Effects of H19/SAHH/DNMT1 on the oxidative DNA damage related to Benzo[a]pyrene exposure

doi:10.21203/rs.3.rs-944681/v1

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Effects of H19/SAHH/DNMT1 on the oxidative DNA damage related to Benzo[a]pyrene exposure

https://doi.org/10.21203/rs.3.rs-944681/v1

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Background: The mechanisms that long noncoding RNA (lncRNA) H19 binding to S-adenosylhomocysteine hydrolase (SAHH) interacted with DNA methyltransferase 1 (DNMT1) and then regulated DNA damage caused by PAHs remain unclear.

Results: We documented that urinary 1-hydroxypyrene (1-OHP) levels were positively associated with blood H19 RNA expression (OR: 1.51, 95% CI: 1.03 - 2.19), but opposite to plasma SAHH activity (OR: 0.63, 95% CI: 0.41 - 0.98) in coke oven workers. Moreover, by constructing various BEAS-2B cell models exposed to Benzo[a]pyrene (BaP), we investigated that H19 binding to SAHH exaggerated DNMT1 expressions and activity. Suppression of H19 enhanced the interaction of SAHH and DNMT1 in BaP-treated cells, decreased OGG1 methylation, reduced oxidative DNA damage and lessened S phase arrest. However, SAHH or DNMT1 single knockdown and SAHH/DNMT1 double knockdown showed the opposite trend.

Conclusions: A H19/SAHH/DNMT1 axis was involved in OGG1 methylation, oxidative DNA damage and cell cycle arrest by carcinogen BaP.

Epigenetics & Genomics

Polycyclic aromatic hydrocarbons

long non-coding RNA H19

Oxidative DNA damage

8-oxoguanine DNA glycosylase 1

S-adenosylhomocysteine hydrolase

DNA methyltransferases

Long noncoding RNAs (lncRNAs), a group of transcription of RNA from non-protein coding regions of the genome, are defined as endogenous cellular RNAs of longer than 200 nucleotides. They are implicated in a number of molecular functions, such as regulators of protein activity, molecular scaffolds, regulation of cell cycle and controllers of chromatin remodelling (1). H19, a multifunctional lncRNA, interacts with other proteins to exert functions as a ribonucleoprotein in tumorigenesis (2). Similar to Zhou J et al.(3), a previous research by our team has revealed that H19 binds to S-adenosylhomocysteine hydrolase (SAHH) and inhibits SAHH function, thus blocking LINE-1 methylation in vitro (4). SAHH has been reported as a highly conserved enzyme capable of reversibly hydrolysing S-adenosylhomocysteine (SAH) and participates in S-adenosylmethionine (SAM)-dependent transmethylation reactions (5). Gene methylation (5-methyl-cytosine), accomplished by the joint action of three SAM-dependent DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), plays a critical role in mammalian development and physiology (6). However, very little is known which type of DNA methyltransferase (DNMT1, DNMT3A or DNMT3B) would play a prominent role in the mechanism of H19 binding to SAHH involved in gene methylation.

The genotoxic potency of PAHs is driven by excessive reactive oxygen species (ROS) production generated in the redox-cycling processes of their metabolic activation which overwhelms their elimination, and thus causing oxidative DNA lesion (7). Base excision repair (BER) is responsible for repairing oxidative lesion (8). Eight-oxoguanine DNA glycosylase 1 (OGG1) acts as the key enzyme participated in the first step of the BER pathway (9). Gene methylation, the important form of epigenetic modification in eukaryote genome, is known to regulate DNA damages caused by PAHs (10). Increased methylation of OGG1 leads to the accumulation of DNA damage and further mutations (11). Although the aspects of genetic alterations in population exposed to PAHs are well-documented (12), abnormal epigenetic regulation is still poorly understood.

Therefore, in order to explore effects of PAHs exposure on peripheral blood H19 RNA expressions and plasma SAHH activity, we performed a cross-sectional observational study among occupational workers. Moreover, Benzo[a]pyrene (BaP), a potent carcinogen of PAHs, can cause a wide range of cell cycle perturbations, including S-phase accumulation, diminished capacity for DNA replication, and inhibition of cell proliferation. We hypothesized that H19/SAHH/DNMTs involved in OGG1 methylation, oxidative DNA damage and cell cycle arrest in human after BaP exposure. To address our objectives, we used human lung epithelial cell lines treated with BaP as a model, and expected to illuminate the molecular mechanism of H19/SAHH/DNMTs regulating OGG1 methylation, oxidative DNA damage and cell cycle arrest by carcinogen BaP.

Effects of PAHs exposure on peripheral blood H19 RNA expressions and plasma SAHH activity among 146 occupational workers

Table 1 summarizes the basic characteristics of study population based on the quartiles of urinary 1-OHP concentrations. There were no significant differences in age, smoking status, drinking status, heating mode, education (P > 0.05); even so, the marginal significances were showed in employment time (P = 0.053). The results indicated that with the rise of urinary 1-OHP levels, the concentrations of urinary 2-NAP, 2-FLU, 9-PHE and ΣOH-PAHs metabolites were also significantly elevated (P < 0.001). In addition, the detailed distributions of peripheral blood H19 RNA expressions and plasma SAHH activity were given in Fig. 1A with violin plots. An elevated median of peripheral blood H19 RNA expressions was showed coupled with the increased quartile of urinary 1-OHP levels (P = 0.479), whereas the median of plasma SAHH activity was slightly reduced (P = 0.844). These findings are consistent with our previous experiments in human lung-derived cells treated with BaP (4).

Table 1

Basic characteristics of participants by urinary 1-OHP among 146 occupational workers ^a
Variable	Urinary 1-OHP (µg/mmol creatinine)					P value ^b
Variable	Total (n = 146)	Quartile 1 (< 0.03) (n = 36)	Quartile 2 (0.03–0.06) (n = 36)	Quartile 3 (0.06–0.11) (n = 37)	Quartile 4 (> 0.11) (n = 37)	P value ^b
General characteristic
Age (years)	40.5 (10)	39.5 (9)	44.0 (11)	43.0 (10)	39.0 (12)	0.144
Employment time (years)	21.0 (12)	20.0 (12)	26.5 (13)	24.0 (10)	19.0 (17)	0.053
Smoking	105 (71.9)	26 (72.2)	27 (75.0)	27 (73.0)	25 (67.6)	0.910
Drinking	73 (50.0)	18 (50.0)	15 (41.7)	20 (54.1)	20 (54.1)	0.685
Central heating	140 (95.9)	33 (91.7)	36 (100.0)	35 (94.6)	36 (97.3)	0.319
Education (years)						0.367
≤ 9	41 (28.1)	10 (27.8)	13 (36.1)	6 (16.2)	12 (32.4)
10–12	57 (39.0)	12 (33.3)	14 (38.9)	15 (40.5)	16 (43.2)
> 12	48 (32.9)	14 (38.9)	9 (25.0)	16 (43.2)	9 (24.3)
PAHs internal exposure biomarker
2-NAP	0.74 (0.77)	0.62 (0.71)	0.70 (0.50)	0.53 (0.58)	1.13 (1.43)	0.001
2-FLU	0.29 (0.27)	0.18 (0.13)	0.27 (0.17)	0.25 (0.29)	0.53 (0.63)	< 0.001
9-PHE	0.09 (0.09)	0.06 (0.03)	0.07 (0.04)	0.09 (0.07)	0.23 (0.22)	< 0.001
ΣOH-PAHs	0.32 (0.29)	0.23 (0.18)	0.27 (0.20)	0.32 (0.20)	0.51 (0.56)	< 0.001
Concentration unit of 2-NAP, 2-FLU, 9-PHE and 1-OHP is µg/mmol creatinine.
^a Data were presented as N (%) or Med (IQR).
^b P-values were calculated from Chi-square test for categorical variables and Kruskal-Wallis H test for numerical variables.

The results of collinearity diagnostics showed that there was no collinearity between the four urinary PAHs metabolites, and also indicated that age was not collinear with employment time (Table S3). Figure 1C represented that peripheral blood H19 RNA expressions were significantly increased when 1-OHP levels were lower than 0.09 µg/mM creatinine, and then maintained on the plateau phase while there is a marginal positive linear association between urinary 1-OHP levels and peripheral blood H19 RNA expressions (4th vs. 1st quartile = 3.44, 95% CI: 1.04–11.44, P _trend =0.058) (Fig. 1B). Nevertheless, the plasma SAHH activity was significantly reduced when 1-OHP levels were lower than 0.06 µg/mM creatinine (Fig. 1C), showing a non-linear association between plasma SAHH activity and urinary 1-OHP concentrations (P _overall = 0.089, P _{non-linearity} = 0.030) (Fig. 1C). However, the results did not reveal that 2-NAP, 9-PHE, 2-FLU and ΣOH-PAHs metabolites have significant effects on the expression of H19 RNA in peripheral blood and plasma SAHH activity (P > 0.05, Table S4).

H19 interacts with SAHH at specific sites and then regulates DNMT1 in BEAS-2B cells exposed to BaP

Previous results have established that H19 interacts with and suppresses SAHH expressions and activity would be enhanced by BaP (4), we performed RIP experiments using four types SAHH-overexpressing (WT-SAHH, M1-SAHH, M2-SAHH and M3-SAHH) cells to illuminate the binding sites between SAHH and H19. Whether exposed BaP or not, we observed an accessorial enrichment of H19 in SAHH-containing complexes in WT-SAHH, M1-SAHH and M2-SAHH cells (Fig. 2A), whereas M3-SAHH cells did not detectably elevate (Fig. 2A, left column 5 & right column 5). After BaP exposure, H19 RNA expressions were inhibited in SAHH-overexpressing cells compare to WT cells (Fig. 2B, right column 3–6), particularly in M3-SAHH cells (Fig. 2B, right column 5). These results reveal that H19 may bind to the 1–300 amino acid chain instead of 300–432 amino acid chain of SAHH.

To better understand which DNMTs is most affected by H19 and SAHH, five types (WT, si-NC, si-H19, si-SAHH, si-H19 + si-SAHH) cells were used to the further experiments (Fig. 2C & 2D). The exaggerated expression (Fig. 2C, right column) and activity (Fig. 2D, right) of DNMT1 were observed in WT cells exposed to BaP, whereas a significant decrease in si-H19, si-SAHH and si-H19 + si-SAHH cells exposed to BaP was observed (Fig. 2C; Fig. 2D). Collectively, these results revealed that H19 binding to SAHH was more likely affected DNMT1 expressions and activity after BaP treatment.

H19 binding to SAHH interacts with DNMT1 in BEAS-2B cells exposed to BaP

As H19 interacts with SAHH and shows characteristics of a ribonucleoprotein particle (4), we reasoned that H19 binding to SAHH interacts with DNMT1 in BEAS-2B cells treated with BaP. In support of the assumption, we accessed Co-IP experiments to examine whether anti-DNMT1 antibody would be able to immunoprecipitate SAHH from complex. As shown in Fig. 3A, Co-IP analysis showed that a nearly 0.5-fold enrichment of SAHH in the DNMT1 antibody complex compared with WT cells. In addition, compared with the WT and si-NC cells, SAHH protein expression in si-H19 cells was significantly increased after BaP exposure (Fig. 3A, right column 3). However, the enrichment of SAHH in DNMT1-containing complex between WT and si-NC cells was similar (Fig. 3A, left column 2 & right column 2).

Additionally, co-localization between endogenous H19, SAHH and DNMT1 was evaluated by confocal microscopy, predominantly in nucleus and perinuclear cytoplasm (Fig. 3B, white arrow). We found that the fluorescence intensity of DNMT1 was increased after BaP exposure for 24 hours (Fig. 3B, column 4). The intracellular co-localization of H19 and SAHH was consistent with the previous results (Fig. 3B, column 5). The increased fluorescence of purple intensity, which displayed the intracellular co-localization of H19 and DNMT1, indicated a stronger steric interaction after BaP exposure for 24 hours, while fluorescence intensity was decreased in si-H19 cells (Fig. 3B, column 6). In addition, the increased fluorescence intensity of SAHH and DNMT1 indicated a stronger steric interaction after BaP exposure for 24 hours and transfected with si-H19 (Fig. 3B, column 7). Moreover, we also observed the increased fluorescence of blue violet, which displayed the intracellular co-localization of H19, SAHH and DNMT1, indicated a stronger steric interaction after BaP exposure for 24 hours (Fig. 3B, column 8). Together, these results suggest that H19 binding to SAHH interacts with DNMT1 in BEAS-2B cells treated with BaP.

H19 /SAHH/DNMT1 regulates OGG1 methylation

In order to test that H19 binding to SAHH might interfere with DNMT1 and then regulate gene methylation in BaP-treated cells, we used eight type cells (WT, si-H19, si-SAHH, si-DNMT1, si-H19 + si-SAHH, si-H19 + si-DNMT1, si-SAHH + si-DNMT1, and si-NC) for BaP treatment. Pearson's correlation tests between methylation values (% 5-mC) each CpG locus in the first exon of OGG1 and mean methylation value of four loci reveal high correlations (Pearson's r ranged from 0.63 to 0.86) (Fig. 4A). Results indicated that after BaP exposure, the levels of OGG1 methylation in BEAS-2B cells increased significantly (Fig. 4B), whereas that in si-H19 cells decreased (Fig. 4B, right column 3). Conversely, SAHH or DNMT1 single knockdown exacerbated OGG1 methylation (Fig. 4B, right column 4 & 5) and SAHH/DNMT double knockdown markedly aggravated the alteration (Fig. 4B, right column 8). Interestingly, although H19 single knockdown obviously reduced OGG1 methylation, H19/SAHH and H19/DNMT1 double knockdown abolished this effect (Fig. 4B, right column 6). However, similar changes in OGG1 methylation were observed in WT and si-NC cells (Fig. 4B, right column 2). Collectively, we concluded that H19/SAHH/DNMT1 regulates OGG1 methylation in BEAS-2B cells treated with BaP.

H19 /SAHH/DNMT1 affects oxidative DNA damage and cell cycle arrest

Given the previous study conducted by our team indicating that OGG1 methylation mediated oxidative DNA damage and cell cycle arrest associated with urinary 1-OHP concentrations in coke oven workers (13), we took an in vitro approach to examine whether H19/SAHH/DNMT1would regulate oxidative DNA damage and cell cycle arrest in accordance with the alteration of OGG1 methylation for BaP exposure. Figure 4C & 4D exhibited the effects H19/SAHH/DNMT1 on the oxidative DNA damage and cell cycle arrest. After transfection, there was no significant change in the levels of 8-OHdG compared with WT cells (Fig. 4C, left column). However, the 8-OHdG levels were significantly increased in cells treated with BaP (Fig. 4C, right column). After BaP exposure, H19 single knockdown slightly reduced 8-OHdG levels (Fig. 4C, right column 3), whereas SAHH or DNMT1 single knockdown significantly elevated 8-OHdG levels (Fig. 4C, right column 4 & 5). After BaP treatment, SAHH/DNMT1 double knockdown further dramatically increased 8-OHdG levels (Fig. 4C, right column 8), whereas H19/SAHH and H19/DNMT1 double knockdown abrogated this effect (Fig. 4C, right column 6 & 7). Furthermore, the results of S phase arrest were parallel to 8-OHdG levels (Fig. 4D). Combined with our previous findings (13) and in vitro data (Fig. 4B), these results strongly support that H19/SAHH/DNMT1would regulate oxidative DNA damage and cell cycle arrest in BEAS-2B cells treated with BaP in keeping with OGG1 methylation alterations.

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 (14, 15), and its upregulated expression profile has been observed in a variety of human malignancies (2). Numerous studies found PAHs are known to trigger lung cancer in animals and humans (16). 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 (17). Additionally, H19 binds to and inhibits SAHH function in vivo and vitro (3, 5). 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 (4).

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 (18). 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 (19). 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 (5, 20).Human SAHH forms a homotetramer consisting of 432 amino acid (aa) and is composed of four identical chemically identical and functionally equivalent subunits (21), 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) (22). 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 (5). 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 (6). 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(23), whereas Ponnaluri V.K. et al demonstrated that SAHH could enhance DNMT1 activity and interact with DNMT1 during S-phase in vitro (24). 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 (25) and abnormal cell cycle distribution, especially S phase arrest (26). BaP, a potent carcinogenic PAHs, can cause cell cycle block, including elevated S-phase cells ratio, weakened DNA replication capacity, and inhibited cell proliferation (27). 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 (28). 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 (13). 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 (29). 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 (30). Previous studies have observed associations between PAHs exposure with global or gene-specific DNA methylation alterations (31). Both PAHs exposure and its related damages (e.g., oxidative stress) have been associated with DNA methylation alterations (31). 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 (32). 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 (33). 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.

Urinary 1-OHP, the vital urinary metabolite for PAHs exposure, was positively associated with peripheral blood H19 RNA expression, while was negatively associated with plasma SAHH activity in occupational workers. BaP, the human carcinogen PAHs, contributed to the binding among H19, SAHH and DNMT1, which synergistically involved in regulating OGG1 methylation, oxidative DNA damage and cell cycle arrest in human lung epithelial cell lines. Hence, we concluded from the study that H19/SAHH/DNMT1 plays an essential role in the OGG1 methylation, oxidative DNA damage and cell cycle arrest.

Participants

A total of 146 occupational workers, who had worked for at least one year in a coke-oven plant in Taiyuan (Shanxi, China), were enrolled in the final analyses. And the workers who had worked on top-, side‐ and bottom‐oven in the coke oven plant were mainly young and middle-aged (Youth age defined by WHO-2017: 15–44 years old). Exclusion criteria included pulmonary diseases, syphilis, cancer, kidney diseases and other factors which may interfere with research. And the participants were not exposed to known occupational carcinogens or dust during the past three months. Our research was approved by the Ethical Review Board of the School of Public Health, Shanxi Medical University, Shanxi, People’s Republic of China. Peripheral blood (5 mL) and morning first urine (20 mL) samples from all study population were collected in the morning of the same day. Plasma, which would be used to test the levels of SAM and SAH, was separated within 4 h after collection and then stored at -80°C.

Determination of PAHs metabolites

The detection of the concentrations of four PAHs metabolites in urine by High performance liquid chromatography (HPLC, Shimadzu Corp, JPN), including 2-hydroxynaphthalene (2-NAP),1-hydroxypyrene (1-OHP), 9-hydroxyphenanthrene (9-PHE) and 2-hydroxyfluorene (2-FLU), has been described in detail in previous studies (34). The quality control data were detailed in Table S1.

Plasma SAHH activity

After pre-treated by 240 uL Trichloroacetic Acid (400 g/L) with an ice bath for 30 min, 1.2 mL plasma was centrifuged at 14000rpm at 4°C for 20 min, and then supernatant was separated and filtered. HPLC-ultraviolet detection was used to measure plasma SAM and SAH concentrations according to a method presented elsewhere (35). We quantified plasma SAM and SAH concentrations in accordance with the peak area with standard solutions of (0, 0.05078, 0.10156, 0.20312, 0.40625, 0.81250, 1.62500 and 3.25000 mg/L for SAM) and (0, 0.03125, 0.06250, 0.12500, 0.25000, 0.50000 and 1.00000 mg/L for SAH) to establish the retention time. The quality control data were detailed in Table S1.

Cell culture

BEAS-2B, a human lung epithelial cell lines, were cultured under 5% CO₂ at 37°C in α-MEM growth medium (GE), supplemented with 100 mg/mL streptomycin, 100 U/mL penicillin and 10% fetal bovine serum (Gibco, Paisley, UK).

Cell transfections

The inhibitions of H19, SAHH and DNMT1 were obtained by siRNA (RiboBio, Guangdong, China) to construct the low-expression cell model. The oligo sequences were shown as below: 5′-CCCACAACAUGAAAGAAAUdTdT-3′ and 5′-dTdTGGGUGUU-GUACUUUCUUUA-3′ for H19, 5′-CAAGCTAACTGAGAAGCAAdTdT-3′ and 5′-dTdTGUUCGAUUGACUCUUCGUU-3′ for SAHH, 5′-GAAGAGACGTAGAGTTACAdTdT-3′ and 5′-dTdTCUUCUCUGCAUCUCAAUGU-3′ for DNMT1. 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 inserted into pCMV-GV141 vector (Genechem, Shanghai, China). For the transfection of siRNA or pCMV-GV141 vector, cells were transfected by Lipofectamine-RNAi MAX or Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. After transfection, RNA or protein was extracted for analysis (Fig. S1A-1E).

Cells treatments

Eight type cells (WT, si-H19, si-SAHH, si-DNMT1, si-H19 + si-SAHH, si-H19 + si-DNMT1, si-SAHH + si-DNMT1, and si-NC) were treated with BaP (Sigma-Aldrich, USA) at a single dose of 16 µM for 24 h to reflect distinct mechanisms. The dose and time in this study were selected based on our preliminary experiments, which suggested that cell viability decrease less than 15% (36).

RNA extraction and Real-time quantitative reverse transcription PCR (RT-PCR)

TRIzon Reagent (CWbiotech, Beijing, China) was used to isolate total RNA from participants’ peripheral blood or cell lines and then cDNA was synthesized by the use of Prime Script RT Master Mix (Takara, Dalian, China) according to the method described in previous researches (4). Quantitative RT-PCR was also performed as described previously. Table S2 lists the RT-PCR primers. The primers for β-actin, H19, SAHH, GAPDH and DNMT1 were used as previously reported (4). The primer for OGG1 was obtained from QuantiTect Primer Assays.

Proteins extraction and Western Blot

The extractions of proteins were obtained by the same method as the previous research from cells (4). Equal amounts of protein were fractionated by different concentrations (7% and 10%) sodium dodecyl sulphate-polyacrylamide gel electrophoresis and subsequently transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked in 5% skim milk with gentle rotation and then incubated overnight at 4°C with the indicated primary antibodies (DNMT1, 1:2000, GeneTex, USA; SAHH, 1:1000, Proteintech, IL, USA; DNMT3A, 1:4000, Proteintech; DNMT3B, 1:500, GeneTex; GAPDH or β-tubulin, 1:4000, CWbiotech). After additional incubation with anti-immunoglobin horseradish peroxidase-linked Ab (1:4000, CWbiotech) for 2 h at 37°C, the levels of immune complexes were visualized and quantified by scanning laser densitometry (Universal Hood Ⅱ, Bio-Rad).

RNA immunoprecipitation (RIP) analysis

RNA immunoprecipitation (RIP) experiments were performed as described previously (4). Reverse transcription was in a 30 mL reaction, followed by RT-PCR (see Table S2 for primer sequences).

DNMT1 activity assay

The DNMT1 activity of cells was quantitated following the manufacturer's protocol (EpiQuik™ DNA Methyltransferase Activity/Inhibition Assay Kit, Epigentek, Germany), and was determined by monitoring the fluorescence product at 450 nm using SpectraMax M2.

Co-immunoprecipitation (Co-IP) analysis

The extractions of proteins were obtained from cells using the same method as the previous research from cells (4). For vitro binding assay, identical amounts of SAHH in extractions of cells and equal volume of Mouse anti-DNMT1 antibody (GeneTex) were added in 1.5 mL tubes and rotated overnight at 4°C. Then 40 µL protein A/G beads (CWbiotech) were added and incubated with rocking for 4 h at 4°C. Protein A/G beads and protein bound to the beads were washed three times with 1 mL of ice-cold PBS to reduce the non-specific binding to the beads. Finally, the proteins bound to beads were analysed by western blot, and probed for SAHH using Rabbit anti-SAHH antibody.

Immunofluorescence analysis

H19 FISH experiments were carried out using Fluorescent In Situ Hybridization Kit (RiboBio) according to the manufacturer’s instructions. The system used H19-Cy3 FISH Probe Mix (1:40, RiboBio) to treat cells overnight at 37°C, avoiding light. After hybridisation, cells were washed twice with hybrid cleaning solution Ⅰ/Ⅱ/Ⅲ (SSC with different concentrations) each for 5 min at 42°C, avoiding light. Following incubating with primary antibodies Rabbit anti-SAHH antibody (1:200, Proteintech) and Mouse anti-DNMT1 antibody (1:200, GeneTex) and fluorescent-labelled secondary antibodies (anti-mouse IgG coupled with Alexa Fluor 488 (1:200, Invitrogen), anti-rabbit IgG coupled with Alexa-Fluor-647 (1:400, Abcam, Cambridge, UK)). To stain the nuclei, cells were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, 1:1000, Solarbio, USA) at room temperature for 10 min, avoiding light. Images were captured using a confocal microscope (FluoView FV10i, Olympus, Japan).

DNA extraction and Gene methylation analysis

Genomic DNA extraction from cultured cells was performed using a Universal Genomic DNA Kit (CWbiotech) following the manufacturer’s instructions. Genomic DNA (500 ng) was subsequently treated with sodium bisulfite by EZ DNA Methylation-Gold Kit (Zymo Research, USA) and amplified by PCR following the manufacturer’s instructions. These PCR amplifications were isolated using the Pyrosequencing Work Station and sequenced by Pyromark Q96 MD pyrosequencing instrument (Qiagen, Germany) per the manufacturer’s instructions. The dispensation order is 5'-ATCGAGATGGCTGATCGATGTCGGTTGATGTATCGT-3', and the analytic sequence of OGG1is 5'-YGGAGAATTGGGGTAYGAAGYGGGGTTTTGATGATTYGTA-3'. We detected DNA methylation levels at four CpG loci of OGG1 in the first exon (Fig. 4A, left top).

Oxidative DNA damage assessment

Total genomic DNA was extracted from cells as described previously. DNA was hydrolysed with nuclease P1 in a water bath at 37°C for 20 min, and then regulated pH to 7.5–8.5 by 1 M Tris. Hydrolytic DNA was incubated with alkaline phosphatase (1 unit / 100 µg DNA) at 37°C for 30 min, and then boiled for 10 min. The content of 8-OHdG was measured following the manufacturer's protocol (8-OHdG ELISA Kit, Abcam), and then the absorbance of samples was detected at 450 nm using SpectraMax M2.

Cell cycle analysis

Cells were collected into 1.5 mL centrifuge tubes for flow cytometry by propidium iodide (PI) staining using DNA Content Quantitation Assay (Cell Cycle) (Solarbio) per the manufacturer’s instructions. Flow cytometry was carried out by the same method as the previous research (37).

Statistical analysis

Statistical representation of data was done by Statistical Package for Social Sciences version 22.0 (IBM SPSS, Chicago, IL, USA) software and SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA).

Continuous data in vitro experiments were summarized as mean ± SD of at least three replicates and compared among means using ANOVA. Post hoc Dunnett analyses were used to analyse the differences between groups when the P value of the ANOVA was < 0.05. Covariance analysis was used to clarify whether there were differences among different types cells independent of exposure. P values < 0.05 were deemed to be statistically significant.

We substituted the concentrations below the LOD with 50% LOD, including four urinary PAHs metabolites, SAM and SAH. Continuous data in the cross-sectional study were expressed as medians (Med) and interquartile ranges (IQR), and compared with Kruskal-Wallis H test. Categorical data in the cross-sectional study were expressed as N (%) and compared with Chi-square test. To investigate the correlations and estimate the potential dose-repose relationship, we used logistic regression models adjusted confounders and restricted cubic spline model, respectively. By assigning the median value in quartiles as a continuous variable, the linear trend across 1-OHP concentration increasing with the quartile can be estimated. The cut points for peripheral blood H19 RNA expressions and plasma SAHH activity were defined as 14.42 and 0.83 in the present study, which were equivalent to the median.

Ethics approval and consent to participate

Our research was approved by the Ethical Review Board of the School of Public Health, Shanxi Medical University, Shanxi, People’s Republic of China.

Consent for publication

Not applicable.

Availability of data and materials

The data sets supporting the results of this article are included within the article and its additional files

Competing interests

The authors have no conflicts of interests to declare.

Funding

We thank all researchers and study population from Steel Group General Hospital of Taiyuan. This project was supported by the grants from National Nature Science Foundation of China [No. 81273041 and 30901180], Research Project Supported by Shanxi Scholarship Council of China [No. HGKY2019053] and Natural Science Foundation of Shanxi Province of China [No. 201701D121146].

Authors' contributions

Ye Fu: Designed research, Performed research, Contributed new reagents or analytic tools, Analysed data, Wrote the paper. Xuejing Li: Designed research, Performed research, Analysed data, Wrote the paper. Baolong Pan: Performed research, Contributed new reagents or analytic tools, Wrote the paper. Yingying Niu: Performed research, Analysed data, Wrote the paper. Bin Zhang: Analysed data, Wrote the paper. Xinyu Zhao: Analysed data, Wrote the paper. Jisheng Nie: Project administration, Wrote the paper. Jin Yang: Funding acquisition, Project administration, Designed research, Performed research, Analysed data, Wrote the paper.

Acknowledgements

Not applicable.

1-OHP, 1-hydroxypyrene; 2-FLU, 2-hydroxyfluorene; 2-NAP, 2-hydroxynaphthalene; 9-PHE, 9-hydroxyphenanthrene; BaP, Benzo[a]pyrene; BER, Base excision repair; DNMT, DNA methyltransferase; lncRNAs, long noncoding RNAs; OGG1, Eight-oxoguanine DNA glycosylase 1; PAHs, Polycyclic aromatic hydrocarbons; ROS, reactive oxygen species; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocysteine hydrolase; SAM, S-adenosylmethionine.

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Effects of H19/SAHH/DNMT1 on the oxidative DNA damage related to Benzo[a]pyrene exposure

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Abstract

Figures

Background

Results

Discussion

Conclusions

Methods

Participants

Determination of PAHs metabolites

Plasma SAHH activity

Cell culture

Cell transfections

Cells treatments

RNA extraction and Real-time quantitative reverse transcription PCR (RT-PCR)

Proteins extraction and Western Blot

RNA immunoprecipitation (RIP) analysis

DNMT1 activity assay

Co-immunoprecipitation (Co-IP) analysis

Immunofluorescence analysis

DNA extraction and Gene methylation analysis

Oxidative DNA damage assessment

Cell cycle analysis

Statistical analysis

Declarations

Abbreviations

References

Supplementary Files

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