缺锌环境下lncRNA UCA1通过竞争性结合miR-193a调控ZIP5反馈调节 细胞内锌水平促进食管癌进展的机制研究

Esophageal cancer is a malignant disease. Studies have been identified that zinc deficiency is a risk factor for esophagus cancer in high-risk areas. Our previous studies showed that zinc transporter ZIP5 could increase intracellular zinc level and promote progression of esophageal cancer which was different from traditional knowledge. In 2017, academician Lin Dongxin published a paper in GASTROENTEROLOGY (IF=18.392) and its conclusion was consistent with our results. Academician Lin demonstrated that ZIP6 could increase intracellular zinc level then promote the invasion of esophageal cancer. There is not yet reported internationally about mechanism of increasing intracellular zinc level and promoting progression of esophageal cancer by special signal pathway in zinc deficiency environment. By using high-throughput sequencing technology, we found that expression of lncRNA UCA1 was affected by zinc deficiency. And lncRNA UCA1 could regulate intracellular zinc level and promote the proliferation and metastasis of esophageal cancer. Based on the KEGG and TCGA database, we know that UCA1 is directly combined with miR-193a and miR-193a is directly combined with 3'UTR in ZIP5. Then, our study is identified that UCA1 regulates intracellular zinc level through regulation ZIP5 by competitive combination with miR-193a and promote esophageal cancer progression in zinc deficiency environment. The study aims to identify new molecular target by using a series of technologies including Cas9, Dual-luciferase reporter assay system, zinc-specific fluorescent probe zinpyr-1 in vivo and vitro.

食管癌是重大恶性疾病，研究证明锌缺乏是高发区食管癌的危险因素。我们前期研究发现锌转运蛋白ZIP5能够升高细胞内锌水平并促进食管癌进展，这与传统认知相悖。2017年林东昕院士在18分杂志发表了ZIP6提高细胞内锌水平增加食管癌侵袭性的论文，与我们的结果一致。然而环境锌缺乏时机体是如何通过特定的信号通路提高细胞内锌水平进而促进食管癌进展？国际上尚未见报道。为此我们利用高通量测序技术，筛选出lncRNA UCA1，发现其表达受缺锌影响且可调节细胞内锌水平促进食管癌增殖和转移，同时利用KEGG、TCGA等数据库发现，UCA1与miR-193a直接结合，而miR-193a与ZIP5的3'UTR直接结合，因此我们假设缺锌时UCA1通过竞争性结合miR-193a调控ZIP5进而调节细胞内锌水平发挥促癌作用。本项目拟从体内、外水平采用Cas9、双荧光素酶系统、锌离子荧光探针等技术研究以期发现新的诊疗靶点。

食管癌发病具有显著的地域特征，高发区的发病率和死亡率高于世界平均水平10倍以上，且危险因素呈现独有的特征。锌缺乏是高发区食管癌发生发展的重要危险因素，但有研究显示锌水平升高也会促进食管癌进展。研究首先通过动物实验验证锌缺乏在食管癌发病过程的促进作用；并从细胞水平探讨锌缺乏时UCA1表达水平升高，并通过竞争性结合miR-193a-3p调控ZIP5反馈调节细胞内锌水平的分子机制；然后通过在食管癌细胞系中过表达UCA1，证明UCA1表达上调对食管癌细胞功能的影响及其分子机制；最后分析食管癌患者的血清锌水平、癌组织中UCA1、miR-193a-3p和ZIP5表达水平之间的关系，明确上述指标与食管癌患者临床特征及预后的关系，探索其转化价值。.锌水平降低能够促进食管癌进展，动物实验显示低锌饲料喂养的小鼠，诱导食管癌发病率显著升高并且食管组织中ZIP5的表达水平显著升高，并且在食管组织进展为食管癌之前ZIP5表达已经升高。体外实验证明锌水平降低时UCA1表达水平升高，miR-193a的表达水平降低，ZIP5表达升高。分别在食管癌细胞中上调和下调UCA1，细胞内锌水平升高和降低，与上调和下调ZIP5的结果一致。通过荧光素酶实验证明了miR-193a分别与UCA1和ZIP5基因mRNA启动子区3’UTR直接结合，锌缺乏时UCA1通过ceRNA途径调控ZIP5的表达。锌水平降低能够促进细胞周期，G0/G1期细胞比例显著降低，CyclinD1的表达显著增加，锌水平降低能够通过增加CyclinD1的表达促进细胞周期，进而促进食管癌进展。同时锌水平降低能够增加放射敏感性，在轻度缺锌时细胞增殖能力不变但细胞周期能够显著变化，对放射线不敏感的G0/G1期细胞比例显著降低，CyclinD1的表达增加，促进细胞周期，放射敏感的G2期细胞比例增加。临床信息显示，食管癌患者血清miR-193b与癌组织中ZIP5表达负相关，并与预后相关。我们还通过一项多中心研究，发现性别、婚姻状况、职业、肿瘤家族史、肿瘤部位、分化程度、病理分期与食管癌生存率有关。.通过多角度探讨食管癌高发区的危险因素，可以为预防和治疗食管癌寻找靶点以及提供依据。