GPR30基因羟甲基化调控在胰岛素诱导子宫内膜异常增生中的作用及机制

Abnormal endometrial proliferation is the pathological base of endometrial hyperplasia as well as adenocarcinoma. Our previous study demonstrated that hyperinsulinemia is one of the main reasons of abnormal endometrial proliferation. IR/IGF-1R is not the only mechanism through which insulin stimulates abnormal proliferation of endometrium. GPR30 is a cell membrane protein which is closely related to abnormal proliferation of endometrium. We found that insulin could stimulate the transcription of GPR30.Insulin could upregulate Tet1 expression which regulates DNA hydroxymethylation. We thus hypothesize that insulin upregualtes GPR30 expression through hydroxymethylation of GPR30 gene promoter by Tet1, thus activates PI3K pathway and stimulates abnormal proliferation of emdometrium. Our study will include 3 parts. ⑴ Analyze the correlation within GPR30 DNA hydroxymethylation/ methylation, GPR30 protein expression, hyperinsulinemia and endometrial abnormal proliferation by immunohistochemical study of clinical cases. ⑵ Investigate the effect of insulin induced GPR30 expression on endometrial cell proliferation using in vitro cell model. ⑶ Explore the mechanism how insulin up-regulates GPR30 expression through hydroxymethylation modification of GPR30 DNA by Tet1. Dot-blotting and BSP are used to investigate the hydroxymethylation of GPR30 DNA by insulin. WB is used to analyze the regulation of Tet1 protein by insulin. Plasmid transfection is used to regulate Tet1 expression. The GPR30 promotor Firefly Luciferase Reporter plasmid and ChIP are used after over-expressing or silencing Tet1 to confirm the regulation of GPR30 DNA by Tet1. GPR30 protein will be over expressed or silenced to study whether cell proliferation is stimulated through Gankyrin -PI3K/AKT pathway after up-regulation of GPR30 by insulin. The mechanism is confirmed in vivo using Psammonys Obesus mice which is the hyperinsulinemia animal model. Our study helps to prevent and treat abnormal endometrial proliferation.

子宫内膜异常增生是子宫内膜增生症和内膜癌的病理基础。我们前期研究发现高胰岛素血症与子宫内膜异常增生密切相关，除胰岛素/胰岛素样生长因子受体外，胰岛素可能还通过其他机制刺激内膜增生，尚待研究。胰岛素可促进子宫内膜细胞膜蛋白GPR30转录，后者与内膜异常增生密切相关；胰岛素还可上调基因羟甲基化酶Tet1表达。由此推测胰岛素通过Tet1羟甲基化修饰GPR30基因启动子促其转录表达，通过下游PI3K通路致内膜异常增生。为证实该假设，本项目采用免疫组化等分析临床标本，明确高胰岛素血症、GPR30基因羟甲基化、GPR30表达与内膜异常增生相关；体外实验验证胰岛素通过GPR30促进内膜细胞增生的作用；BSP、hMeDIP-Seq等阐明胰岛素通过Tet1羟甲基化修饰GPR30基因促其表达的机制；信号通路阻断剂等验证胰岛素上调GPR30后通过下游PI3K通路促进细胞增生。为有效防治子宫内膜异常增生提供依据。

子宫内膜异常增生是子宫内膜增生症和内膜癌的病理基础。结合文献报道和我们前期研究，我们发现胰岛素抵抗在子宫内膜增生过长、不典型性增生到内膜癌的全过程持续存在。我们前期研究发现高胰岛素血症与子宫内膜异常增生密切相关，然而阻断胰岛素受体或胰岛素样生长因子1 受体（IR/IGF-1R）后，胰岛素促进细胞增生的作用仅部分受到抑制。因此，除胰岛素直接刺激细胞增生以外，还存在其他机制促进子宫内膜异常增生。本项目从胰岛素抵抗介导的慢性炎症通过羟甲基化修饰调控雌激素受体表达进而增加子宫内膜癌雌激素敏感性方面进行研究。我们发现慢性炎性微环境可通过表观遗传学机制诱导子宫内膜雌激素受体（包括核受体和膜受体）表达增加子宫内膜癌细胞雌激素敏感性。在子宫内膜局部，巨噬细胞是慢性炎症的重要调控者。CD68+CD163+巨噬细胞浸润数量随着病程进展而逐渐增多，且与ERα表达水平呈正相关；并从巨噬细胞浸润微环境中筛选出差异细胞因子IL-17A,体外研究显示IL-17A可增加TET1表达增加ERα基因启动子5-羟甲基化修饰，进而启动ERα的转录和表达，从而导致子宫内膜异常增生。同样，有胰岛素抵抗的子宫内膜癌标本中TET1和GPER均高表达，且呈正相关性，提示胰岛素介导的GPER高表达与羟甲基化相关，分子机制水平研究揭示了胰岛素通过羟甲基化修饰上调GPR30表达增加子宫内膜雌激素敏感性是其诱导子宫内膜癌发生的另一条可能机制。本项目有助于了解慢性炎症在激素相关子宫内膜病变中的作用，阐明慢性炎性微环境调控雌激素受体增加子宫内膜雌激素敏感性的作用机制，为子宫内膜异常增生的预防和治疗方案的选择提供新的思路。