Abstract:
Objective To investigate the effects of benzoapyrene (BaP) on the expression of long non-coding RNA (lncRNA) H19 and S-adenosylhomocysteine hydrolase (SAHH) in human bronchial epithelial cells (16HBE), and to assess the relationship between H19 and SAHH.
Methods A batch of 16HBE cells were exposed to BaP at 1, 2, 4, 8, 16, and 32 μmol/L for 24 h, and a control group (culture medium only) and a solvent control group were established. Another batch of 16HBE cells were exposed to 16μmol/L BaP for 1, 2, 4, 8, 12, and 24 h, and a control group (0 h) was also established. The relative expression of H19 was detected by reverse transcriptionPCR (RT-PCR). The protein expression of SAHH was detected by Western blot. The distribution of H19 and SAHH were detected by lncRNA fluorescence in situ hybridization (FISH) combined with laser scanning confocal microscopy.
Results The expression levels of H19 were not different between the solvent control group and the untreated group. The relative expression levels of H19 in the 16HBE cells were gradually elevated with higher BaP concentrations and extended exposure time, according to the results of RT-PCR. The expression levels of H19 were significantly higher in the 16HBE cells exposed to BaP at concentrations of 8, 16, and 32 μmol/L for 24 h than those of the solvent control group (Ps < 0.05), and higher in the 16HBE cells exposed to 16 μmol/L BaP for 4, 8, 12, and 24 h than those of the 0 h group (Ps < 0.05). The relative expression levels of H19 reached a peak at 32 μmol/L BaP, which increased 78.0% compared with the solvent control group. For 16 μmol/L BaP exposure, the relative expression levels of H19 reached a peak at 24 h, which increased 48.3% compared with the 0 h group. The protein expression levels of SAHH in 16HBE were not different between the solvent control group and the untreated group, and significantly decreased with higher BaP concentrations and extended exposure time (P < 0.05), according to the results of Western blot. The protein expression levels of SAHH reached a bottom at 32 μmol/L BaP, which was 37.2% lower than that of the solvent control group. For 16 μmol/L BaP exposure, the protein expression levels of SAHH reached a bottom at 24 h, which was 33.1% lower than that of the 0 h group. The immunofluorescence results showed that both H19 and SAHH were distributed in cytoplasm and nucleus, predominantly in perinuclear cytoplasm and nuclear membrane. In the 16HBE cells exposed to 16 μmol/L BaP for 24 h, more H19 were detected in perinuclear cytoplasm, while less SAHH were detected as compared to the 0 h group, and the immunofluorescence intensity of H19 and SAHH co-localized in cytoplasm and nucleus was increased.
Conclusion Exposure to BaP could increase the relative expression levels of H19 in 16HBE cells, while decrease the protein expression levels of SAHH. Co-localization of H19 and SAHH in 16HBE cells suggests an interaction between H19 and SAHH.
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