ABSTRACT
BACKGROUND: Lung cancer is the most revenant and deadly tumors around the world. Here we aimed to explore the effects of hepatocyte nuclear factor 1B (HNF1B) and PCDHA13 overexpression on PI3K/AKT phosphorylation and malignant biological behavior in lung cancer A549 cells. METHODS: HNF1B and PCDHA13 were amplified, and their overexpression plasmids were constructed for transfection. RT-PCR was used to detect the mRNA levels of HNF1B and PCDHA13. Cell proliferation and cell apoptosis were detected by clone formation experiments and flow cytometry, respectively, while cell invasion was studied by Transwell assay. The expression of survivin, PCNA, Caspase-3, Caspase-9, VEGF, and fibronectin was detected using immunoblotting, as was PI3K/AKT phosphorylation. RESULTS: The level of HNF1B mRNA expression was significantly higher in the pcNDA-HNF1B group than in the control group (P<0.05), and the level of PCDHA13 mRNA expression in the pcNDA-PCDHA13 group was also significantly increased (P<0.05). The clone formation rate and cell invasion count in pcNDA-HNF1B or pcNDA-PCDHA13 transfected groups were significantly reduced in comparison with the control group, which were further validated with the protein expression levels of survivin, PCNA, VEGF, and fibronectin (P<0.05). However, the apoptosis rate, and the cleaved caspase3/caspase3 and cleaved caspase9/caspase9 protein expression ratios were all significantly increased (P<0.05). Cells transfected with pcNDA-HNF1B or pcNDA-PCDHA13 showed decreased levels of PI3K/AKT phosphorylation (P<0.05). CONCLUSIONS: Overexpression of HNF1B and PCDHA13 inhibits the phosphorylation of PI3K/AKT and hinders the malignant biological behavior of lung cancer A549 cells.
ABSTRACT
Objective The mechanisms of PCDHA13 promoter methylation in breast cancer have not yet been elucidated at present. This study was to investigate the role of PCDHA13 gene promoter methylation in the development of breast cancer.Methods The methylation state of PCDHA13 gene promoter in human breast cancer tissues was detected by MassARRAY mass spectrum methylation sequencing. 100μmol/L 5-Aza was prepared with culture medium. The ZR-75-1 cells with 60% cell confluence were added to the final concentration of 5 μmol/L(low concentration group) and 10 μmol/L(high concentration group) 5-Aza, and the control group was only added culture medium. Detection of methylation status of PCDHA13 gene promoter in human breast cancer cells by bisulfite sequencing and methylation-specific PCR, and analysis of methylation status and mRNA expression of PCDHA13 gene by semi-quantitative RT-PCR. Western blot, MTT and DAPI staining were used to detect the effect of 5-Aza treatment on proliferation and apoptosis of breast cancer ZR-75-1 cells.Results The methylation degree of PCDHA13 gene promoter in the 1, 4-6, 9, 10 and 11 CpG loci in breast cancer tissues was significantly higher than that in normal breast group \[(0.2639±0.1575) vs (0.1612±0.1706), (0.2509±0.1377) vs (0.1688±0.0992), (0.4204±0.2087) vs (0.2621±0.1731), (0.3761±0.1407) vs (0.2824±0.1486), (0.3922±0.1294) vs (0.3072±0.1496)\], and the difference was statistically significant (P0.05). The expression of PCDHA13 mRNA of ZR-75-1 cells was loss in control group, but the expression of PCDHA13 mRNA was reversed after treated with 5-Aza, and the expression of PCDHA13 mRNA was significantly higher in high concentration group than that in low concentration group(P>0.05). After treated with 5-Aza for 24, 48 and 72 hours, the growth inhibition rates were lower in low concentration group than that in high concentration group (P>0.05). The morphology of the nuclei was basically normal and there was no apoptosis occurred in ZR-75-1 cells. But after treated with 5-Aza, some ZR-75-1 cells showed nuclear condensation, chromatin agglutination and heavy coloration.Conclusion This study showed that the low expression or loss of mRNA is associated with hypermethylation of the PCDHA13 gene promoter in breast carcinoma. The PCHDA13 gene expression can be reversed by 5-Aza in ZR-75-1 cells. The re-expression of PCHAD13 not only inhibit the proliferation of cells, but also promote apoptosis. Abnormal methylation of PCDHA13 may become a potential tumor marker for breast cancer.
