5-Aza-2-Deoxycytidine To Improve The Sensitivity Of Endometrial Carcinoma KLE Cells To Medroxyprogesterone Acetate

Objective:To study the sensitivity of KLE cells to the gestational hormone medroxyprogesterone acetate by DNMT inhibitor5-Aza-2-deoxycytidine,and to preliminarily explore the action mode and possible mechanism of 5-Aza-2-deoxycytidine in cells,so as to provide a new treatment idea and theoretical basis for patients with endometrial cancer who are not ideal in clinical treatment with medroxyprogesterone acetate.Methods:First,the tissue DNA of normal people and endometrial cancer patients were extracted as templates,and methylated with ammonium sulfite to design an experimental protocol for methylation-specific PCR from the amount of primers,the amount of templates,and annealing.The optimal conditions for DNA methylation detection were determined in terms of temperatures and numbers of cycles.Then under the optimal conditions,the DNA methylation of normal and endometrial cancer tissues was detected.Endometrial cancer cell lines KLE and Ishikawa were cultured in vitro.After treatment of the cell line with DNNT inhabitor drug,the DNA methylation status and PRB protein expression of the cells were measured before and after treatment,and the optimal drug concentration and drug treatment time were determined.Combined application of 5-Aza-2-deoxycytidine and medroxyprogesterone acetate to treat cell lines,and then observe the inhibition rate of endometrial cancer cells.Detection of mRNA expression of DNA methyltransferase in cells before and after administration by quantitative real-time PCR.Results:1.Using methylation-specific PCR technology to successfully detect the DNA methylation of endometrial cancer patients and healthy population tissues.Under the optimal reaction conditions,the experimental results are obvious.When the methylation-specific primer completely matches the DNA template,a corresponding band appears on the gel electrophoresis,which proves that the tissue is methylated.On the contrary,when the non-methylated primer completely matches the DNA template,a corresponding one also appears,the band proves that the tissue is not methylated;when both the M band and the U band appear,it proves that the tissue has been partially methylated,and part of the methylation is classified as methylation.2.Methylation was detected by methylation-specific PCR in 43cases of intrauterine carcinoma,including 13 cases of complete methylation,23 cases of partial methylation,and 7 cases of non-methylation.Meanwhile,the methylation of 71 normal tissues was detected,among which 0 cases were completely methylated,10 cases were partially methylated,and 61 cases were non-methylated.DNA methylation status in cancer tissues was statistically different from normal tissues?p<0.0001?,while non-methylation status in normal tissues was statistically significant compared with endometrial cancer tissues?p<0.0001?.3.Culture the endometrial cancer cell lines KLE and Ishikawa in vitro,and detect the DNA methylation of the PRB gene of KLE cell line and Ishikawa cell line before and after treatment with5-Aza-2-deoxycytidine.The PRB of KLE cells was demethylated,but in the Ishikawa cell line with moderate progesterone receptor B expression,DNA methylation remained almost unchanged.4.Western Blot detection results of the treatment of KLE cells progesterone receptor B protein expression showed that after different concentrations of KLE cell lines were treated,both in the 24 h,48 h,72 h group,2.5 mM concentration group were the best concentration.We then compared the time at this concentration between groups,and found that the group with a concentration of 2.5 mM for 48 h was the best time and the best concentration,which was statistically different from the other two groups?p<0.01?.5.The MTT detected the cytostatic rate of KLE cells after5-Aza-2-deoxycytidine treatment.The results showed that compared with24 h and 72 h,the 48 h cytostatic rate after 5-Aza-2-deoxycytidine treatment was the highest.The rate of cell inhibition after medroxyprogesterone acetate treatment of KLE showed that 72h and 10^-5mol/L was the best group.Compared with the other groups,the cell inhibition rate was the largest.The 5-Aza-2-deoxycytidine with medroxyprogesterone acetate combined treatment of KLE cell inhibition rate showed that the combination treatment group had the highest cell inhibition rate compared with the 5-Aza-2-deoxycytidine control group and medroxyprogesterone acetate control group with statistical differences?p<0.001?.6.Fluorescent quantitative PCR was used to detect the mRNA content of DNA methyltransferase in KLE cells after5-Aza-2-deoxycytidine treatment.Detection of the content of DNA methyltransferase in the cells of the KLE control group and the optimal drug concentration treatment group without drug treatment,compared with the KLE control group,the mRNA expreesion of DNMT 1 was reduced by about 2 times?p<0.01?;compared with the KLE control group,the mRNA expression of DNMT 3a decreased by about 3 times?p<0.001?,while the expression of DNMT 3b compared with the KLE control group had no significant change in mRNA expression,which was not statistically significant.Conclusion:1.The DNA methylation may related to the occurrence of endometrial cancer which was determined by detecting the DNA methylation of endometrial cancer patients and healthy women tissues.2.DNMT inhibitor 5-Aza-2-deoxycytidine can effectively improve the sensitivity of endometrial cancer KLE cells to gestational hormone drugs.The mechanism may be by inhibiting the activity of DNMT 1 and DNMT 3a DNA methyltransferases,or by regulating the ratio of DNMT1/DNMT 3b or DNMT 3a/DNMT 3b.