miR-99a调节靶基因PLK1和EIF2C2表达影响膀胱癌生物学行为的分子机制研究

Bladder cancer is one of urinary tract malignant tumors in China and it has high mortality. It is specially important and urgent to find new therapeutic targets. Applicant's preliminary study found that there was the lower miR-99a expression in bladder cancer than that in normal bladder tissue. Bioinformatics software was used to predict that PLK1 and EIF2C2 should be the target genes of miR-99a. It was detected that PLK1 and EIF2C2 had more highly expressed in bladder cancer than those in normal tissue. The expressions of PLK1 and EIF2C2 increased with the higher degree of malignant bladder cancer. Further experiments about inhibition and overexpression of miR-99a combined with the Pearson correlation analysis showed that the miR-99a expression was negatively correlated with the expression of PLK1 and EIF2C2. On this basis, the subject will be thoroughly exploring the molecular mechanism of miR-99a in bladder cancer progression. Applicants plan to observe the changes of biological characteristics of bladder cancer cells through in vitro intervention of miR-99a expression, revealing the role of miR-99a in the progression of bladder cancer. Biological functions and animal experiments were used to confirm miR-99a regulating target genes EIF2C2, PLK1 and downstream proteins of PLK1. It was analyzed that the relationship of the proteins and the change of biological characteristics of bladder cancer cells. It will clarify the role of miR-99a in molecular mechanism of bladder cancer progression and provide new ideas for targeted therapy of bladder cancer.

膀胱癌是我国泌尿系恶性肿瘤之一，死亡率高。寻找新的治疗靶点，具有特殊的重要性和迫切性。申请者前期研究发现miR-99a在膀胱癌中较正常组织中低表达，通过生物信息学软件预测miR-99a两个重要的靶基因PLK1和EIF2C2，并检测PLK1和EIF2C2在膀胱癌中较正常组织中高表达，随着膀胱癌恶性度增加而增加。进一步抑制和过表达miR-99a实验结合Pearson相关分析显示miR-99a表达与PLK1和EIF2C2的表达负相关。在此基础上，申请者拟通过体外干预miR-99a表达观察膀胱癌细胞生物学特性的改变，揭示miR-99a在膀胱癌进展中的作用；通过生物学功能实验和动物实验证实miR-99a对靶基因EIF2C2和PLK1及其下游蛋白的调控作用，分析它们与膀胱癌细胞生物学特性的改变关系，阐明miR-99a在膀胱癌进展中的分子作用机理，为膀胱癌靶向治疗提供新的思路。

膀胱癌是我国泌尿系恶性肿瘤之一，死亡率高。寻找新的治疗靶点，具有特殊的重要性和迫切性。申请者前期研究发现miR-99a在膀胱癌中较正常组织中低表达，通过生物信息学软件预测miR-99a两个重要的靶基因PLK1和EIF2C2，并检测PLK1和EIF2C2在膀胱癌中较正常组织中高表达，随着膀胱癌恶性度增加而增加。进一步抑制和过表达miR-99a实验结合Pearson相关分析显示miR-99a表达与PLK1和EIF2C2的表达负相关。.经过系统实验研究发现：1.在正常膀胱组织中miR-99a的表达比膀胱癌组织中的表达水平高，且随着肿瘤分期增高miR-99a的表达有显著下降。2.miR-99a在膀胱上皮细胞和膀胱癌细胞中均有表达，在正常膀胱癌上皮SV-HUC-1细胞中表达高于在膀胱癌5637，T24和UM-UC-3中表达水平。在SV-HUC-1中转染miR-99a inhibitor后细胞增殖能力增强。在5637，T24和UM-UC-3中转染miR-99a mimics后细胞增殖能力降低、侵袭和转移能力也降低。3.使用荧光素报告酶检测系统，通过共转染靶基因野生型/突变型荧光素酶报告载体和miR-99a mimics/ inhibitor，检测荧光素酶活性强度，证实PLK1和EIF2C2是miR-99a的靶基因；然后观察转染miR-99a inhibitor后，PLK1和EIF2C2表达变化、周期相关蛋白CDC25B和Wee1活性。4.构建裸鼠膀胱癌动物模型， 通过裸鼠接种miR-99a mimics转染的膀胱癌细胞实验，发现肿瘤组织变小PLK1和EIF2C2表达降低、周期相关蛋白CDC25B和Wee1活性降低；构建裸鼠膀胱癌原位模型，使用脂质体-miR-99a和慢病毒-miR-99a分别膀胱内灌注，观察膀胱癌肿瘤体积减小，肿瘤组织增殖能力降低。.通过该项研究明确miR-99a对在膀胱癌的发生发展中起抑制作用，证明miR-99a负性调控靶基因EIF2C2和PLK1及其下游蛋白CDC25B、Wee1表达及活性，miR-99a负性调控膀胱癌的细胞增殖、侵袭转移能力。体内验证miR-99a负性调控PLK1和EIF2C2影响膀胱癌进展，并发现miR-99a膀胱灌注在膀胱癌治疗中发挥一定作用。在国内外期刊中投稿6篇文章，在国际SCI源期刊上已发表论文3篇，协助培养硕士和博士生3人