Effect of 5′-Aza-2′ -deoxycytidine on methylation, mRNA and protein expression of CDX2 gene in colorectal cancer HT-29 and LoVo cells

Authors：Xu C W; Ge C; Wang L P; Fang Y; Zhang Y P^*

Source：World Chinese Journal of Digestology, 2014, 22(10): 1423-1430.

DOI：10.11569/wcjd.v22.i10.1423

• 5&prime
• -Aza-2&prime
• -deoxycytidine
• CDX2 gene
• DNA methylation
• HT-29
• LoVo

Aim: To investigate the effect of 5′-Aza-2′-deoxycytidine (5′-Aza-CdR), a methylation inhibitor, on the methylation, mRNA and protein expression of the CDX2 gene in colorectal ancer cell lines HT-29 and LoVo. Methods: HT-29 and LoVo cells were treated with different dosages of 5′-Aza-CdR. After treatment, CDX2 gene methylation was determined by Methylight assay, and CDX2 mRNA and protein expression was detected by realtime PCR and Western blot, respectively. Results: Methylight assay showed that CDX2 gene methylation was not reversed by 5′-Aza- CdR. The expression levels of CDX2 mRNA were increased in both HT-29 (1.000 ± 0.000, 0.973 ± 0.024, 1.014 ± 0.019 and 1.094 ± 0.020, respectively) and LoVo cells (1.000 ± 0.000, 0.966 ± 0.038, 1.050 ± 0.029 and 1.007 ± 0.019, respectively) cells treated with 5′-Aza-CdR for different durations. Western blot analysis indicated that 5′-Aza-CdR treatment could recover the CDX2 protein expression in both HT-29 (0.454 ± 0.049, 0.501 ± 0.041, 0.340 ± 0.050 and 0.531 ± 0.046, respectively) and LoVo (0.527 ± 0.037, 0.415 ± 0.037, 0.432 ± 0.040 and 0.626 ± 0.046, respectively) cells. The effect of 5′-Aza-CdR on CDX2 mRNA and protein expression was not doseor time-dependent, but the expression levels of CDX2 mRNA and protein differed significantly in HT-29 (mRNA: F = 25.146, P = 0.000; protein: F = 9.700, P = 0.005) and LoVo cells (mRNA: F = 5.470, P = 0.024; protein: F = 17.701, P = 0.001). Conclusion: CDX2 mRNA and protein expression is not affected by DNA methylation in HT-29 and LoVo cells.