Benzo(a)pyrene promotes the malignant progression of malignant-transformed BEAS-2B cells by regulating YTH N6-methyladenosine RNA binding protein 1 to inhibit ferroptosis.

Benzo[a]pyrene (BaP) is associated with the development of lung cancer, but the underlying mechanism has not been completely clarified. Here, we used 10 µM BaP to induce malignant transformation of human bronchial epithelial BEAS-2B cells, named BEAS-2B-T. Results indicated that BaP (6.25, 12.5 and 25 µM) treatment significantly promoted the migration and invasion of BEAS-2B-T cells. Meanwhile, BaP exposure inhibited ferroptosis in BEAS-2B-T, ferroptosis-related indexes Fe2+, malondialdehyde (MDA), lipid peroxidation (LPO) and reactive oxygen species (ROS) decreased significantly. The protein level of ferroptosis-related molecule transferrin receptor (TFRC) decreased significantly, while solute carrier family 7 membrane 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) increased significantly. The intervention of ferroptosis dramatically effected the migration and invasion of BEAS-2B-T induced by BaP. Furthermore, the expression of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) was markedly increased after BaP exposure. YTHDF1 knockdown inhibited BEAS-2B-T migration and invasion by promoting ferroptosis. In the meantime, the contents of Fe2+, MDA, LPO and ROS increased significantly, TFRC was markedly increased, and SLC7A11, FTH1, and GPX4 were markedly decreased. Moreover, overexpression of YTHDF1 promoted BEAS-2B-T migration and invasion by inhibiting ferroptosis. Importantly, knockdown of YTHDF1 promoted ferroptosis and reduced BEAS-2B-T migration and invasion during BaP exposure, and overexpression of YTHDF1 increased migration and invasion of BEAS-2B-T by inhibiting ferroptosis during BaP exposure. RNA immunoprecipitation assays indicated that the binding of YTHDF1 to SLC7A11 and FTH1 markedly increased after YTHDF1 overexpression. Therefore, we concluded that BaP promotes the malignant progression of BEAS-2B-T cells through YTHDF1 upregulating SLC7A11 and FTH1 to inhibit ferroptosis. This study reveals new epigenetic and ferroptosis markers for preventing and treating lung cancer induced by environmental carcinogens.

FTO mediates bisphenol F-induced blood-testis barrier impairment through regulating ferroptosis via YTHDF1/TfRc and YTHDF2/SLC7A11 signal axis.

Bisphenol F (BPF) has been extensively utilized in daily life, which brings new hazards to male reproductive health. However, the specific functional mechanism is still unclear. Both cell and animal models were utilized for exploring the role of RNA methylation and ferroptosis and its underlying mechanisms in male reproductive injury induced by BPF. In animal model, BPF severely destroyed the integrity of the blood-testis barrier (BTB) and induced ferroptosis. Furthermore, BPF significantly affected the barrier function of TM4 cells and promoted ferroptosis. Importantly, ChIP assays revealed that BPF inhibited AR transcriptional regulation of FTO and FTO expression was downregulated in TM4 cells. Overexpression of FTO prevented the impairment of BTB by inhibiting ferroptosis in TM4 cells. Mechanistically, FTO could significantly down-regulate the m6A modification level of TfRc and SLC7A11 mRNA through MeRIP experiment. RIP experiments showed that YTHDF1 can bind to TfRc mRNA and promote its translation while YTHDF2 could bind to SLC7A11 mRNA and reduce its mRNA stability. Therefore, our results suggest that FTO plays a key role in BPF induced male reproductive toxicity through YTHDF1-TfRc axis and YTHDF2-SLC7A11 axis and may provide new ideas and methods for the prevention and treatment of male reproductive diseases associated with environmental pollutants.

FTO-Nrf2 axis regulates bisphenol F-induced leydig cell toxicity in an m6A-YTHDF2-dependent manner.

Studies have shown that Bisphenol F (BPF) as an emerging bisphenol pollutant also has caused many hazards to the reproductive systems of humans and animals. However, its specific mechanism is still unclear. The mouse TM3 Leydig cell was used to explore the mechanism of BPF-induced reproductive toxicity in this study. The results showed BPF (0, 20, 40 and 80 µM) exposure for 72 h significantly increased cell apoptosis and decreased cell viability. Correspondingly, BPF increased the expression of P53 and BAX, and decreased the expression of BCL2. Moreover, BPF significantly increased the intracellular ROS level in TM3 cells, and significantly decreased oxidative stress-related molecule Nrf2. BPF decreased the expression of FTO and YTHDF2, and increased the total cellular m6A level. ChIP results showed that AhR transcriptionally regulated FTO. Differential expression of FTO revealed that FTO reduced the apoptosis rate of BPF-exposed TM3 cells and increased the expression of Nrf2, MeRIP confirmed that overexpression of FTO reduced the m6A of Nrf2 mRNA. After differential expression of YTHDF2, it was found that YTHDF2 enhanced the stability of Nrf2, and RIP assay showed that YTHDF2 was bound to Nrf2 mRNA. Nrf2 agonist enhanced the protective effect of FTO on TM3 cells exposure to BPF. Our study is the first to demonstrate that AhR transcriptionally regulated FTO, and then FTO regulated Nrf2 in a m6A-modified manner through YTHDF2, thereby affecting apoptosis in BPF-exposed TM3 cells to induce reproductive damage. It provides new insights into the importance of FTO-YTHDF2-Nrf2 signaling axis in BPF-induced reproductive toxicity and provided a new idea for the prevention of male reproductive injury.

RNA binding protein YTHDF1 mediates bisphenol S-induced Leydig cell damage by regulating the mitochondrial pathway of BCL2 and the expression of CDK2-CyclinE1.

Bisphenol S (BPS) causes reproductive adverse effects on humans and animals. However, the detailed mechanism is still unclear. This research aimed to clarify the role of RNA binding protein YTHDF1 in Leydig cell damage induced by BPS. The mouse TM3 Leydig cells were exposed to BPS of 0, 20, 40, and 80 µmol/L for 72 h. Results showed that TM3 Leydig cells apoptosis rate markedly increased in BPS exposure group. Meanwhile, the apoptosis-related molecule BCL2 protein level decreased significantly, and Caspase9, Caspase3, and BAX increased significantly. Moreover, the cell cycle was blocked in the G1/S phase, CDK2 and CyclinE1 were considerably down-regulated in BPS exposure groups, and the protein level of RNA binding protein YTHDF1 decreased sharply. Furthermore, after overexpression of YTHDF1, the cell viability significantly increased, and the apoptosis rate significantly decreased in TM3 Leydig cells. In the meantime, BCL2, CDK2, and CyclinE1 were significantly up-regulated, and BAX, Caspase9, and Caspase3 were significantly down-regulated. Conversely, interference with YTHDF1 decreased cell proliferation and promoted apoptosis. Importantly, overexpression of YTHDF1 alleviated the cell viability decrease induced by BPS, and interference with YTHDF1 exacerbated the situation. RIP assays showed that the binding of YTHDF1 to CDK2, CyclinE1, and BCL2 significantly increased after overexpressing YTHDF1. Collectively, our study suggested that YTHDF1 plays an essential role in BPS-induced TM3 Leydig cell damage by regulating CDK2-CyclinE1 and BCL2 mitochondrial pathway at the translational level.

IFI44L as a novel epigenetic silencing tumor suppressor promotes apoptosis through JAK/STAT1 pathway during lung carcinogenesis.

Numerous evidence showed that the occurrence and development of lung cancer is closely related to environmental pollution. Therefore, new environmental response predictive markers are urgently needed for early diagnosis and screening of lung cancer. Interferon-induced protein 44-like (IFI44L) has been shown to be related in a variety of tumors, but its function and mechanism during lung carcinogenesis still have remained largely unknown. In this study, gene expression and methylation status were analyzed through online tools and malignant transformation models. Differentially expressed cell models and xenograft tumor models were established and used to clarify the gene function. RT-qPCR, western blotting, immunohistochemistry, and co-immunoprecipitation (Co-IP) were used to explore the mechanism. Results showed that IFI44L was dramatically downexpressed during lung carcinogenesis, and its low expression may be attributed to DNA methylation. Overexpression of IFI44L obviously inhibited cell growth and promoted apoptosis. After knockdown of IFI44L expression, the proliferation ability was remarkably increased and the apoptosis was significantly reduced. Functional enrichment showed that IFI44L was involved in apoptosis and JAK/STAT1 signaling pathway, and was highly correlated with downstream molecules. After overexpression of IFI44L, the expression of P-STAT1 and downstream molecules XAF1, OAS1, OAS2 and OAS3 were significantly increased. After knockdown of STAT1 expression, the pro-apoptotic effect of IFI44L was reduced. Co-IP results showed that IFI44L had protein interaction with STAT1. Results proved that IFI44L promoted STAT1 phosphorylation and activated the JAK/STAT1 signaling pathway by directly binding to STAT1 protein, thereby leading to cell apoptosis. Our study revealed that IFI44L promotes cell apoptosis and exerts tumor suppressors by activating the JAK/STAT1 signaling pathway. It further suggests that IFI44L has clinical therapeutic potential and may be a promising biomarker during lung carcinogenesis.

TET1 involved in bisphenol A induced TM3 Leydig cell toxicity by regulating Cav3.3 hydroxymethylation.

Bisphenol A (BPA), an important environmental pollutant, is known to damage reproductive development. However, the underlying epigenetic mechanism in Leydig cells during BPA exposure has not been explored in detail. In this study, TM3 Leydig cells were treated with BPA (0, 20, 40 and 80 µM) for 72 h. The differentially expressed TET1 cell model was constructed to explore the mechanism of BPA-induced cytotoxicity. Results showed that BPA exposure significantly inhibited cell viability and increased apoptosis of TM3 Leydig cells. Meanwhile, the mRNA of TET1, Cav3.2 and Cav3.3 decreased significantly with the increase of BPA exposure. Importantly, TET1 significantly promoted proliferation of TM3 Leydig cells and inhibited apoptosis. Differentially expressed TET1 significantly affected BPA-induced toxicity in TM3 Leydig cells. Notably, TET1 elevated the mRNA levels of Cav3.2 and Cav3.3. MeDIP and hMeDIP confirmed that TET1 regulated the expression of Cav3.3 through DNA hydroxymethylation. Our study firstly presented that TET1 participated in BPA-induced toxicity in TM3 Leydig cells through regulating Cav3.3 hydroxymethylation modification. These findings suggest that TET1 acts as a potential epigenetic marker for reproductive toxicity induced by BPA exposure and may provide a new direction for the research on male reproductive damage.

TET1 mediated male reproductive toxicity induced by Bisphenol A through Catsper-Ca2+ signaling pathway.

Bisphenol A (BPA) exposure has many adverse effects on the reproductive system in animals and humans. Ten-eleven translocation 1 (TET1) is closely related to a variety of biological processes through regulating the dynamic balance of DNA demethylation and methylation. However, the role and mechanism of TET1 during BPA induced reproductive toxicity are largely unknown. In this study, mouse spermatogonia cell line GC-2 was treated with BPA in the final concentration of 0, 20, 40 and 80 µM for 72 h. The cell model of differential TET1 gene expression was established to explore the role and mechanism. We found that the growth rate of GC-2 cells, and the intracellular calcium level decreased significantly with the increase of BPA dose, while TET1 and Catsper1-4 expression level decrease with a dose-dependent relationship. Furthermore, TET1 overexpression promoted the proliferation of GC-2 cell, the increase of calcium ion concentration, and the expression level of Catsper1-4, while knockdown of TET1 leads to the opposite results. Mechanistically, TET1 expression promoted the hydroxymethylation of Catsper1-4 and reduced their methylation level. In addition, the expression level of Catsper1-4 was positively correlated with TET1 gene expression level in semen samples of the population. Our study revealed for the first time that TET1 gene regulates the expression of related molecules in the Catsper calcium signal pathway through its hydroxymethylation modification to affect the calcium level, thereby participating in the process of BPA induced damage. These results indicated that TET1 gene may be a potential biomarker of BPA induced male reproductive toxicity.

Gene expression network related to DNA methylation and miRNA regulation during the process of aflatoxin B1-induced malignant transformation of L02 cells.

Aflatoxin is a secondary metabolite secreted by Aspergillus flavus, parasitic Aspergillus, and other fungi through the polyketone pathway, and it can be detected in many foods. Aflatoxin has strong toxicity and carcinogenicity, and many studies have shown that aflatoxin is highly associated with liver cancer. In the present study, malignant transformation of L02 cells was induced by aflatoxin B1 (AFB1), and the gene expression, miRNA expression, and methylation level were detected by high-throughput sequencing. The gene and miRNA expression results showed that 2547 genes and 315 miRNAs were changed in the AFB1-treated group compared with the DMSO group. Among them, RSAD2 and SCIN were significantly upregulated, whereas TRAPPC3L and UBE2L6 were significantly downregulated. Has-miR-33b-3p was significantly upregulated, whereas Has-miR-3613-5p was significantly downregulated. The methylation results showed that 2832 CpG sites were methylated on the promoter or coding DNA sequence (CDS) of the gene, whereas the expression of DNMT3a and DNMT3b was significantly upregulated. Moreover, hypermethylation occurred in TRAPPC3L, CDH13, and SPINK13. The results of GO and KEGG pathway analyses showed that significantly changed genes and miRNAs were mainly involved in tumor formation, proliferation, invasion, and migration. The results of network map analysis showed that Hsa-miR-3613-5p, Hsa-miR-615-5p, Hsa-miR-615-3p, and Hsa-miR-3158-3p were the key miRNAs for malignant transformation of L02 cells induced by AFB1. In addition, the expression of ONECUT2, RAP1GAP2, and FSTL4 was regulated by DNA methylation and miRNAs. These results suggested that the gene expression network regulated by DNA methylation and miRNAs may play a vital role in AFB1-induced hepatocellular carcinoma.

Identification of SRY-box 30 as an age-related essential gatekeeper for male germ-cell meiosis and differentiation.

Although important factors governing the meiosis have been reported in the embryonic ovary, meiosis in postnatal testis remains poorly understood. Herein, we first report that SRY-box 30 (Sox30) is an age-related and essential regulator of meiosis in the postnatal testis. Sox30-null mice exhibited uniquely impaired testis, presenting the abnormal arrest of germ-cell differentiation and irregular Leydig cell proliferation. In aged Sox30-null mice, the observed testicular impairments were more severe. Furthermore, the germ-cell arrest occurred at the stage of meiotic zygotene spermatocytes, which is strongly associated with critical regulators of meiosis (such as Cyp26b1, Stra8 and Rec8) and sex differentiation (such as Rspo1, Foxl2, Sox9, Wnt4 and Ctnnb1). Mechanistically, Sox30 can activate Stra8 and Rec8, and inhibit Cyp26b1 and Ctnnb1 by direct binding to their promoters. A different Sox30 domain required for regulating the activity of these gene promoters, providing a "fail-safe" mechanism for Sox30 to facilitate germ-cell differentiation. Indeed, retinoic acid levels were reduced owing to increased degradation following the elevation of Cyp26b1 in Sox30-null testes. Re-expression of Sox30 in Sox30-null mice successfully restored germ-cell meiosis, differentiation and Leydig cell proliferation. Moreover, the restoration of actual fertility appeared to improve over time. Consistently, Rec8 and Stra8 were reactivated, and Cyp26b1 and Ctnnb1 were reinhibited in the restored testes. In summary, Sox30 is necessary, sufficient and age-associated for germ-cell meiosis and differentiation in testes by direct regulating critical regulators. This study advances our understanding of the regulation of germ-cell meiosis and differentiation in the postnatal testis.

DNA methylation and hydroxymethylation associated with gene expression regulatory network during 3-methylcholanthrene induced lung cell malignant transformation.

3-methylcholanthrene (3-MCA) is a typical representative PAH. It has strong toxicity and is a typical chemical carcinogen. However, the epigenetic mechanisms underlying 3-MCA-induced tumourigenesis are largely unknown. In this study, a model of the 3-MCA-induced malignant transformation of human bronchial epithelial (HBE) cells was established successfully. The profiles of gene expression and DNA methylation and hydroxymethylation were obtained and analysed with an Illumina HiSeq 4000. A total of 707 genes were found to be significantly up-regulated, and 686 genes were found to be significantly down-regulated. Compared to control cells, 8545 mRNA-associated differentially methylated regions and 15,121 mRNA-associated differentially hydroxymethylated regions in promoters were found to be significantly altered in transformed cells. By using mRNA expression and DNA methylation and hydroxymethylation interaction analysis, 99 differentially expressed genes were identified. Among them, CA9 and EGLN3 were verified to be significantly down-regulated, and CARD6 and LCP1 were shown to be significantly up-regulated, and these genes mainly participated in cell growth, migration and invasion, indicating that these genes were key genes involved in the 3-MCA-induced malignant transformation of HBE cells. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that a large number of differentially expressed genes (DEGs) were involved mainly in RNA polymerase II transcription factor activity, chemical carcinogenesis, base-excision repair (BER), cytokine-cytokine receptor interactions, glycerolipid metabolism, steroid hormone biosynthesis, cAMP signalling pathways and other signalling pathways. Our study suggested that characteristic gene alterations associated with DNA methylation and hydroxymethylation could play important roles in environmental 3-MCA-induced lung carcinogenesis.

Epigenetic silencing of TET1 mediated hydroxymethylation of base excision repair pathway during lung carcinogenesis.

The methylcytosine dioxygenase Ten-eleven translocation 1 (TET1) is an important regulator for the balance of DNA methylation and hydroxymethylation through various pathways. Increasing evidence has suggested that TET1 probably involved in DNA methylation and demethylation dysregulation during chemical carcinogenesis. However, the role and mechanism of TET1 during lung cancer remains unclear. In this study, we found that TET1 expression was significantly down-regulated and the methylation level was significantly up-regulated in 3-methylcholanthrene (3-MCA) induced cell malignant transformation model, rat chemical carcinogenesis model, and human lung cancer tissues. Demethylation experiment further confirmed that DNA methylation negatively regulated TET1 gene expression. TET1 overexpression inhibited cell proliferation, migration and invasion in vitro and in vivo, while knockdown of TET1 resulted in an opposite phenotype. DNA hydroxymethylation level in the promoter region of base excision repair (BER) pathway key genes XRCC1, OGG1, APEX1 significantly decreased and the degree of methylation gradually increased in malignant transformed cells. After differential expression of TET1, the level of hydroxymethylation, methylation and expression of these genes also changed significantly. Furthermore, TET1 binds to XRCC1, OGG1, and APEX1 to maintain them hydroxymethylated. Blockade of BER pathway key gene alone or in combination significantly diminished the effect of TET1. Our study demonstrated for the first time that TET1 expression is regulated by DNA methylation and TET1-mediated hydroxymethylation regulates BER pathway to inhibit the proliferation, migration and invasion during 3-MCA-induced lung carcinogenesis. These results suggested that TET1 gene can be a potential biomarker and therapy target for lung cancer.

Diagnostic and prognostic value of the BEX family in lung adenocarcinoma.

Previous studies have demonstrated that members of the brain-expressed X-linked (BEX) family participate in a wide range of biological functions in normal and tumor tissues. However, their role and clinical significance in lung adenocarcinoma (LUAD) remains unclear. The present study investigated The Cancer Genome Atlas data and revealed that the BEX family was downregulated in LUAD tissues compared with adjacent non-cancerous tissues. Additionally, analysis of LUAD cohorts from the Oncomine database revealed similar results. Furthermore, the expression of BEX members was significantly decreased in several LUAD cell lines compared with normal lung epithelial cells in vitro. The aforementioned data mining and in vitro results suggested that the BEX family may be involved in the development of LUAD. Furthermore, receiver operating characteristic curve analysis revealed that BEX members exhibited high sensitivity and specificity for the diagnosis of patients with LUAD. The low expression levels of BEX1, BEX4 and BEX5 were associated with certain pathologic features, particularly in advanced LUAD. Survival analysis demonstrated that BEX members, particularly BEX4, were involved in the prognosis of patients with LUAD at early and late clinical stages. The results obtained in the current study suggested that BEX members may serve as potential tumor biomarkers for the diagnosis and prognosis of patients with LUAD.

Author Correction: Identification of TC2N as a novel promising suppressor of PI3K-AKT signaling in breast cancer.

After publication of this article, it came to the attention of the authors that their names had been reordered. Professor. Jia Cao and Prof. Jin-yi Liu are the co-corresponding authors, and Prof. Jin-yi Liu should be the last author.

Epigenetic inactivation of LHX6 mediated microcystin-LR induced hepatocarcinogenesis via the Wnt/ß-catenin and P53 signaling pathways.

Microcystins (MCs) have been shown to be carcinogenic by animal and cellular experiments and found to be associated with the development of human hepatocellular carcinoma (HCC) through epidemiological studies. However, the molecular mechanism of microcystin-LR (MC-LR) induced HCC is still unclear. This study is determined to clarify the role and mechanism of LHX6 in MC-LR-induced hepatocarcinogenesis. Using the previously established MC-LR-induced malignant transformation model in L02 cells, we screened out LHX6, homeobox gene that was significantly changed. We found that LHX6 was significantly down-regulated in MC-LR treated L02 cells and the liver tissue of rats treated for 35 weeks with 10 µg/kg body weight of MC-LR. Expression of LHX6 in human tumor tissue was significantly down-regulated in high MC-LR-exposure group. LHX6 was hypermethylated in MC-LR treated L02 cells and up-regulated after treatment with 10 µM of 5-aza-2'-deoxycytidine. Furthermore, overexpression of LHX6 inhibited proliferation, invasion and migration of malignantly transformed L02 cells in vitro and in vivo, while knockdown of LHX6 resulted in an opposite phenotype. In addition, we found that up-regulation of P53 and Bax resulted in apoptosis, and that down-regulation of CTNNB1 and MMP7 led to migration of MC-LR treated L02 cells. Blockade of P53 and CTNNB1 by its inhibitor significantly diminished the effect of LHX6. These genes were working together during the process of MC-LR-induced hepatocarcinogenesis. Our study demonstrated for the first time that LHX6 gene expression is regulated by DNA methylation and can inhibit the proliferation, invasion and migration through Wnt/ß-catenin and P53 signaling pathways during the MC-LR-induced hepatocarcinogenesis. This result may suggest that LHX6 gene can be used as a potential target gene and a biomarker for liver cancer treatment.

Identification of TC2N as a novel promising suppressor of PI3K-AKT signaling in breast cancer.

Although TC2N has proven to be an oncogene in lung cancer, its biological function and molecular mechanisms in other cancer still remains unclear. Here, we investigate in breast cancer that TC2N expression is sharply overexpressed in breast cancer specimens compared with normal breast specimens, and the low TC2N expression was associated with advanced stage, lymphatic metastasis, larger tumors and shorter survival time. Upregulation of TC2N significantly restrains breast cancer cell proliferation in vitro and tumor growth in vivo. Mechanistically, TC2N blocks AKT signaling in a PI3K dependent and independent way through weakening the interaction between ALK and p55γ or inhibiting the binding of EBP1 and AKT. To sum up, these results unmask an ambivalent role of TC2N in cancer, providing a promising inhibitor for PI3K-AKT signaling.

Epigenetic silencing of ALX4 regulates microcystin-LR induced hepatocellular carcinoma through the P53 pathway.

Recent studies have shown that microcystin-LR (MC-LR) is one of the principal factors that cause liver cancer. Previously we have found that Aristaless-like Homeobox 4 (ALX4) was differentially expressed in MC-LR-induced malignant transformed L02 cells. However, the expression regulation, role and molecular mechanism of ALX4 during the process of liver cancer induced by MC-LR are still unclear. The expression of ALX4 was detected by quantitative reverse-transcription PCR and Western blot in MC-LR induced malignantly transformed cell and rat models. Methylation status of ALX4 promoter region was evaluated by methylation-specific PCR and bisulfite genomic sequencing. The anti-tumor effects of ALX4 on MC-LR induced liver cancer were identified in vitro and in vivo. ALX4 expression was progressively down-regulated in MC-LR-induced malignantly transformed L02 cells and the MC-LR exposed rat models. ALX4 promoter regions were highly methylated in malignantly transformed cells, while treatment with demethylation agent 5-aza-dC significantly increased ALX4 expression. Functional studies showed that overexpression of ALX4 inhibits cell proliferation, migration, invasion and metastasis in vitro and in vivo, blocks the G1/S phase and promotes the apoptosis. Conversely, knockdown of ALX4 promotes cell proliferation, migration and invasion. Mechanism study found that ALX4 exerts its antitumor function through the P53 pathway, C-MYC and MMP9. More importantly, ALX4 expression level showed a negative relation with serum MC-LR levels in patients with hepatocellular carcinoma. Our results suggested that ALX4 was inactivated by DNA methylation and played a tumor suppressor function through the P53 pathway in MC-LR induced liver cancer.

TC2N, a novel oncogene, accelerates tumor progression by suppressing p53 signaling pathway in lung cancer.

The protein containing the C2 domain has been well documented for its essential roles in endocytosis, cellular metabolism and cancer. Tac2-N (TC2N) is a tandem C2 domain-containing protein, but its function, including its role in tumorigenesis, remains unknown. Here, we first identified TC2N as a novel oncogene in lung cancer. TC2N was preferentially upregulated in lung cancer tissues compared with adjacent normal lung tissues. High TC2N expression was significantly associated with poor outcome of lung cancer patients. Knockdown of TC2N markedly induces cell apoptosis and cell cycle arrest with repressing proliferation in vitro, and suppresses tumorigenicity in vivo, whereas overexpression of TC2N has the opposite effects both in vitro and in vivo. Using a combination of TCGA database and bioinformatics, we demonstrate that TC2N is involved in regulation of the p53 signaling pathway. Mechanistically, TC2N attenuates p53 signaling pathway through inhibiting Cdk5-induced phosphorylation of p53 via inducing Cdk5 degradation or disrupting the interaction between Cdk5 and p53. Moreover, the blockade of p53 attenuates the function of TC2N knockdown in the regulation of cell proliferation and apoptosis. In addition, downregulated TC2N is involved in the apoptosis of lung cancer cells induced by doxorubicin, leading to p53 pathway activation. Overall, these findings uncover a role for the p53 inactivator TC2N in regulating the proliferation and apoptosis of lung cancer cells. Our present study provides novel insights into the mechanism of tumorigenesis in lung cancer.

TMEM196 hypermethylation as a novel diagnostic and prognostic biomarker for lung cancer.

Emerging evidences have revealed tumor-specific gene methylation is considered to be a promising non-invasive biomarker for many different types of cancers. This study was determined whether TMEM196 gene hypermethylation and downregulation are considered to be promising biomarkers for early diagnosis and prognosis in lung cancer. Methylation status was detected with methylation-specific PCR. Kaplan-Meier survival curves and Cox regression analysis were used to determine the significance of prognosis. TMEM196 gene was hypermethylated in 68.1% (64/94) of lung cancer tissues, 52.8% (67/127) of plasma and 55.2% (79/143) of sputum samples, but unmethylated (0/50) in normal tissues. TMEM196 methylation in plasma and sputum samples was significantly correlated with that in the corresponding paired tumor tissues (r = 0.750, r = 0.880, P < 0.001). TMEM196 aberrant methylation in cancer tissues, plasma and sputum DNA was significantly associated with age and pathological type (P < 0.05). TMEM196 high methylation could robustly distinguish lung cancer patients (AUC = 0.905) from normal subjects and patients with TMEM196 high methylation have a significantly poorer survival than those with low level from The Cancer Genome Atlas (Wilcoxon P < 0.001). Multivariate models showed TMEM196 methylation is an independent prognostic marker in lung cancer. Furthermore, the overall survival of patients with low TMEM196 expression was significantly poorer than that of TMEM196-high patients (P < 0.001, log-rank test). Low TMEM196 expression in tumor tissues was found to predict poorer survival (HR = 3.007; 95%CI, 1.918-4.714). Our study provided new insights into the clinical importance and potential use of TMEM196 methylation and expression as novel early diagnostic and prognostic biomarkers for human lung cancers.

Bisphenol A induced male germ cell apoptosis via IFNß-XAF1-XIAP pathway in adult mice.

Bisphenol A (BPA) impairs male fertility by acting as an endocrine disruptor. However, the mechanisms by which BPA cause reproductive toxicity are not fully elucidated. Here, we explored the role of XAF1, a novel pro-apoptosis molecule, in BPA-induced abnormal spermatogenesis and the transcriptional regulation mechanism of BPA-induced XAF1. BPA exposure detrimentally impacted spermatogenesis by inducing excessive germ cell apoptosis. XAF1 was upregulated in germ cells after BPA exposure, which was involved in the apoptosis pathway. In addition, the expression levels of XIAP and XAF1 were inversely correlated after BPA exposure. Knockdown of XAF1 expression partially inhibited the apoptosis of GC-2 cells, suppressed the activation of caspase 3 and improved the BPA-induced XIAP expression. Moreover, IFNß expression levels were significantly upregulated after BPA exposure both in vitro and in vivo, and these levels were positively related to the expression of XAF1. Furthermore, IFNß knockdown reduced the expression of XAF1 and increased the expression of XIAP in BPA-treated GC-2 cells. Together, these data indicated that BPA triggers male germ cell apoptosis in mice via the IFNß-XAF1-XIAP pathway, which may contribute to BPA-induced testis toxicity.

A commercial Roundup® formulation induced male germ cell apoptosis by promoting the expression of XAF1 in adult mice.

Roundup® is extensively used for weed control worldwide. Residues of this compound may lead to side effects of the male reproductive system. However, the toxic effects and mechanisms of Roundup® of male germ cells remain unclear. We aimed to investigate the apoptosis-inducing effects of Roundup® on mouse male germ cells and explore the role of a novel tumor suppressor XAF1 (X-linked inhibitor of apoptosis-associated factor 1) involved in this process. We demonstrated that Roundup® can impair spermatogenesis, decrease sperm motility and concentration, and increase the sperm deformity rate in mice. In addition, excessive apoptosis of germ cells accompanied by the overexpression of XAF1 occurred after Roundup® exposure both in vitro and in vivo. Furthermore, the low expression of XIAP (X-linked inhibitor of apoptosis) induced by Roundup® was inversely correlated with XAF1. Moreover, the knockdown of XAF1 attenuated germ cell apoptosis, improved XIAP expression and inhibited the activation of its downstream target proteins, caspase-3 and PARP, after Roundup® exposure. Taken together, our data indicated that XAF1 plays an important role in Roundup®-induced male germ cell apoptosis. The present study suggested that Roundup® exposure has potential negative implications on male reproductive health in mammals.