RGMb通过BMP信号通路促进子宫内膜腺细胞的增殖

RGMb蛋白是反义导向分子（repulsive guidance molecule，RGM）家族成员之一，可在细胞水平上介导骨形态发生蛋白（bone morphogenetic protein，BMP）的信号通路。大量研究报道，RGMb参与调控细胞的增殖、分化、凋亡以及细胞间的黏附能力。本研究旨在探讨RGMb对子宫内膜腺细胞的作用及其分子机制。应用siRNA与过表达技术处理子宫内膜腺细胞系（Ishikawa），采用qPCR与Western印迹技术确定其转染效率，以及BMP相关信号通路（MAPK与Smad）成员的表达。结果显示：下调RGMb基因的表达显著降低了p-ERK1/2（1.861 ± 0.1864 vs 0.885 ± 0.0869，P=0.0090）与p-Smad1/5/8（1.624 ± 0.1238 vs 1.093 ± 0.0890，P=0.0253）的表达水平。过表达RGMb基因显著升高了p-ERK1/2（1.237 ± 0.1114 vs 2.089 ± 0.1658，P=0.0130）与p-Smad1/5/8（1.139 ± 0.0562 vs 1.98 ± 0.1449，P=0.0056）的表达水平。而RGMb基因下调和过表达对p-P38 MAPK蛋白表达水平均无显著影响（P>0.05）。采用CCK-8和qPCR技术检测RGMb对Ishikawa细胞增殖活力及增殖相关基因的影响。结果显示：转染80 nmol/L RGMb siRNA显著降低Ishikawa细胞的增殖活力（0.479 ± 0.0271 vs 0.3487 ± 0.0094，P=0.0104），同时降低增殖相关基因CCND1（1 ± 0.0366 vs 0.6719 ± 0.0236，P=0.0017）和CDK2（1 ± 0.0370 vs 0.853 ± 0.0135，P=0.0202）表达水平；转染1 μg/mL RGMb基因过表达质粒显著提高Ishikawa细胞增殖活力（0.283 ± 0.0030 vs 0.3714 ± 0.0140，P=0.0001），同时增加CCND1（1 ± 0.0178 vs 1.375 ± 0.0356，P=0.0007）和CDK2（1 ± 0.0188 vs 1.376 ± 0.0513，P=0.0023）基因的表达水平。以上结果表明，RGMb 可能通过p-ERK1/2与p-Smad1/5/8影响Ishikawa细胞增殖活力，为进一步研究RGMb调控子宫机能的分子机制提供科学依据。

RGMb Enhanced the Proliferation of Endometrial Gland Cells through BMP Pathway

The RGMb protein is a member of the repulsive guidance molecule (RGM) family, which mediates the signaling pathway of bone morphogenetic protein (BMP) at the cellular level. Many studies have reported that RGMb is involved in the regulation of cell proliferation, differentiation, apoptosis and intercellular adhesion. The present study aims to investigate the effect of RGMb on endometrial gland cells and its molecular mechanism. RGMb siRNA and overexpression were applied to treat the endometrial gland cell line (Ishikawa). The efficiency of transfection and the expression of BMP-related signaling pathways (MAPK and Smad) were determined by qPCR and Western blotting. The results showed that down-regulation of RGMb expression significantly decreased the expression levels of p-ERK1/2 (1.861 ± 0.1864 vs 0.885 ± 0.0869, P=0.0090) and p-Smad1/5/8 (1.624 ± 0.1238 vs 1.093 ± 0.0890, P=0.0253), and RGMb overexpression significantly increased the expression levels of p-ERK1/2 (1.237 ± 0.1114 vs 2.089 ± 0.1658, P=0.0130) and p-Smad1/5/8 (1.139 ± 0.0562 vs 1.98 ± 0.1449, P=0.0056). Neither down-regulation or over-expression of the RGMb gene effected the expression level of p-P38 MAPK (P>0.05). CCK-8 and qPCR were used to detect the effect of RGMb on cell proliferation. The results showed that 80 nmol/L RGMb siRNA significantly decreased the proliferation of Ishikawa cells (0.479 ± 0.0271 vs 0.3487 ± 0.0094, P=0.0104), and the expression levels of proliferation-related genes CCND1 (1 ± 0.0366 vs 0.6719 ± 0.0236, P=0.0017) and CDK2 (1 ± 0.0370 vs 0.853 ± 0.0135, P=0.0202); The proliferation rate of Ishikawa cells was significantly increased (0.283 ± 0.0030 vs 0.3714 ± 0.0140, P=0.0001), as well as the expression levels of CCND1 (1 ± 0.0178 vs 1.375 ± 0.0356, P=0.0007) and CDK2 (1 ± 0.0188 vs 1.376 ± 0.0513, P= 0.0023),when transfected with 1 μg/mL RGMb overexpression plasmids. All these results indicate that RGMb might affect the proliferation of Ishikawa cells by p-ERK1/2 and p-smad1/5/8, which provides a scientific basis for further study in the regulation mechanism of RGMb in uterine function.