In this cross-sectional study, we found that urinary 1-OHP levels were positively associated with peripheral blood H19 RNA expression in occupational workers, while urinary 1-OHP levels were negatively associated with plasma SAHH activity. Using human lung epithelial cell lines (BEAS-2B) with BaP treatment as a model, we observed that H19 binding to SAHH exaggerate DNMT1 expressions and activity with BaP treatment. Suppression of H19 enhanced the interaction of SAHH and DNMT1 induced by BaP. And H19/SAHH/DNMT1 may regulate OGG1 methylation, oxidative DNA damage and S phase arrest in BEAS-2B cells exposed to BaP. As far as we know, this represents the first case that a H19/SAHH/DNMT1 axis involving in OGG1 methylation, oxidative DNA damage and cell cycle arrest by carcinogen PAHs both in human and cells.
LncRNA H19 has been closely related with human several diseases in recent studies (Chen et al., 2020; Wang et al., 2020), and its upregulated expression profile has been observed in a variety of human malignancies (Wang et al., 2019). Numerous studies found PAHs are known to trigger lung cancer in animals and humans (Petit et al., 2019). 1-OHP has been widely used as a biomarker of PAHs exposure and a urinary metabolite of pyrene in occupationally exposed population, which accounts for 1.2% - 13.4% of PAHs exposure in coke-oven plant (Olujimi et al., 2018). Additionally, H19 binds to and inhibits SAHH function in vivo and vitro (Zhou et al., 2015; Oksana et al., 2013). In this cross-sectional study, a positive correlation was also observed between urinary 1-OHP levels and peripheral blood H19 RNA expression in occupational workers, while a negative correlation between urinary 1-OHP levels and plasma SAHH activity was observed. Our previous vitro experiments exposed to BaP also yielded the consistent results (Fu et al., 2018).
One-carbon metabolism comprises complex biological networks in which input nutrients are processed through a series of chemical reactions to cycle carbon units. The produced metabolites are then made available for important processes including cellular biosynthesis, methylation, regulation of redox status and amino-acid homeostasis (Fedra et al., 2017). Essentially, one-carbon metabolism involves three pathways: the folate and methionine cycles, and the transsulfuration pathway. The folate and methionine cycles overlap upon the synthesis of methyltetrahydrofolate (MTHF) necessary for the generation of methionine through methylation of homocysteine (Tibbetts and Appling, 2010). Methionine is then converted into the fundamental metabolite SAM, the universal cellular methyl donor required for DNA, RNA, protein, and lipid methylation. SAHH is an enzyme, which catalyses the hydrolysis of SAH which is formed after the donation of the methyl group of SAM to a methyl acceptor in methylation reaction (Oksana et al., 2013; Li et al., 2013).Human SAHH forms a homotetramer consisting of 432 amino acid (aa) and is composed of four identical chemically identical and functionally equivalent subunits (Porcelli et al., 2000), each with three domains: a small C-terminal domain (386 - 432 aa), a cofactor-binding domain (197 - 351 aa) and a substrate-binding domain (1 - 181 aa and 355 - 385 aa) (Beluzić et al., 2008). Nonetheless, we know little about the amino acid residues involved in the catalytic mechanism of SAHH. And then, WT-SAHH (1 - 432 aa), M1-SAHH (1 - 150 aa), M2-SAHH (151 - 300 aa), M3-SAHH (300 - 432 aa) mutant versions of SAHH were constructed to explore association between amino acid sites of SAHH catalytic activity and the function of lncRNA H19. Whether exposed BaP or not, we observed an accessorial enrichment of H19 in SAHH-containing RNPs in WT-SAHH, M1-SAHH and M2-SAHH cells, whereas M3-SAHH cells did not detectably elevate. Furthermore, H19 RNA expressions were inhibited in SAHH-overexpressing cells compare to WT cells, particularly in M3-SAHH cells. Collectively, H19 may bind to the 1 - 300 amino acid chain of SAHH to regulate oxidative DNA damage and cell cycle arrest exposed to BaP.
Otherwise, SAHH offers a single way in which catalyses the reversible SAH hydrolysis to relief from SAM-dependent methylation inhibition in eukaryotes (Oksana et al., 2013). Gene methylation, maintained strictly by the action of three DNMTs (DNMT1, DNMT3A and DNMT3B) in normal cells, is a key controller in a vast array of biological processes, such as DNA replication and repair (Oksana et al., 2004). Thus, the increase of SAHH activity would affect SAM-dependent DNMTs, which leads to the change of gene methylation. However, not all DNMTs are sensitive to SAHH. In one report, H19/SAHH/DNMT3B circuit regulates genome-wide methylation in human tumour cell models(Zhong et al., 2017), whereas Ponnaluri V.K. et al demonstrated that SAHH could enhance DNMT1 activity and interact with DNMT1 during S-phase in vitro (Ponnaluri et al., 2018). In this experiment, we found that whether treated cells with BaP or not, H19 or SAHH single knockdown and H19/SAHH double knockdown attenuated DNMT3A (Fig. S1F) and DNMT3B protein expressions (Fig. S1G). However, H19 or SAHH single knockdown and H19/SAHH double knockdown attenuated DNMT1 protein expressions and activity after BaP exposure. Moreover, the interaction of SAHH with DNMT1 would be strengthened by BaP and inhibition of H19 enhanced the interaction of SAHH and DNMT1 induced by BaP. Furthermore, co-localization between endogenous H19, SAHH and DNMT1 was observed in nucleus and perinuclear cytoplasm. Our investigations revealed that H19/SAHH might regulate BaP-induced abnormal gene methylation via binding to DNMT1 and exaggerate its expression and activity.
PAHs have attracted much attention due to their carcinogenicity and its metabolism mechanisms occurred by cytochrome P450-mediated oxidase system may lead to oxidative DNA damage (Campo et al., 2020) and abnormal cell cycle distribution, especially S phase arrest (Andrysik et al., 2007). BaP, a potent carcinogenic PAHs, can cause cell cycle block, including elevated S-phase cells ratio, weakened DNA replication capacity, and inhibited cell proliferation (Hruba et al., 2010). Accordingly, we conducted the vitro experiment with BEAS-2B cells treated with BaP as a simulated condition and hope to provide insights into the epigenetic regulation mechanism of H19/SAHH/DNMT1. Moreover, 8-OHdG, a ROS-induced DNA base modification, is a sensitive and stable biomarker in the evaluation of DNA damage by oxidation factors (Davalli et al., 2018). Previous study also examined that a significantly elevated 8-OHdG levels (OR = 2.63, 95% CI = 1.04 - 6.66) and S phase arrest (OR = 2.76, 95% CI = 1.18 - 6.45) was associated with high levels of urinary 1-OHP in 385 study population (Fu et al., 2019). In this experiment, we have observed that suppression of H19 reduced oxidative DNA damage and recovered S phase arrest, while suppression of SAHH and DNMT1 shows the opposite trend. It was validated that H19 binding to SAHH interacts with DNMT1 to regulate oxidative DNA damage and cell cycle arrest in human lung epithelial cell lines after BaP exposure.
Further, BER is thought to be the vital guardian pathway participated in the removal of the common oxidative lesion 8-OHdG, which is initiated by OGG1 involving in the first step of this repair process (Castillejos et al., 2000). OGG1 is bifunctional enzyme: it is able to remove 8-OHdG paired with C and therefore distinguish between 8-OHdG and the vast majority of normal bases (Klungland and Bjelland, 2007). Previous studies have observed associations between PAHs exposure with global or gene-specific DNA methylation alterations (Herbstman et al., 2012). Both PAHs exposure and its related damages (e.g., oxidative stress) have been associated with DNA methylation alterations (Herbstman et al., 2012). Moreover, exposure to some PAHs in PM10 have reported to show a higher methylation level in specific DNA repair genes, such as OGG1 and APEX (Alvarado-Cruz et al., 2016). Findings from the previous reports were consistent, which workers highly exposed to PAHs had an OGG1 Pos.4 hypermethylation in comparison with the low-exposed employees (Hernandez-Cortes et al., 2018). Based on the aforementioned information, it led us to be curious about the role of OGG1 methylation regulated by H19/SAHH/DMNT1 plays in oxidative DNA damage and cell cycle arrest related to BaP exposure. As expected, the significant rise occurred in OGG1 methylation with BaP treatment, whereas the decrease was observed when H19 was downregulated. Conversely, SAHH or DNMT1 single knockdown exacerbated OGG1 methylation. Interestingly, although H19 single knockdown obviously reduced OGG1 methylation, H19/SAHH and H19/DNMT1 double knockdown abrogated this effect while SAHH/DNMT1 double knockdown aggravated the alteration. It is further confirmed that H19/SAHH/DNMT1 can enhance the suppression of SAM-dependent biological methylation in BaP-treated cells, thereby reducing OGG1 methylation. As stated above, oxidative DNA damage and S phase arrest was consistent to OGG1 methylation. It also prompted that the alterations OGG1 methylation may be related with oxidative DNA damage and S phase arrest in BaP-treated cells. These parallel results indicated that H19/SAHH/DNMT1 plays a critical role in oxidative DNA damage and cell cycle arrest by PAHs.
In summary, our results support the hypothesis that H19/SAHH/DNMT1 axis may be involved in OGG1 methylation, oxidative DNA damage and cell cycle arrest by carcinogen BaP. The results in this study suggest the possible oxidative DNA damage and cell cycle arrest might reciprocally influence each other and form a vicious cycle, leading to greater severity DNA damage and even cancer. Further research should be aimed at a more detailed study of the specific mechanisms by which methylation occurs and plays a role in DNA damage.
Further, we have only studied oxidative DNA damage and cell cycle arrest in human lung epithelial cell lines, but have not shown any results on reactive oxygen species and χH2AX, which are the main markers of oxidative DNA damage. So, we will continue to have studied the effects of the parameters on ROS levels and expression of χH2AX in BEAS-2B cell line and lung cancer cell line, and published the research results in succession.