PLCE1基因调控miR-106b~25基因簇宿主基因MCM7促进食管鳞癌细胞增殖的作用及分子机制研究

Esophageal squamous cell carcinoma (ESCC) is a disorder of cell cycle and proliferation, looking for the key molecules involved in regulating the cell cycle is also the breakthrough point for the treatment of carcinoma. Our previous research found that: the expression of phospholipase C epsilon 1 (PLCE1) mRNA and protein was extremely higher in esophageal cancers tissues and the overexpressed PLCE1 can promote the proliferation of ESCC cells; the results from the high-resolution miRNA microarray analysis indicated that PLCE1 can upregulate the expression of miR-106b~25 clusters; the mini-chromosome maintenance protein 7 (MCM7), the host gene of miR-106b~25 clusters, was overexpressed in esophageal cancers tissues compared with normal tissues, and was positively correlated with PLCE1expresion. Moreover, the co-immunoprecipitation analysis exhibited that the PLCE1 can combine with MCM7 in nucleus of ESCC cell lines. Based on our previous results, we suspected that the combination of PLCE1 and MCM7 in nucleus can make the phosphorylation of the MCM7 and abnormal activation, which induce the cell cycle out of control and promote the abnormal proliferation of esophageal cancer cells. To validate our suspection, in this project we firstly evaluated the relationship between the expression of PLCE1 and MCM7 and the tumor proliferation; secondly, using the GST pull down、co-IP and yeast two hybrid experiment, we will clarify and validate the action scope of PLCE1 and MCM7, meanwhile, building the mutants which is lack of connection scope and MCM7 phosphorylation locus mutant to verify the effects and molecular mechanisms of combination of PLCE1 and MCM7 in esophageal cancer proliferation; finally, using the transplant tumor in nude mice, we will illuminate the inhibition effects of targeting PLCE1-MCM7 on esophageal cancer proliferation, and above all, we will find a novel molecular target and strategy for the treatment of esophageal carcinoma.

食管癌是细胞周期紊乱而增殖异常疾病，寻找靶向调控癌细胞周期的关键分子仍是治疗的突破点。前期研究发现：PLCE1在食管癌中高表达且可促进癌细胞增殖；miRNA芯片提示PLCE1可显著上调miR-106b~25基因簇的表达；MCM7为该基因簇宿主基因，其在食管癌中高表达且与PLCE1呈正相关，IP显示两者可在核内互相结合。综上推测：PLCE1入核后可与MCM7结合并使其磷酸化而异常激活，进而促进食管癌细胞周期紊乱和异常增殖。为此，本项目拟首先在食管癌组织中分析PLCE1与MCM7表达及与肿瘤增殖的关系；其次，使用GST-pull down、CoIP、酵母双杂交等明确和验证二者作用域，构建结合域缺失突变体及MCM7磷酸化位点突变等明确二者结合对食管癌细胞增殖的影响及机制；最后采用裸鼠移植瘤等方法阐明在体干预PLCE1-MCM7对食管癌细胞增殖的抑制作用，以期为食管癌的靶向治疗提供新的靶点和策略。

肿瘤是细胞周期紊乱而增殖异常疾病，寻找靶向调控癌细胞周期的关键分子仍是治疗的突破点。本课题主要聚焦研究PLCE1与MCM7之间的互相关系，进而揭示促进食管癌细胞周期失控和异常增殖的分子机制。（1）免疫组化检测发现MCM7蛋白在汉族、哈萨克族、维吾尔族食管鳞癌组织中明显高表达，且其高表达与患者淋巴结转移、分化及不良预后相关；且PLCE1和MCM7蛋白表达在食管鳞癌组织中呈正相关。（2）细胞生物学实验显示干扰PLCE1及MCM7后，食管鳞癌细胞的增殖水平明显受到抑制；MDC染色、AO染色及细胞免疫荧光结果表明干扰PLCE1及MCM7后细胞内自噬体数量均增加，自噬蛋白LC3的表达明显上调，流式细胞检测发现干扰PLCE1及MCM7后细胞凋亡上升。（3）食管癌细胞系中PLCE1、miR-106b~25、MCM7、E2F1高表达且表达呈正相关性。敲除PLCE1表达可减低miR-106b~25、MCM7的表达。敲除或沉默PLCE1表达能抑制E2F1蛋白磷酸化并减低其在胞核中的分布。（4）E2F1、PKCα与PLCE1可相互结合，PKCα促进E2F1蛋白磷酸化，E2F1能结合在MCM7基因启动子区上促进miR-106b~25和MCM7表达；（5）食管癌细胞中PLCE1可促进MCM7苏氨酸磷酸化及其与MCM家族复合物MCM2-7的结合。PLCE1、RIOK2、MCM7三者间存在内、外源性相互结合，在食管鳞癌细胞系中PLCE1通过RIOK2促进MCM7苏氨酸的磷酸化。PLCE1通过RIOK2促进MCM7与MCM2-7内源性和外源性结合，进一步验证发现PLCE1可通过RIOK2促进MCM复合物在核内的积聚及复制起始复合物的形成，进而促进细胞周期由G1进展为S期，导致细胞恶性增殖。通过以上研究初步阐明了PLCE1通过miR-106b~25基因簇宿主基因MCM7调控食管癌细胞DNA复制与增殖的分子机制，为PLCE1促进食管癌细胞恶性增殖提供新机制，同时明确靶向干预PLCE1/MCM7信号通路对在体食管癌细胞增殖的抑制作用，为食管癌的诊疗提供新的靶点和策略。