潜在抑癌基因eIF4E3二重调控Snail/E-cad轴的翻译表达抑制宫颈癌EMT和侵袭转移

eIF4E3, a strong and potetial tumor suppressor gene neglected for many years, was discovered in 2013 to specially suppress the translation of eIF4E target genes and to selectively initiate the translation of other genes.However, its role in most tumors has not been studied. After studying eIF4E for many years, we pre-tested eIF4E3 and priliminarily found that eIF4E3 down-regulated Snail but up-regulated E-cad translation and suppressed the invasion of cervical cancer cells. On the basis of our priliminary finding and other’s discovery, we assumed that “eIF4E3 doubly regulates the translation of Snail/E-cad axis to suppress EMT, invasion and metastasis of cervical cancer”. To confirm the assumption, we shall construct eIF4E3 wild-type and mutant plasmids, transfect the plasmids into cervical cancer cells to establish stable cell lines, make nude mice xenograft, observe human cervical tumor tissues and follow up patients, to study the effect of eIF4E3 over-expression and reduced expression on cervical cancer at cell, animal and human body levels; we shall also use sucrose density gradient ultracentrifugation polysome isolation, rabbit reticulocyte translation-dual luciferase report system, co-immunoprecipitation, RT-qPCR ，WB and so on to explore the molecular mechanisms，in which eIF4E3 suppresses Snail translation initiated by eIF4E and directly initiate the translation of E-cad. The results from this project will firstly discern the role of eIF4E3 in suppression of cervical cancer and its mechanism, and open a new thinking for the treatment and prevention of metastasis of cervical cancer.

2013年后才发现埋没多年的eIF4E3是对抗eIF4E的潜在抑癌基因，但其在绝大多数肿瘤中的作用未明。我们在多年研究eIF4E的基础上向前摸索发现eIF4E3下调eIF4E靶基因Snail的翻译但上调E-cad基因的翻译并抑制宫颈癌侵袭，提出“eIF4E3二重调控Snail/E-cad轴的翻译表达抑制宫颈癌EMT和侵袭转移”的假说。为了验证假说，我们构建eIF4E3野生型和突变体质粒、转染及建立稳定细胞株和裸鼠移植瘤，检测人体肿瘤组织和随访病人，在细胞、动物、人体三层面研究eIF4E3过表达或缺失表达对宫颈癌影响；用蔗糖密度梯度高速离心分离多核糖体、兔网织红细胞-双荧光素酶翻译报告系统、免疫共沉淀、RT-qPCR、WB等方法研究eIF4E3抑制eIF4E起始Snail翻译及直接起始E-cad翻译的分子机制。本结果将率先阐明eIF4E3抑制宫颈癌的作用及机制，为宫颈癌转移防治开拓新思路。

eIF4E3可能是对抗著名翻译起始因子eIF4E的重要潜在抑癌基因。但至今eIF4E3的研究报道极少，其在绝大多数肿瘤中的作用未明。我们在多年研究eIF4E的基础上向前摸索发现eIF4E3下调eIF4E靶基因Snail的翻译但上调E-cad基因的翻译并抑制宫颈癌侵袭，提出“eIF4E3二重调控Snail/E-cad轴的翻译表达抑制宫颈癌EMT和侵袭转移”的假说。为了验证假说，我们构建了eIF4E3野生型和突变体质粒、转染两种宫颈癌细胞株Hela（HPV18+）和C33a（HPV-）及建立稳定细胞株，检测eIF4E3及相关分子的mRNA和蛋白水平改变，检测癌细胞增殖、克隆形成、细胞周期、凋亡、侵袭等改变，检测eIF4E3等在人体宫颈肿瘤组织中的表达改变及随访病人，在细胞、人体层面研究eIF4E3对宫颈癌影响。结果：①eIF4E3在宫颈癌细胞中呈低表达，瞬时和稳定转染eIF4E3后，癌细胞增殖率、克隆形成率、EMT、侵袭率显著下降，凋亡率上升。②在宫颈炎、CINⅡ-CINⅢ、未转移宫颈癌、转移宫颈癌组织中eIF4E3蛋白阳性率依次为90.38% 、66.67%、55.56%、40.00%，其表达强度与宫颈肿瘤级别相关系数为-0.500。③建立两个eIF4E3突变体质粒C69a和W86a。经检测，这两个突变体都不能结合mRNA，失去翻译起始的作用，同时不能抑制宫颈癌细胞的生物学行为。④转染eIF4E3后，癌细胞分布在G0/G1期下降，S期上升，G2/M期下降，同时细胞增殖核抗原（PCNA）和Cyclin B1下调，而Cyclin A1和p27变化不明显。⑤转染eIF4E3后，癌细胞的E-cad mRNA 水平不改变，但蛋白水平升高；Rip实验显示eIF4E3结合E-cad mRNA；在稳定转染eIF4E3基因的癌细胞中敲减E-Cadherin，细胞增殖率、克隆形成率、EMT、侵袭率显著显著回升，细胞周期逆转，凋亡率下降。⑥转染eIF4E3后，癌细胞的凋亡率升高，Bax、Fas表达升高，Bcl-2表达下降。结果清楚表明： eIF4E3是宫颈癌的抑癌基因，通过下调PCNA和cyclin B1抑制增殖,通过直接起始E-cad翻译表达抑制EMT、侵袭转移，通过下调Bcl-2及上调Bax和Fas，激活内、外凋亡途径促进宫颈癌细胞凋亡。结果验证了科学假说，为宫颈癌防治打下了理论基础。