Study On The Methylation Status Of TFPI-2 Gene In Acute Myeloid Leukemia And The Inhibitory Mechanism Of Decitabine In K562 Cell

Aim:Detect the methylation status of tissue factor pathway inhibitor-2(TFPI-2)gene in acute myeloid leukemia(AML) and study the effect of demethylation of Decitabine and its inhibitory effect to K562 cell to provide the experimental basis for the clinical application of Decitabine in treating acute myeloid leukemia and seeking for new molecular target gene for leukemia.Methods:1.We used the MS-PCR methods to detect the methylation status of TFPI-2 gene in 33 new-diagnosed AML patients and 16 IDA patients.2.Using the methods of CCK-8 and trypan blue to detect the K562 cell proliferation after treated with Decitabine for different concentrations and time.3.We examined the change of methylation status、expression of m RNA and protein of TFPI-2 gene in K562 cell with MS-PCR 、 RT-PCR and Westen-Blot methods.Results:1.The percentage of TFPI-2 promoter methylation in new-diagnosed AML patients was 63.63%(21/33),respectively, none of the control group patientsshowed methylation,and there was statistical significance between them(P<0.05).2.Through global analysis, the CCK-8 results showed that the comparison between different groups of Decitabine and time and their interaction had statistical significance(P<0.05). The further compartive analysis showed that at the same time, the survival rate of K562 cells declined as the concentration of Decitabine increasing,which had statistically significant difference between the concentration of various groups(P <0.05),and at the same concentration of Decitabine, the survival rate of K562 cells also declined as the time prolonged and there had a statistically significant difference between the time groups(P<0.05)4.Through global analysis,the Typan blue counting results showed that the comparison between different groups of Decitabine and time and their interaction had statistical significance(P < 0.05). The further compartive analysis showed that the counting of K562 cells declined as the concentration of Decitabine increasing. However, after treating 24 hours,different groups of various concentration of Decitabine compared with no treatment group, there was no statistical significance(P>0.05), and after treating 48 hours and 72 hours, the counting of K562 cells declined as the concentration of Decitabine increasing, which compared to the no treatment group had statistical significance(P <0.05). At the same concentration of Decitabine, the counting showed the upward trend;the counting of 48 hours and 72 hours groups are both more than the 0 hour group and had statistical significance(P<0.05),while there had no statistical significance between the 24 hours group and 0 hour group(P>0.05).5. TFPI-2 gene was hypermethylation in K562 cell. Decitabine can adverse the status and be dependent with concentration.After treated with Decitabine,the expression of m RNA and protein level of TFPI-2 gene increased and were dependent with concentration, group of different concentration had statistically significance(P<0.05).Conclusion:1. Aberrant methylation of TFPI-2 gene is a frequent epigenetic event of AML.2. The aberrant methylation of TFPI-2 gene is a major cause of its dow--ngrade in K562 cell.3. Decitabine can adverse the methylation status of TFPI-2 gene in K562 cell and increase the expression of m RNA and protein of the gene and may have some effect on inhibiting the proliferation of K562 cell.