Gou Jinhai, Hu Tingwenyi, Li Lin, Xue Luqi, Zhao Xia, Yi Tao, Li Zhengyu
Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China; and Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, Sichuan Province, PR China.
Department of Gynecology and Obstetrics, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China; and Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, Sichuan Province, PR China; and Sichuan Key Laboratory of Gynecologic Oncology, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China.
Reprod Fertil Dev. 2019 Apr;31(5):932-940. doi: 10.1071/RD18314.
In a previous study we found the expression of epithelial-mesenchymal transition (EMT) biomarkers, including E-cadherin and N-cadherin, was significantly altered in uterine endometrium during embryo implantation via regulation by microRNA (miRNA)-429 and protocadherin-8 (Pcdh8). As a natural continuation of the previous study, the aim of the present study was to explore the role of EMT during embryo implantation and the potential activity of twist basic helix-loop-helix transcription factor 2 (Twist2) in regulating embryo implantation. A pregnancy model was established by naturally mating adult female ICR mice with fertile males. A pseudopregnancy model was established by mating fertile female ICR mice with vasectomised males. An invitro model of embryo implantation was established by the coculture of Ishikawa and JAR spheroids. Endometrial tissue during the peri-implantation period was collected, as were Ishikawa cells, JAR cells and cocultured cells. The expression of EMT markers (E-cadherin, N-cadherin, vimentin and cytokeratin) and Twist2 was detected invivo and invitro using the western blot analysis during embryo implantation. The expression of N-cadherin and vimentin (mesenchymal markers) was upregulated in the invitro implantation model, with downregulation of E-cadherin and cytokeratin (epithelial markers) expression. The expression of N-cadherin, vimentin and Twist2 increased significantly at the implantation sites at the time of implantation (Day 5), whereas the expression of E-cadherin and cytokeratin decreased. Location of Twist2 during embryo implantation was detected by immunohistochemistry (IHC), which revealed that it was extensively expressed in endometrial glandular epithelium and luminal epithelium at implantation sites on Day 5. The effect of the expression of Twist2 on embryo implantation was evaluated by suppressing Twist2 using Twist2-short interference (si) RNA in invivo and invitro models. The numbers of implanted embryos and the implantation rate were compared invivo and invitro. Western blot analysis showed that suppression of Twist2 led to upregulation of E-cadherin and cytokeratin, accompanied by downregulation of N-cadherin and vimentin (P<0.05). The number of implanted embryos after Twist2-siRNA interference was lower than in normal pregnancy (mean (±s.d.) 2.4±0.5 vs 6.8±1.3 respectively; P<0.05). These findings suggest the involvement of EMT in embryo implantation. The suppression of Twist2 could suppress embryo implantation by regulating EMT.
在之前的一项研究中，我们发现上皮-间质转化（EMT）生物标志物（包括E-钙黏蛋白和N-钙黏蛋白）的表达在胚胎植入期间的子宫内膜中通过微小RNA（miRNA）-429和原钙黏蛋白-8（Pcdh8）的调控发生了显著改变。作为之前研究的自然延续，本研究的目的是探讨EMT在胚胎植入过程中的作用以及 twist 碱性螺旋-环-螺旋转录因子2（Twist2）在调节胚胎植入方面的潜在活性。通过将成年雌性ICR小鼠与可育雄性自然交配建立妊娠模型。通过将可育雌性ICR小鼠与输精管切除的雄性交配建立假孕模型。通过 Ishikawa 细胞和 JAR 球体共培养建立胚胎植入的体外模型。收集植入期周围的子宫内膜组织、Ishikawa 细胞、JAR 细胞和共培养细胞。在胚胎植入过程中，使用蛋白质免疫印迹分析在体内和体外检测EMT标志物（E-钙黏蛋白、N-钙黏蛋白、波形蛋白和细胞角蛋白）和 Twist2 的表达。在体外植入模型中，N-钙黏蛋白和波形蛋白（间充质标志物）的表达上调，而 E-钙黏蛋白和细胞角蛋白（上皮标志物）的表达下调。在植入时（第5天），植入部位的 N-钙黏蛋白、波形蛋白和 Twist2 的表达显著增加，而 E-钙黏蛋白和细胞角蛋白的表达下降。通过免疫组织化学（IHC）检测 Twist2 在胚胎植入过程中的定位，结果显示其在第5天植入部位的子宫内膜腺上皮和腔上皮中广泛表达。通过在体内和体外模型中使用 Twist2 短干扰（si）RNA 抑制 Twist2 来评估 Twist2 表达对胚胎植入的影响。比较体内和体外植入胚胎的数量和植入率。蛋白质免疫印迹分析表明，抑制 Twist2 导致 E-钙黏蛋白和细胞角蛋白上调，同时 N-钙黏蛋白和波形蛋白下调（P<0.05）。Twist2-siRNA 干扰后植入胚胎的数量低于正常妊娠（分别为平均（±标准差）2.4±0.5 与 6.8±1.3；P<0.05）。这些发现表明EMT参与了胚胎植入。抑制 Twist2 可通过调节EMT来抑制胚胎植入。
