HnRNP F提高Snail mRNA稳定性促进EMT参与膀胱癌演进

Invasion and metastasis are the important factors related to the prognosis of bladder cancer. Its mechanism is complex and still receiving much more attention. Our previous study has found that the expression of HnRNP F was significantly increased in bladder cancer, and could enhance the ability of tumor cell proliferation and invasion. When HnRNP F was silenced, the stability of Snail mRNA and EMT related markers could be inhibited. Thus, we put forward the hypothesis: HnRNP F promotes the EMT involved in bladder cancer evolution by enhancing Snail mRNA stability. On the basis of our previous findings, this project intends to use immunohistochemistry to detect the expression of HnRNP F in clinical samples and analyze its clinical significance, observe the alterations of HnRNP F on the biological behaviors of bladder cancer cells in vitro and in vivo, and the regulation effect on EMT. Furthermore, the interaction mode and function domain between HnRNP F and Snail mRNA will be examined through the means of RIP, Snail mRNA stability test and Snail mRNA truncated method. Our aim, to clarify the biological functions of HnRNP F protein in bladder cancer and elucidate the mechanism that HnRNP F regulates the process of EMT in bladder cancer by affecting Snail expression, will provide new ideas for the mechanisms of tumor occurrence, progression and the development of EMT in bladder cancer.

浸润和转移是影响膀胱癌预后的重要因素，其机制复杂多样，仍是研究的热点。我们前期发现核内不均一核糖核蛋白F(HnRNP F)在膀胱癌中表达升高，可促进肿瘤细胞增殖和侵袭；沉默HnRNP F能抑制Snail mRNA稳定性和EMT相关标志物的表达，由此提出“HnRNP F提高Snail mRNA稳定性促进EMT参与膀胱癌演进”的研究假说。本项目拟在前期基础上，利用免疫组化技术检测临床样本中HnRNP F蛋白的表达并分析其临床意义；从体内外明确HnRNP F对膀胱癌细胞生物学行为的影响及对EMT调控效应；通过RIP、RNA稳定性检测和Snail mRNA截短等方法明确HnRNP F蛋白与Snail mRNA相互作用方式及作用结构域；明确HnRNP F蛋白在膀胱癌中的生物学功能并阐明HnRNP F影响Snail表达调控膀胱癌EMT的分子机制，为膀胱癌的发生、发展及EMT诱导的机制提供新思路。

浸润和转移是影响膀胱癌预后的重要因素，其机制复杂多样，仍是研究的热点。HnRNP F在不同的肿瘤中表达水平不一。本项目开展前，国内外尚未见HnRNP F蛋白与膀胱癌的相关文献报道。因此，我们在前期通过荧光差异蛋白组学方法筛选出HnRNP F蛋白在膀胱癌组织中表达明显升高的基础上，对包含有103例膀胱癌及癌旁组织的组织芯片进行免疫组化分析，提示HnRNP F在膀胱癌中具有促癌作用；并建立HnRNP F稳定上调和沉默的人膀胱癌细胞株，体内外观察发现HnRNP F可增加进膀胱癌细胞转移、侵袭能力及EMT转化；同时，在分子水平上观察到沉默HnRNP F表达后，膀胱癌细胞侵袭能力和EMT转化能力受到抑制，而且Snail表达明显降低，提示HnRNP F蛋白可能通过调节Snail表达诱导膀胱癌细胞发生EMT，但具体的分子机制尚不清楚。我们后续通过实验结合生物信息学预测，提示Snail mRNA稳定性可能受到AREs结合蛋白的调控。我们又利用RIP预实验初步证实HnRNP F能够与Snail mRNA相互结合；进一步，又发现HnRNP F沉默后，Snail mRNA的稳定性显著降低，说明HnRNP F能够调节Snail mRNA稳定性来影响其表达，进一步我们通过荧光素酶报告实验及Snail mRNA的3'UTR含有3个AREs的突变体及截短实验证实HnRNP F通过结合Snail mRNA的3'UTR的AREs调控Snail mRNA稳定性进而调控EMT参与膀胱癌进展；此外，我们还对HnRNP F对膀胱癌细胞增殖的机制进行了探讨，发现HnRNP F与TPX2蛋白相互结合且具有正相关表达关系，可以且调控TPX2蛋白表达从而改变周期相关蛋白cyclin D1和p21的表达量进而促进细胞周期进程加快，从而导致细胞增殖；另一方面还发现PI3K/AKT信号通路可通过FOXO1的磷酸化作用影响其与HnRNP F的启动子的结合，进而调控HnRNP F的表达；以上结果发表SCI收录期刊论著3篇。本项目按照研究计划顺利进行，其中有三位参与研究工作的硕士研究生顺利毕业，达到预期研究目标。本项目有望为膀胱癌的发生、 发展及 EMT 诱导的机制提供新思路，在膀胱癌的进展的病理机制方面有重要的理论意义，HnRNP F蛋白有望成为治疗膀胱癌的一个潜在的药物筛选靶标。