长链非编码RNA MALAT1调控胃平滑肌细胞表型转换在糖尿病胃轻瘫中的作用及机制

Recent studies found that gastric smooth muscle cell (SMC) phenotypic switch mediated the development of diabetic gastroparesis (DGP); however, the mechanism leading to the phenotypic switch of SMCs remains unknown. Our previous study found that long noncoding (Lnc) RNA MALAT1 was up-regulated in the gastric tissues of DGP rats. High glucose intervention up-regulated LncRNA MALAT1, down-regulated miRNA-449a and reduced the expression of contractile phenotype markers in human gastric SMCs. Furthermore, database analysis suggested that Krupple-like factor 4 (KLF4) is the target gene of miRNA-449a. We therefore hypothesized that during diabetes development, MALAT1 is up-regulated and competitively binds to miR-449a, and increases the target gene KLF4 expression, thereby preventing the transcription complex myocardin/SRF from binding to the smooth muscle-specific gene promoter region, inducing SMCs switching to synthetic phenotype, and eventually reduces gastric contractility. In the present study, firstly, we will use MALAT1 and miRNA-449a silenced and overexpressed cell models to investigate the effect and mechanism of MALAT1 on gastric SMC phenotype. Secondly, we plan to establish DGP mouse models with wild-type and MALAT1 knock-out mice to verify that MALAT1-mediated SMC phenotype switch participates in the development of DGP. Thus, our study will demonstrate the hypothesis at molecular, cell, tissue and overall level, providing new targets for the prevention and treatment of DGP.

研究发现胃平滑肌细胞（SMC）表型转换介导了糖尿病胃轻瘫（DGP）的发病，但SMC表型转换的机制不明。我们前期研究发现：DGP大鼠胃长链非编码（Lnc）RNA MALAT1上调，高糖干预胃SMC可上调MALAT1、下调miRNA-449a、减少SMC收缩标志蛋白表达；数据库分析提示Krupple样因子4（KLF4）是miR-449a的靶基因。故提出假说：糖尿病时，高糖诱导MALAT1上调，竞争性结合miR-449a，使靶基因KLF4表达增加，阻止转录复合物myocardin/SRF与平滑肌特异基因结合，促SMC向合成型转换，胃平滑肌收缩力下降。拟采用MALAT1及miR-449a沉默、过表达细胞模型，研究MALAT1对胃SMC表型的作用机制；建立MALAT1敲除小鼠DGP模型，验证MALAT1在DGP中的作用。从而在分子、细胞、组织和整体水平上论证假说，为DGP防治提供新的靶点。

糖尿病胃轻瘫（DGP）是糖尿病（DM）的常见并发症。我们前期研究表明，长链非编码RNA（lncRNA）转移相关肺腺癌转录本1（MALAT1）的表达与DGP密切相关。然而，MALAT1在DGP发病机制中的作用仍不清楚。本研究中，我们旨在阐明MALAT1在DGP发病中的作用。首先，我们通过lncRNA测序分析获取lncRNAs表达谱；然后，我们检测DGP模型小鼠和糖尿病患者胃组织中MALAT1的表达；最后，我们利用短发夹RNA技术，RNA免疫沉淀和双荧光素酶报告基因检测深入研究MALAT1在高糖（HG）诱导人胃平滑肌细胞（HGSMC）增殖、迁移、表型转换和卡巴胆碱诱导的细胞内Ca2+变化中的作用及机制。结果表明MALAT1在 DGP 模型小鼠、糖尿病患者胃组织以及HG条件下的HGSMC中表达均上调；MALAT1敲低影响HG条件下HGSMC活力、增殖、迁移及表型转换；此外，研究表明MALAT1通过吸附miR-449a，调节HGSMC中Delta样配体1（DLL1）的表达；MALAT1/miR-449a/DLL1通路任一环节改变都会影响HG条件下HGSMC的增殖、迁移、表型转换及卡巴胆碱诱导的胞内Ca2+浓度变化。综上，我们研究阐明了DGP发病机制中的一个新的调节信号通路：MALAT1/miR-449a/DLL1轴。