The Impact Of Knockdown Dnmt1 And Dnmt3b On MEFs Apoptosis, Cycle, Global Methylation Level

The incomplete reprogramming of donor cells is considered to be the main reason for low efficiency of somatic cell nuclear transfer. Correct expression of methyltransferase 1(Dnmt1) and methyltransferase 3b(Dnmt3b) in early embryos have important influence on future development. Studies found that the two enzymes express less in preimplantation embryos than in somatic cell nuclear transfered embryos. So the hypothesis is that knockdown both Dnmt1 and Dnmt3 b to induce demethylation in donar cells may improve epigenetic programming in SCNT(somatic cell nuclear transfer). In cancer cells, interfere both DNMT1 and DNMT3 b can dramatically reduce the global methylation level, while what would happen on the somatic cells when double knockdown the two methyltransferases has not been reported. Here we used si RNA to knockdown DNMT1 and DNMT3 b on mouse embryonic fibroblasts(MEFs) to investigate changes on MEFs cell apoptosis, cell cycle and global methylation level. The experimental groups were randomly divided into 6 groups: si-Dnmt1 alone(80n M), si-Dnmt3 b alone(80n M), a combination of si-Dnmt1 and si-Dnmt3b(80+80n M), NC-Dnmt1/Dnmt3 b group(80n M), NC-Dnmt1+Dnmt3b(80+80n M), and a blank control group. Using Real-time quantitative PCR to detect Dnmt1, Dnmt3 b and PCNA(proliferating cell nuclear antigen) m RNA expression; western blot detecting DNMT1 and DNMT3 b protein changes; apoptosis kits to test cell apoptosis; cell cycle kit to detect the cell cycle change; a global DNA methylation quantification kit was used to characterise the global DNA methylation level. The main results were as follows:1. Total RNA was extracted for q-PCR to quantify the m RNA expression of DNA methyltransferases(DNMTs) 24 h post-transfection. Compared with negative control(NC), the Dnmt1 m RNA of si Dnmt1 alone group decreased 69.6%, Dnmt3 b m RNA of si Dnmt3 b alone reduced 63.1%; Dnmt1 m RNA decreased 60.1%, Dnmt3 b m RNA reduced 63.4% on the double knockdown group. Interfering Dnmt1 significantly reduces the DNMT1 protein about 48% on separate interference group(P<0.01). The DNMT3 b protein reduced 20% on double interference group(P<0.01).2. The PCNA m RNA level reduced significantely on Dnmt3 b knockdown group(P<0.05). Double knockdown Dnmt1 and Dnmt3 b significantely decreased m RNA level of PCNA(P<0.01).3. Flow cytometry detecting cell apoptosis 48 h post-tranfection found that compared with NC group, apoptosis increased significantly(P<0.05) on si Dnmt3 b alone group, middle-late apoptosis and total apoptosis significantly increased(P<0.01) on Dnmt1 and Dnmt3 b double knockdown group. Detecting protein p53 found it increased significantly(P<0.05) on double interference group.4. Cell cycle of the double interference group with increased cells at G1 and G2 phase, S phase cells reduced(P=0.087) 48 h post-transfection without significant difference.5. Compared with negative control group, the methylation level of si Dnmt1 alone were not decreased siginificantely, while knockdown Dnmt3 b induces demethylation of MEFs significantely(P<0.01). The global methylation of double knockdown group reduced 44%(P<0.01).