Relationships Between Icariin and Anti-Apoptotic miRNA-21 in Mouse Blastocyst Development In vitro

Abstract

In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium (mCZB) as control group. The experimental group (Ica group) was supplemented with 0.6 μg mL−1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21: pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group ((85.14±6.57)% vs. (72.04±11.58)%; (82.50±7.11)% vs. (66.80±11.70)%, respectively, P<0.01). The total cell number of blastocysts had extreme difference between Ica group and control group ((61.40±9.64) vs. (46.23±4.50), P<0.01). The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group ((1.47±0.51) vs. (2.94±0.66); (2.40±0.27)% vs. (6.25±0.62)%, respectively, P<0.01). Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 (P<0.01), down-regulated pro-apoptotic Caspase3 (P<0.05) and PTEN (P<0.01), up-regulated anti-apoptotic Bcl-2 (P<0.01). In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-apoptotic genes and down-regulated pro-apoptotic genes. These apoptosis-related genes were regulated by miRNA-21.