多梳蛋白复合体1重要组分RNF2在膀胱癌侵袭转移中的作用和机制

Multiple molecular alterations have been described in urothelial carcinoma of the bladder (UCB). RNF2, an important subunit of polycomb repressor complex 1 (PRC1), was reported to have a clinicopathologic/prognostic significance in UCB patients and involved in tumor aggressiveness in our recent study. However, its oncogenic role in UCB aggressiveness and underlying molecular mechanisms is.unclear. In the present study, we investigated whether Silence of RNF2 by shRNAs or enforced overexpression of RNF2 in UCB cells is important for cell growth,invasive and/or metastatic capacity. Using a Human Tumor Metastasis PCR Array containing cell metastasis-related genes and Immunohistochemistry, we determine the potential downstream targets regulated by RNF2 is NR4A3. Moreover, the critical downstream target of RNF2 will be determined by rescue experiments. We then plan to confirm whether this epigenetically transcription suppressing of RNF2 and/or its PRC1 subunits (RING1A and Bmi1) is mediated by Snail and/or Runx1, so as to examine underling molecular mechanism that links the RNF2 to the tumor suppressor gene. Furthermore, we plan to identify the key negative regulator of.the RNF2 using bioinformatics analyses and further validated its role for RNF2 overexpression in UCB tissues and cells.

膀胱癌（UCB）的发生发展是一个多基因改变的复杂过程。最近，我们发现多梳蛋白复合体1重要组分RNF2表达上调与UCB侵袭转移和预后密切相关。但RNF2在UCB侵袭转移中的作用和机理还不清楚。本项目拟首先探讨RNA干扰和外源性过表达RNF2对UCB细胞侵袭转移生物学行为的影响；通过Real-time PCR芯片检测和免疫组化筛选出RNF2作用的靶基因为NR4A3，进一步，在基因表达回复实验中验证NR4A3为RNF2作用的关键肿瘤抑制基因；应用ChIP和荧光素酶报告基因等实验，探讨和确证RNF2及其复合物成员（RING1A和Bmi1）是否通过转录因子Snail或Runx1募集并沉默NR4A3；IP实验分析它们之间的结合与作用方式。综合分析并确定RNF2沉默NR4A3的分子机制。同时利用生物信息学方法，分析可导致RNF2在UCB中上调的关键因子，并分别在组织和细胞中确证其介导RNF2过表达的作用

膀胱癌（UCB）转移复发是目前膀胱癌的治疗难点。前期实验结果证明RNF2的表达升高与膀胱癌患者的不良预后密切相关，通过本课题资助，我们探索了RNF2基因在UCB侵袭转移中的作用和功能，RNF2的表达水平与UCB细胞上皮-间质转化密切相关，其作用机制是RNF2调控下游关键靶基因NR4A3，这种调控机制依赖于转录因子Snail。进一步，RNF2和NR4A3的蛋白表达水平可以联合作为分子标记物来预测膀胱癌患者的不良预后。此外，我们还初步探索了膀胱癌的免疫微环境及与膀胱癌患者预后的关系；发现中心体蛋白72过度表达是诊断UCB的一个可靠分子标记物，而且与UCB细胞侵袭转移水平密切相关；探究了抑癌基因Nkx2.8在膀胱癌细胞侵袭转移中的作用和机制。本课题的完成将对全面认识UCB转移复发机制，促进UCB防治工作提供部分实验研究的依据。