NMMHC-IIA蛋白调控CTNNB1基因转录促胃癌细胞失巢凋亡抵抗及腹膜转移机制

Peritoneal metastasis of gastric cancer (GC) always indicates very poor survival, and its molecular mechanism is still far from being uncovered. In this study, we had found that NMMHC-IIA expression was stepwise increased in nontumorous gastric mucosa, primary gastric cancer tissues and peritoneal metastatic foci by two-dimensional fluorescent differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF MS). We also found its expression was negatively associated with GC patients' survival. Then, we knocked down and up-regulated NMMHC-IIA's expression in gastric cancer cell lines by TALEN-mediated knockdown and lentivirus-mediated gene transfection. Unexpectedly, we found the expression of CTNNB1 was postively associated with the expression of NMMHC-IIA. Moreover, the number of gastric cancer cells' anoikis in soft agar increased after knocking down their expression of NMMHC-IIA. Besides, we found NMMHC-IIA largely located in the nucleus of gastric cancer cells, and specifically recognized CTNNB1 promoter to regulate its transcription. We also found staurosporine could inhibite S1943 phosphorylation of nuclear NMMHC-IIA and the expression of CTNNB1. Based on above results and literature we read, we hypothesized that NMMHC-IIA promoted anoikis resistance and peritoneal metastasis of gastric cancer cells by regulating CTNNB1 transcription. We planned to use cell experiments and orthotropic GC models to confirm whether NMMHC-IIA promoted anoikis resistance and peritoneal metastasis of gastric cancer cells by increasing CTNNB1 expression. We also wanted to know the nuclear localization signal and DNA binding domains of NMMHC-IIA. Lastly, we would confirm whether staurosporine could inhibit peritoneal metastasis of gastric cancer cells in orthotropic GC models. This study is helpful to uncover the new mechanisms of gastric cancer peritoneal metastasis and good for looking for drug targets.

胃癌腹膜转移提示患者预后极差，转移机制尚待深入。前期发现NMMHC-IIA在胃正常上皮、胃癌原发灶和腹膜转移灶间递增表达；TALEN质粒和慢病毒构建NMMHC-IIA敲低和过表达稳转细胞株并发现CTNNB1表达与NMMHC-IIA水平呈正相关，且敲低NMMHC-IIA促进胃癌细胞失巢凋亡；NMMHC-IIA存在核定位，可识别CTNNB1启动子区、促进其转录；星孢菌素可抑制核NMMHC-IIA蛋白S1943磷酸化和CTNNB1转录。结合文献及前期实验提出假设，NMMHC-IIA调控CTNNB1转录促进胃癌细胞失巢凋亡抵抗及腹膜转移。后续拟通过细胞实验和原位种植瘤模型验证NMMHC-IIA 是否通过上调CTNNB1表达增强失巢凋亡抵抗促进胃癌腹膜转移；明确NMMHC-IIA核定位信号及DNA结合域；通过原位种植瘤模型验证星孢菌素体内抑癌效果。本研究有助于探索胃癌腹膜转移新机制，寻找药物靶点。

腹膜转移预示胃癌患者预后不良，其机理尚不完全清楚。本研究采用2-DIGE、MALDI-TOF/TOF质谱和单细胞转录组检测正常胃粘膜、胃癌原发灶和腹膜转移灶间差异蛋白表达；用慢病毒shRNA和转录激活因子样效应核酸酶技术敲除胃癌细胞系内肌球蛋白重链9 (MYH9)表达；免疫荧光，免疫透射电子镜检、染色质分离、免疫共沉淀和染色质免疫沉淀分析、双荧光素酶报告基因检测、琼脂糖-寡核苷酸pull-down实验、流式细胞术和细胞失巢凋亡检测等实验研究胃癌细胞系内MYH9诱导β-catenin (CTNNB1)核转录相关机制；裸鼠和条件性转基因小鼠体内进一步验证蛋白间表达相关性和MYH9促癌功能。结果示MYH9在胃癌腹膜转移灶组织中显著高表达，并与胃癌患者预后差相关；MYH9主要通过四个核定位信号聚集于胃癌细胞核内；MYH9识别CTNNB1启动子特定DNA结合域，并与肌球蛋白轻链9、β-肌动蛋白和RNA聚合酶II相互作用促进CTNNB1转录，增加胃癌细胞失巢凋亡抵抗能力；Staurosporine通过减少核MYH9 S1943磷酸化抑制CTNNB1转录、Wnt/β-catenin信号转导和胃癌进展。细胞核MYH9诱导的CTNNB1表达可作为胃癌腹膜转移潜在干预靶点。