PLCE1靶向调控miR-106b影响新疆哈族食管癌细胞自噬和凋亡的分子机制研究

The Kazakh population, in Xinjiang, is characterized by having higher incidence of esophageal carcinoma than those in the general population of China. Inducing autophagic cell death has been a novel promising cancer therapeutic strategy. In our previous research, we have found that the expression level of PLCE1 was extremely higher in ESCC tissues. We also proved that PLCE1 could inhibit the autophagy and apoptotic in ESCC cells by targeting Beclin1 and RBL2. The Agilent miRNA microarrays analysis indicated that the miR-106b was the downstream target of PLCE1 and was notable upregulated; the biology prediction algorithms revealed that Beclin1 and RBL2 were the potential target gene for miR-106b. Therefore, we would hypothesize that PLCE1 could suppress the expression of Beclin1 and RBL2 through upregulating the miR-106b expression, which resulted in the inhibition of cell autophagy and apoptotic of ESCC cell lines. To verify the hypothesize, firstly, we will analyze the relationship between the expression of PLCE1 and miR-106b and the condition of the autophagy and apoptotic in tumor tissues respectively. Secondly, using knockdown of PLCE1 or miR-106b expression by shRNA or miRNA inhibitor and inducing overexpression of it, we will research the influence on activity of autophagy and apoptotic in cancer cells, and clarify the cellular function of miR-106b target on Beclin1 and RBL2. Finally, we will investigate the therapeutic action of promoting the autophagy and apoptotic in ESCC via intervene the PLCE1/miR-106b signaling pathway in vivo. Depending on all above studies and experiments, we hope find a novel diagnostic/prognostic marker and molecular therapeutic targets of Kazakh esophageal carcinoma.

新疆哈族食管癌死亡率远高于全国平均水平，诱导癌细胞自噬性死亡已成肿瘤治疗新策略。我们前期发现磷脂酶C1（PLCE1）在食管癌中高表达并可下调Beclin1和RBL2来抑制食管癌细胞自噬和凋亡；miRNA表达谱芯片提示PLCE1可显著上调miR-106b的表达，生物信息学预测miR-106b潜在靶基因为Beclin1和RBL2，综上我们推测：PLCE1可能通过miR-106b抑制其靶基因的表达，进而抑制食管癌的自噬和凋亡。为验证假说，本项目拟首先在食管癌组织中分析PLCE1与miR-106b表达及肿瘤自噬和凋亡的关系；其次，通过诱导和阻断PLCE1和miR-106b表达，研究细胞自噬活性和凋亡的改变，并明确miR-106b与Beclin1和RBL2的靶向作用关系；最后，构建动物模型阐明干预PLCE1/miR-106b通路促进食管癌自噬和凋亡的治疗作用，以期为哈族食管癌治疗提供新的靶点。

诱导癌细胞自噬性死亡是肿瘤治疗的新策略之一。根据前期研究，我们提出了食管癌易感基因磷脂酶C1（PLCE1）可能通过miR-106b抑制其靶基因Beclin1和RBL2的表达，进而抑制食管癌的自噬和凋亡的假说。通过本项目的实施，我们发现并证实：（1）与正常食管粘膜组织相比，食管鳞癌组织中PLCE1蛋白与miR-106b-5p的表达均显著增高，二者呈正相关关系；而自噬相关蛋白Beclin 1、RBL2的表达均显著降低，并与PLCE1蛋白与miR-106b-5p的表达呈负相关，Beclin 1的表达与食管鳞癌分化和临床分期有关，可能是影响汉族ESCC患者预后的独立危险因素。（2）运用Affymetrix GeneChip® miRNA 3.0 Arrays芯片发现PLCE1可显著上调miR-106b-5p，通过实时定量PCR验证发现沉默PLCE1后miR-106b表达显著下调，增强miR-106b表达可部分逆转PLCE1表达抑制所导致的食管癌细胞增殖能力降低、凋亡和自噬增加。（3）PLCE1过表达可下调食管鳞癌细胞的自噬和凋亡水平，促进食管细胞的恶性转化；食管癌Eca109细胞裸鼠移植瘤模型中，PLCE1基因被同抑制时，裸鼠成瘤体积与瘤重量显著降低，血管生成相关蛋白表达显著降低，凋亡相关蛋白和的表达量显著增高。（4）生物信息学及双荧光素酶报告基因试验证实RBL2和Beclin-1分别为miR-106b的靶基因，功能学实验证实miR-106b可靶向抑制Beclin-1及RBL2的表达，进而显著促进食管鳞癌细胞的增殖并抑制食管癌细胞的自噬和凋亡。综上所述，我们认为PLCE1对调控食管癌细胞自噬和凋亡的分子机制可能是通过调控miR-106b-5p及其靶基因Beclin-1和RBL2实现的。因此，对PLCE1通过miR-106b-5p参与自噬抑制的分子机制的深入研究，可能为食管鳞癌个性化治疗提供新的思路和潜在的靶点。在本项目实施过程中，我们发现MCM7为miR-106b的宿主基因，那么PLCE1基因与MCM7之间是否存在着调控关系进而调控miR-106b的表达，其确切分子机制有待进一步的研究。通过本项目的实施，培养研究生6名，发表相关SCI论文15篇，获得授权国家专利1项，科技奖励1项。