靶向调控PLCE1基因的microRNAs在新疆哈族食管癌侵袭转移中的作用与机制研究

The incidence of the Esophageal squamous cell carcinoma (ESCC) in Kazakh population is higher than that of the general population of China. As we all known, invasion and metastasis are important factors affecting the prognosis of patients with ESCC. In our previous study, we have confirmed that the polymorphisms within the phospholipase C epsilon 1 (PLCE1) contribute to Kazakh ESCC susceptibility and also found that the elevated expression of PLCE1 was associated with lymph node metastasis and advanced TNM stages of ESCC. Furthermore, our study also confirmed that the expression of miR34, targeting in regulating the PLCE1expression by using bioinformatics predictions, showed a significantly differences between the ESCC tissues and matched normal esophageal tissues. The research aims at investigating the molecular mechanism of the microRNAs(miRNAs) targeting in regulating the PLCE1 and inhibiting the invasion and metastasis of Kazakh ESCC. Firstly, an initial screening of the miRNAs, targeting in regulating the PLCE1expression, will be conducted by the conbination of investigating the miRNA expression profiling between the Kazakh ESCC cell lines with different metastatic potentials using microarrays and bioinformatics predictions. Secondly, we will examine the expression of PLCE1 and miRNAs initial screening and and analyze the correlativity of them. Simultaneously, we will use the luciferase reporter assay system to confirm the direct relationship between them. Finally, using the cell invasion model in vitro and the mouse xenograft model in vivo, we will observe and reveal that the miRNAs inhibit the invasion and metastasis of Kazakh ESCC through regulating the PLCE1 gene and affacting the MAPK signalling pathway. The findings of our study will provide the novel targets for treatment of invasion and metastasis of Kazakh ESCC.

新疆哈族食管癌发病率远高于全国平均水平。侵袭转移是影响患者预后的重要因素。我们前期研究发现，PLCE1基因不仅与哈族食管癌的发病密切相关，其表达上调还可促进食管癌的侵袭和转移；同时还发现miR34等的表达水平在癌与正常粘膜中存在显著差异，且是生物信息学预测的可调控PLCE1的miRNA分子。为深入探讨靶向调控PLCE1的miRNA在食管癌侵袭转移中的分子机制，本项目拟首先运用miRNA表达谱芯片检测不同转移潜能的食管癌细胞并结合生物信息学初步筛选出可靶向调控PLCE1基因的miRNA；其次，检测筛选出的miRNA与PLCE1在组织中的表达及相关性分析，并利用荧光素酶报告系统确认两者的直接作用关系；最后，通过体外细胞侵袭和裸鼠体内移植瘤模型，观察并揭示靶向调控PLCE1的miRNA分子通过MAPK信号通路影响食管癌细胞侵袭能力和原位瘤转移能力的作用与机制，为哈族食管癌转移的诊疗提供新靶点。

我国是全球食管鳞癌发病率和死亡率最高的国家之一，新疆哈萨克族食管癌的死亡率远高于全国平均水平和同地区的其他民族。本次研究中我们对可能靶向调控PLCE1基因的miRNAs进行预测，发现miR-145及miR-34a为可能靶向调控PLCE1基因的分子，并且确认PLCE1与miR-145和miR-34a分别可能存在一定的负向调控关系。通过荧光素酶实验验证了在食管鳞癌细胞系中miR-145及miR-34a可靶向调控PLCE1的表达；通过一系列体外实验我们发现miR-145、miR-34a可显著抑制食管鳞癌细胞的增殖，促进食管鳞癌细胞的凋亡，并可通过调控细胞的骨架从而抑制食管鳞癌细胞的迁移能力，增强miR-145或miR-34a表达且转染PLCE1 siRNA介导的PLCE1基因沉默能更加显著的降低食管鳞癌的增殖和侵袭能力。同时，对表达不同miRNA及PLCE1水平的细胞系进行凋亡相关蛋白（cleaved-PARP、Bax、Bcl-2等）及EMT相关蛋白（E-cad、Vimentin）进行检测，也验证了体外细胞生物功能学试验的结果。通过对食管癌患者癌组织及癌旁组织进行检测，我们发现miR-145在食管癌组织中表达显著降低（癌组织41.92±9.089，正常组织104.4±13.00，P = 0.0002），miR-145的表达与食管癌患者淋巴结转移（N0：66.54±32.47，N1-3：25.89±9.95，P = 0.0002）和临床分期密切相关（T1/2：54.01±21.86，T3/4：31.03±15.77，P = 0.0381）。食管鳞癌组织样本中miR-145 mRNA 表达与PLCE1蛋白存在着明显的负相关关系（r = -0.472， P = 0.008）。食管鳞癌组织中 miR-34a 表达与癌旁组织相比，明显降低（p=0.0022），在食管鳞癌组织中miR-34a表达与 PLCE1呈负相关关系（r= -0.4658，p=0.0189）。通过以上研究结果我们发现，在食管鳞癌中，miR-145及miR-34a可靶向调控PLCE1，抑制食管癌细胞的生长、侵袭及转移，促进其凋亡，本研究多角度阐述miR-145与miR-34a在食管鳞癌中的生物学行为，为食管鳞癌的临床治疗提供了理论基础。