The Effects Of DNMT3A On The Pathogenesis Of Adenomyosis In Vivo

Objective: Our previous study has found that the mRNA and protein levels of DNMT3 A were strikingly lower in the ectopic endometriums in adenomyosis(AM) when compared with nomal endometriums, and the similar results were also observed in their corresponding ESCs isolated from uterine endometriums of AM and control individuals, and restoration of DNMT3 A expression in ESCs inhibited the cell proliferation and promote cell apoptosis. However, whether these similar effects would also be observed in vivo remain unknown. Herein, we further explore the potential roles of dysregulated DNMT3 A expression in the pathogenesis of AM in vivo.Methods: The ectopic ESCs(n=10) and control ESCs(n=7) were isolated, purified and cultured. The expression levels of DNMT3 A protein in these cells were analyzed by Western blot. We set up normal ESCs(normal group) and ectopic ESCs(ectopic group); and we selected an ESC cell with extremely low expression of DNMT3 A in the ectopic group to construct the transgenic DNMT3A-overexpressing and control ESC cells with our previously constructed DNMT3A-overexpression lentivirus(GV205Flag-DNMT3A) and control lentivirus(GV205Flag), respectively. 17-beta estrogen sustained release tablets were embeded in the subcutaneous of nude mice to establish high estrogen physiological cycle, then normal ESCs, ectopic ESCs of AM, GV205Flag-DNMT3 A and GV205 Flag transgenic ESCs were inoculated into 4 groups of nude mice subcutaneously, respetively, and each group contrained 12 mice; then compared the proliferation of seeded cells dynamically.Results:(1) The protein levels of DNMT3 A were strikingly lower in ectopic ESCs compared with eutopic ESCs from control sample.(2) The DNMT3A-Flag fusion protein was detected in the transgenic GV205Flag-DNMT3 A ESCs, suggesting the lentivurses were infected successfully.(3) Immunohistochemical assays of the cell implantation site tissues in the 4 groups was positive for vimentin protein, which was characteristic protein marker expressed only in stromal cells.(4) The average volume of GV205Flag-DNMT3 A ESC group was 15.375±9.546mm3 smaller than that of GV205 Flag ESCs(16.75 ±9.907mm3), even though there is no significant difference(P>0.05). Additionally, the average volume of eutopic ESCs from control individuals was 18.417±8.218mm3, smaller significantly than the ectopic ESCs group from AM patients whose average volume was 32±16.548mm3(P<0.05). Furthermore, the average volume of ectopic group was significantly higher than that in both the negative group and the positive group(P<0.05).Conclusions:(1) Overexpression of DNMT3 A could inhibit the proliferation of ESCs, reduce growth of the tumor-like tissue, providing the bases to continue studying the roles of DNMT3 A in ESCs of AM in the whole animal level.(2) In this experiment, we injected human ESCs of AM in the subcutaneous tissues of nude mice under artificially regulated menstrual cycle, providing a meaning reference for in vivo study in some other similar diseases.