cell transformation.

2.5. Clustering analysis of DEGs

Based on this method, the abovementioned identified DEGs from the different experimental groups were divided into 6 clusters with different expression trends, with 409, 573, 553, 804, 768, and 438 genes in clusters 1, 2, 3, 4, 5 and 6, respectively (Fig. 5A-B). The DEGs in cluster 2/3 are considered to be the consequence of the combined effects of radon exposure and TP53 knockout, and they were selected for further analysis. Therefore, genes of clusters 2 and 3 were subjected to P53-related protein interaction network analysis using the STRING database. The analysis revealed that a total of 76 genes fell into the above network (Fig. 5C). In addition, functional enrichment analysis indicated that these 76 genes were significantly associated with several significant biological functions, such as "apoptotic mitochondrial alterations", "mtDNA damage", "cell-cycle checkpoint", "regulation of autophagy" and "response to hypoxia" (Fig. 5D). On the other hand, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis predicted that these genes could be associated with "apoptosis", "p53 signaling pathway", "mitophagy", and "mTOR and Wnt signaling pathway" (Fig. 5E). As shown in Fig. 5F, expression analysis of these 76 genes in TCGA LUAD and LUSC databases showed that 8/11 genes significantly upregulated in both LUAD and LUSC databases clustered in cluster 3 (Table S2). Additionally, 6/11 genes notably downregulated in LUAD and LUSC databases were presented in cluster 2, including BTG2, an anti-proliferative tumor suppressor gene (Table S3). Based on these analysis, BTG2 was selected for further analysis to uncover the synergistic effects of radon exposure and TP53 KO on BEAS-2B cells.

2.6. BTG2 mediates the p53-triggered MOMP

qPCR analysis demonstrated that the mRNA expression levels of BTG2 was downregulated in the Rn, TP53^-/- and TP53^-/- + Rn groups, and more pronounced in the TP53^-/- + Rn group (Fig. 6A). At the protein level, the western blot results showed a similar trend in the expression levels of BTG2 (Fig. 6B). In vivo, exposure of mice to radon dose-dependently downregulated BTG2, as evidenced by immunohistochemistry (IHC) staining and semi-quantitative analysis (Fig. 6C-D). To further evaluate the effect of BTG2 on MOMP in radon-exposed and TP53^-/- cells, BTG2 was knocked down in TP53 WT cells, while it was overexpressed in TP53^-/- cells. As shown in Fig. 6E-G, BTG2 silencing significantly attenuated cell apoptosis and enhanced MMP in TP53 WT cells. However, BTG2 overexpression abrogated the effects of TP53 knockdown on the reduced apoptosis rate and elevated MMP. In addition, BTG2 silencing in TP53 WT cells significantly inhibited MOMP, as evidenced by BAX downregulation and Cyt-C upregulation in mitochondria, as well as the opposite changes in cytoplasm. However, BTG2 overexpression in TP53-/- cells promoted the accumulation of BAX and the release of Cyt-C from mitochondria (Fig. 6H-M).

Bioinformatics analysis using the TCGA database showed that BTG2 downregulation was associated with tumor stage and invasion depth in both LUAD and LUSC (Figure S3A-F). Furthermore, BTG2 downregulation was also observed in TP53-mutant LUAD samples when compared with WT TP53 samples (Figure S3G), as well as in several lung cancer datasets in the GEO database (Figure S3H-G). Additionally, survival analysis based on TCGA LUAD dataset and the KMPlotter online tool revealed that patients with high BTG2 expression levels exhibited better prognosis (Figure S4A-B). Correlation analysis revealed that BTG2 expression was significantly correlated with multiple cell senescence-related genes and oncogenes both in TCGA-LUAD (Figure S4C-D). and TCGA-LUSC datasets (Figure S4E-F). Moreover, BTG2 expression was found to correlate with several infiltrated immune cells, including hematopoietic stem cells, endothelial cells, cancer-associated fibroblast, macrophages and other cells (Figure S4G-H).

2.7. TP53 downregulation was regulated by m⁶A demethylase ALKBH5

Moreover, the upstream mechanism of the TP53 down-regulation reduced by radon exposure was explored. The heatmap reveals that m⁶A demethylase ALKBH5 was downregulated in radon-exposed cells based on the high-throughput sequencing results (Fig. 7A). Based on 10 GEO datasets and TCGA LUAD/LUSC datasets, ALKBH5 was found upregulated in 3 datasets while downregulated in 5 datasets (Figure S6). The global m⁶A modification in radon-exposed cells was found higher than that in control cells (Fig. 7B). Based on the correlation analysis, TP53 expression was found to significantly correlate with ALKBH5 in TCGA LUAD (Figure S5A) and LUSC (Figure S5B) datasets, as well as GTEx lung dataset (Figure S5C). Also, ALKBH5 expression was found reduced in radon-exposed cells and lung cancer cells (Figure S5D). A predicted m⁶A modification site was found within TP53 mRNA (Figure S5E), and the site was validated by meRIP-qPCR analysis (Fig. 7C). After attenuating ALKBH5 by transfected with shALKBH5 plasmid or treated with ALKBH5 inhibitor IOX1 in BEAS-2B and H1299 cells, P53 expression was significantly downregulated (Fig. 7D-F). The further RNA stability assay indicated that knockdown ALKBH5 promotes the degradation of TP53 mRNA both in BEAS-2B and H1299 cells (Fig. 7G-H). Also, the MOMP was evaluated in ALKBH5 downregulated cells and results revealed that BAX was significantly accumulated in cytoplasm, while Cyt-C was accumulated in mitochondria (Fig. 7I-L). On the contrary, Cyt-C was down-regulated in cytoplasm and BAX was down-regulated in mitochondria after knocking down ALKBH5 (Fig. 7I-L). These results revealed that ALKBH5 regulates TP53 expression in an m⁶A-dependent manner, and its downregulation could inhibit MOMP in lung cells.

It has been reported that high-energy α particles generated from radon decay can lead to the accumulation of multiple gene mutations, thus contributing to cancer development. Previous animal experiments demonstrated that radon inhalation could induce lung injury, fibrosis, and epithelial-mesenchymal transition (EMT) in mice [30]. Our previous studies have shown lung damage after radon exposure [31, 32]. Herein, the results show the accumulation of MDA and reduction of SOD activity in radon-exposed cells in a dose-dependent manner. Mutations in the P53 gene are a common defect in lung cancer of various types. Previous population survey data suggested that residential and occupational radon exposure could contribute to increased mutation rates of the TP53 gene in human lung tumors [16, 33]. Consistently, the downregulation of P53 protein was also found in the lung tissue of radon-exposed mice in this study. Also, mtDNA copy number was significantly increased in radon-exposed mice, indicating radon exposure can induce the dysfunction of mitochondria. Many recent studies have confirmed that mitochondrial dysfunction is one of the causes of cancer development [34, 35]. The functional state of mitochondria is often closely associated with its outer membrane permeability. Apoptosis includes the extrinsic and intrinsic pathways, which are mainly regulated by proteins of the BCL2 family in mitochondria [36]. Most stimuli can induce cell apoptosis through the mitochondrial pathway, known as MOMP. Cancer cells usually block cell apoptosis by upregulating the anti-apoptotic BCL2 protein, thereby inhibiting the MOMP pathway [37]. The binding of BAX pro-apoptotic protein to OMM can trigger MOMP, which in turn promotes the release of Cyt-C from mitochondria into the cytoplasm. An increased accumulation and release of BAX and Cyt-C, respectively, were also observed in the mitochondria in the radon-exposed cells. It has been reported that cytoplasmic P53 can interact with BCL2 protein family members to promote MOMP [38, 39]. Consistently, P53 knockdown significantly promoted the changes in MOMP, thus supporting its role in cell apoptosis.

Based on the “mfuzz” analysis, BTG2 fell in cluster 2, which suggests that radon exposure and TP53 knockout have synergistic effects to facilitate malignant transformation. BTG2 was originally identified as a P53-inducible gene, whose expression could be increased under the induction of P53 when DNA was damaged [40]. It has been also reported that BTG2 is a direct transcriptional target of P53 [40, 41] and regulates several cellular processes, including cell proliferation, differentiation, and apoptosis [41, 42]. Herein, BTG2 fell in cluster 2, which suggests that radon exposure and TP53 knockout have synergistic effects to promote malignant transformation. BTG2 is an early response gene expressed in response to a variety of stimuli and can regulate cell differentiation, proliferation, and apoptosis [43]. Previous studies demonstrated that decreased BTG2 expression was associated with reduced overall survival in patients with breast cancer and early-stage non-small cell lung cancer [44, 45]. Our analysis also shows the differential expression of BTG2 in TP53 mutant and nonmutant LUAD tissues. Another study showed that PUMA/BBC3 directly regulates mitochondrial depolarization and cell death, while BTG2 accumulation can further enhance this effect by promoting p53 mitochondrial localization [46]. Based on the overexpression and knockdown of both genes, we confirmed that the BTG2-MOMP pathways play important roles in the radon-induced lung cancer process.

N6-methyladenosine (m⁶A) is the most abundant internal modification of RNA in eukaryotic cells, which can affect multiple aspects of RNA metabolism, including RNA processing, nuclear output, RNA translation, and decay [47]. More and more studies have shown that the RNA demethylase ALKBH5 plays a key role in various human malignant tumors, mainly through the post-transcriptional regulation of oncogenes or tumor suppressors [48]. A previous study revealed that ALKBH5 can inhibit YAP activity to suppress tumor growth and metastasis [49]. On the contrary, another study revealed that ALKBH5 overexpression is an unfavorable prognostic factor in NSCLC, and ALKBH5–IGF2BPs axis can promote proliferation and tumorigenicity of NSCLC cells [50]. The evidence within the context of pulmonary oncology appears to exhibit a degree of contradiction, which may suggest a dependency on the specific subtype of lung carcinoma. The specificity of lung cancer subtypes may influence the pathobiological characteristics and clinical manifestations of the disease, resulting in a heterogeneous response to uniform therapeutic strategies across different subtypes [51]. Here, we found global m⁶A methylation level was increased, while ALKBH5 was downregulated in radon-exposed lung cells. Further studies reveal that TP53 down-expression is regulated by ALKBH5 in a m⁶A-dependent manner. A previous study has also found that m⁶A modification was found within TP53 mRNA in melanoma, which can be regulated by YTHDF2 and promote its degradation, thereby accelerating tumor tumorigenesis [52].

In conclusion, long-term radon exposure can reduce p53 expression and increase mitochondrial copy number. TP53 knockout and long-term radon exposure have a synergistic effect on mitochondrial outer membrane permeability changes and malignant transformation. Mechanically, the downregulation of m⁶A demethylase ALKBH5 induced by radon exposure leads to the decreased expression of TP53, which in turn promotes the malignant transformation of lung cells through the BTG2-mediated MOMP pathway.

4.1. Radon-exposed mouse model

The animal model of lung injury by radon inhalation was established as previously described [53, 54]. Briefly, a total of 32 male BALB/c mice were randomly divided into the following four groups: The control group (control) and the experimental groups were exposed to radon for 10 h/day, 6 days/week, and up to 189 h/377 h/755 h, which were equivalent to cumulative doses of 30/60/120 Working Level Months (WLM), respectively. All animals were housed in the Laboratory Animal Center of Soochow University and maintained according to the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals[55] and the ARRIVE guidelines[56]. The experimental protocol was approved by the Laboratory Animal Ethics Committee of the Experiment Animal Center of Soochow University (approval no. 201503A095). Mice in each group were exposed to 100,000 Bq/m³ radon in a multi-functional ecological radon chamber. Each mouse was exposed to radon for 12 h per day for up to 189, 377, and 755 h. The above hours were equivalent to cumulative exposure doses of 30, 60, and 120 WLM, corresponding to mice in the low, medium, and high dose groups, respectively. Mice were anesthetized with 60 mg/kg pentobarbital (intraperitoneal injection) when exposed to radon. During the exposure period, mice had free access to food, water, and activity. Mice of each group were euthanized via CO2 inhalation within 24 h after reaching the exposure dose. The lung tissue from each mouse was then dissected. Subsequently, 10 mg of the left and right lung tissue was used for genomic DNA extraction, while the remaining tissues were bisected. One section was cryopreserved at -80℃ and the other one was fixed with 4% paraformaldehyde and was used for pathological examination.

4.2. Immunohistochemistry (IHC)

Tissue sections of 0.3 µm thickness were deparaffinized and hydrated for staining. Following antigen retrieval, sections were blocked with 5% goat serum and 3% hydrogen peroxide. Subsequently, the sections were processed for IHC staining, following incubation with specific primary antibodies against B-cell translocation gene 2 (BTG2; dilution, 1:100) and BCL2 (dilution 1:100; both from ProteinTech Group, Inc., USA). The tissues were then incubated with a goat anti-rabbit secondary antibody for 20 min, followed by labeling with streptavidin-HRP for 30 min at room temperature. Following washing, DAB was applied as a chromogen and the sections were counterstained with hematoxylin and dehydrated. The average optical density was quantified using the ImageJ plugin IHC profiler.

4.3. Oxidative Stress Detection

The activities of superoxide dismutase (SOD), and the levels of the lipid peroxidation product malonaldehyde (MDA) in the lung tissues of mice were determined according to the manufacturer's instructions (Nanjing JianchengBioeng Inst., China). The data are expressed as units of activity or nanomoles per milligram of protein.

4.4. Measurement of mtDNA copy number

Total DNA was isolated using a DNA Isolation Kit (Qiagen, Dusseldorf, Germany). The mtDNA copy number was measured using the Relative Human Mitochondrial DNA Copy Number Quantification qPCR Assay Kit (Zhong Qiao Xin Zhou Biotechnology Co., Ltd., China) according to the manufacturer's instructions. Briefly, for each genomic DNA sample, two qPCR reactions were prepared, one with mtDNA primer stock solution and one with human single copy reference primer stock solution. Each qPCR reaction contained a 2 ng genomic DNA template, 2 µl primer stock solution, 10 µl 2X qPCR master mix, and nuclease-free H₂O to a final volume of 20 µl. The results were calculated using the comparative cycle threshold (ΔΔCt) method.

4.5. Western blot analysis

Total proteins were extracted from cells using RIPA lysing buffer (Beyotime Institute of Biotechnology, China) and the protein concentration was determined using an Enhanced BCA Kit (Beyotime Institute of Biotechnology, China). Total protein extracts (30 µg) were separated by SDS-PAGE and were then transferred onto a PVDF membrane (MilliporeSigma, Billerica, MA). Following blocking with 5% BSA, the membrane was incubated at 4°C overnight with primary antibodies against BAX (dilution, 1:1,000), Cyt-C (dilution, 1:1,000), GAPDH (internal reference for cytoplasmic extracts; dilution, 1:1,000) and Cyt-C oxidase subunit 4I1 (COX4I1; internal reference for nuclear extracts; dilution, 1:1,000; all from ProteinTech Group, Inc., USA). Following incubation with primary antibodies, the membranes were incubated with HRP-labeled secondary antibodies. The protein bands were visualized using the ECL substrate and the GeneTools GBox (Syngene, Frederick, MD, USA) system. The intensity of each band was quantified using ImageJ software (NIH). GAPDH served as the internal control.

4.6. In vitro long-term radon exposure cell model

A model of malignant transformation in BEAS-2B cells induced by long-term exposure to radon was successfully established, as previously described [57]. Briefly, by employing transcription activator-like effector nuclease (TALEN) technology, we have engineered a BEAS-2B cell line with the TP53 gene specifically knocked out. Then, normal and TP53 knockout (KO) BEAS-2B cells were seeded into 0.4-µM pore size Transwell membrane. Subsequently, the membrane was placed into an in vitro cell exposure system. Following cell adhesion, cells were exposed to 20,000 Bq/m³ radon for 20 min per passage into an HD-3 type multi-functional radon chamber. Cells were subjected to continuous and repeated exposure to radon for 30 generations. A total of four groups are set: i. Sham-exposed control BEAS-2B cells (BEAS-2B group); ii BEAS-2B cells exposed to radon for 30 passages (Rn group); iii. TP53 KO BEAS-2B cells (TP53^-/- group); and iv. TP53 KO BEAS-2B cells were exposed to radon for 30 passages (TP53^-/- + Rn group).

4.7. Cell proliferation assays

To assess cell cycle distribution, cells were trypsinized and were then fixed with 70% ethanol at -20°C overnight. The fixed cells were washed twice with PBS, followed by incubation with 10 µg/ml RNase A and 50 µg/ml propidium iodide (PI; Beyotime Institute of Biotechnology, China) for 30 min. The cell cycle was analyzed using BD FACS Canto II and FlowJo V10.3 software (FlowJo LLC, Ashland, OR, USA). For the 5-ethynyl-2’-deoxyuridine (EdU) cell proliferation assay, cells were seeded into 6-well plates and then treated with 10 µM EdU for 2 h. Following fixing with 4% paraformaldehyde and permeabilization with 0.3% Triton X-100 in PBS, cells were incubated with a click reaction solution (Beyotime Institute of Biotechnology, China). Images were captured within 24 h under an inverted fluorescent microscope and analyzed using NIH ImageJ software (Version 1.8.0).

4.8. Detection of cell apoptosis

The proportion of apoptotic cells was determined using an Annexin V-FITC/PI detection kit (Beyotime Institute of Biotechnology, China), according to the manufacturer's instructions. The cells were trypsinized into single cells, followed by incubation with 5 µl Annexin V-FITC and 5 µl PI at room temperature for 20 min in the dark. Data from at least 10,000 cells/sample were then collected and analyzed with a flow cytometer (BD FACS CantoII; BD Biosciences, USA).

4.9. Soft-agar colony formation assay

Approximately 2,000 cells were seeded onto 1.5-ml top agar (DMEM supplemented with 10% FBS and 0.35% agarose) and on a 2.5-ml bottom layer of solidified bottom agar (DMEM supplemented with 10% FBS and 0.6% agarose) in 6-well culture plates and cultured for three weeks. Colonies were stained with crystal violet solution and their number was then counted. Images of cell colonies from each time point were captured under an inverted microscope and the number of cell colonies was calculated to evaluate the malignant characteristics of cell lines. Cells were counted in replicates of three independent biological experiments.

4.10. Cell migration assays

For the wound healing assay, cells were seeded into 6-well plates and allowed to grow until they reached 100% confluency. Subsequently, cells were cultured in fresh FBS-free medium and the cell monolayers were scratched with a pipette tip. Images at different time points were captured under an inverted microscope and the wound closure rate was analyzed using NIH ImageJ software (Version 1.8.0). For the Transwell migration assay, cells in 1 ml FBS-free medium were seeded into the upper chamber of the Transwell membrane (Corning, Inc., USA). In addition, the lower chamber was supplemented with a 2 ml complete medium. Following incubation at 37°C for 24 h, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet solution. Subsequently, the cells in the upper chamber of the Transwell insert were removed with a cotton swab. Images of the migrated cells were captured under an inverted microscope and were then assessed using NIH ImageJ software (Version 1.8.0).

4.11. Assessment of mitochondrial membrane potential (MMP)

MMP was assessed using an enhanced JC‑1 Mitochondrial Membrane Potential assay kit (Beyotime Institute of Biotechnology, China). Briefly, cultured cells were incubated with 1 ml of the prepared JC-1 working solution at 37°C for 20 minutes. After washing twice with JC-1 staining buffer, cells were digested to prepare a single cell suspension. Green (JC-1 monomer) and red (JC-1 polymer) fluorescent signals in the samples were detected using a flow cytometer.

4.12. High-throughput sequencing

Total RNA was extracted from a different group of cells. Subsequently, high-throughput mRNA sequencing analysis was performed using the Illumina HiSeq 4000 platform (Illumina, Inc.), according to the standard operating procedures. The mRNA sequencing libraries were prepared using the Illumina TruSeq RNA Kit (Illumina, Inc.). mRNAs with different expression profiles between samples were normalized to fragments per kilobase of transcript per million mapped reads. Differential expression analysis between groups was performed using the DEGseq package. P < 0.05 and |log2 (fold change between groups)| ≥1 were set as the screening criteria for differentially expressed gene (DEG) identification. The sequencing raw data was uploaded to the SRA repository (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA917102).

4.13. Cluster analysis of expression profile

Cluster analysis was performed using the "Mfuzz" R package. To obtain genes synergistically affected by TP53 KO and radon treatment, genes that were consistently upregulated or downregulated in BEAS-2B cells, radon-exposed cells, or radon-exposed TP53-deficient cells were finally extracted.

4.14. Bioinformatics analysis

Enrichment analysis was performed by inputting the gene list into the DAVID online tool (https://david.ncifcrf.gov/). Protein-protein interaction (PPI) network was analyzed using the STRING online tool (https://cn.string-db.org/). The Cancer Genome Atlas (TCGA) lung cancer datasets were downloaded from the Xena database (https://xena.ucsc.edu/), while the lung cancer proteomic datasets from the CPTAC database (https://proteomics.cancer.gov/programs/cptac). Furthermore, several lung cancer datasets were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Finally, survival analysis was carried out using a Kaplan-Meier plotter (http://kmplot.com/analysis/) [58].

4.15. qPCR assay

Following cell centrifugation, total RNA was extracted from 5×10⁶ cells with 1 ml TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Subsequently, total RNA (~ 1.5 µg) was reverse transcribed into complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer’s protocol. qPCR was performed using the FastStart Universal SYBR Green Master (ROX) Kit (Roche Diagnostics, Switzerland) on the QuantStudioTM 6 Flex RT-qPCR System (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA). GAPDH served as the internal reference gene. The qPCR primer sequences used are listed in Table S1.

4.16. Plasmid construction and transfection

The full-length BTG2 and BCL2 coding sequences (CDS) were amplified using Pfu DNA Polymerase (Sangon Biotech Co. Ltd., China) and were then ligated into the pCDH vector to construct the pBTG2 and pBCL2 plasmids. In addition, short hairpin RNA clones (shRNAs) targeting BTG2 and BCL2 were designed using the BLOCK-iT™ RNAi Designer. Subsequently, the shRNA clones were annealed and ligated into the pGreen vector to construct the shBTG2 and shBCL2 plasmids. The TP53 WT and KO cells were transfected with the above overexpression and silencing plasmids using Lipofectamine 6000 reagent (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions.

4.17. m⁶A related assays

m⁶A sites within TP53 mRNA were predicted according to the sequence-based RNA adenosine methylation site predictor (SRAMP) online tool (http://www.cuilab.cn/sramp) [59]. m⁶A site-specific primers were designed for meRIP-qPCR. The m⁶A modification in BEAS-2B cells was measured by using the Magna MeRIP m⁶A Kit (cat. no. 17-10499, Millipore Sigma, MA, USA) according to the manufacturer’s instructions.

To validate the regulation effect of ALKBH5 on TP53, ALKBH5 was attenuated cells transfected by shALKBH5 or treated by ALKBH5 inhibitor IOX1 (MedChemExpress, NJ, USA).

4.18. RNA stability assay

Cells transfected with shALKBH5 and shNC were seeded into a 6-well plate. These cells were collected at 0, 1, 2, 3, 4 and 5 h after being treated with actinomycin D (ACTD, final concentration, 10 µg/ml). Total RNA was isolated using TRIzol® reagent for qPCR to detect the TP53 mRNA levels. The TP53 mRNA decay rate was determined using a non-linear regression curve fitting (one-phase decay).

4.19. Statistical analysis

All statistical analyses were performed with GraphPad Prism V8.0 and R software. For simple comparisons between two groups, when the statistical data obeyed normality and the variance was homogeneous, a t-test was carried out. When the statistical data obeyed the normality but the variance was unequal, a corrected t-test was used. Finally, when data did not obey the normal distribution, a non-parametric Wilcoxon rank sum test was applied. One-way ANOVA was used to compare the differences among multiple groups followed by Tukey's post hoc test for pairwise comparisons between groups after the results showed differences. P < 0.05 was considered to indicate a statistically significant difference.

We have confirmed through in vitro experiments that BTG2 can regulate MOMP and mediate TP53 deletion and radon exposure induced malignant transformation of lung cells, but there is a lack of in vivo experiments to verify the function of BTG2. In addition, changes in MOMP require further evidence at the cellular level, such as detecting mitochondrial permeability transition point (MPMP).

Author Contributions: Conceptualization, Jin Wang and Jian Tong; Formal analysis, Jin Wang, Kaifan Liu, Jiawen Li and Qian Xu; Funding acquisition, Hailin Tian and Jianxiang Li; Methodology, Jin Wang and Qian Xu; Project administration, Hailin Tian; Resources, Qian Xu; Software, Jin Wang and Lirong Tan; Supervision, Jianxiang Li; Validation, Lirong Tan; Visualization, Jin Wang, Lirong Tan, Kaifan Liu, Jiawen Li, Bin Chen, and Lijun Fang; Writing – original draft, Jin Wang and Qian Xu; Writing – review & editing, Jian Tong, Hailin Tian, and Jianxiang Li.

Funding: This study was supported by the National Natural Science Foundation of China (81172707 and 81673126) and Science and Technology Plan Project of Jiangsu Province (BZ2023008). The study was also supported by Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, MOE Key Laboratory of Geriatric Diseases and Immunology as well as the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Institutional Review Board Statement: The animal protocol was approved by the Laboratory Animal Ethics Committee of the Experimental Animal Center of Soochow University (approval no. 201503A095). All animals were cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Data Availability Statement: All data generated and described in this article are available from the corresponding web servers, and are freely available to any scientist wishing to use them for noncommercial purposes, without breaching participant confidentiality. All codes and R-packages used in the study are publicly available and have been disclosed in Methods or are available from the corresponding authors on reasonable request.

Conflicts of Interest: The authors declare no conflict of interest.

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No competing interests reported.

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m 6 A demethylase ALKBH5 regulates TP53 to promote malignant transformation of lung cells induced by radon exposure via BTG2-mediated mitochondrial pathway

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Abstract

Figures

1. Introduction

2. Results

2.1. Long-term radon exposure promotes lung tissue damage in mice

2.2. Radon exposure and TP53 KO induces malignant transformation of BEAS-2B cell

2.3. Radon exposure and TP53 KO induces MOMP in BEAS-2B cell

2.4. Radon exposure and TP53 KO can alter the gene expression profile in BEAS-2B cells

2.5. Clustering analysis of DEGs

2.6. BTG2 mediates the p53-triggered MOMP

2.7. TP53 downregulation was regulated by m6A demethylase ALKBH5

3. Discussion

4. Materials and Methods

4.1. Radon-exposed mouse model

4.2. Immunohistochemistry (IHC)

4.3. Oxidative Stress Detection

4.4. Measurement of mtDNA copy number

4.5. Western blot analysis

4.6. In vitro long-term radon exposure cell model

4.7. Cell proliferation assays

4.8. Detection of cell apoptosis

4.9. Soft-agar colony formation assay

4.10. Cell migration assays

4.11. Assessment of mitochondrial membrane potential (MMP)

4.12. High-throughput sequencing

4.13. Cluster analysis of expression profile

4.14. Bioinformatics analysis

4.15. qPCR assay

4.16. Plasmid construction and transfection

4.17. m6A related assays

4.18. RNA stability assay

4.19. Statistical analysis

5. Limitations of the study

Declarations

References

Additional Declarations

Supplementary Files

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2.7. TP53 downregulation was regulated by m⁶A demethylase ALKBH5

4.17. m⁶A related assays