核糖修饰小激活RNA分子治疗膀胱癌的体内实验研究

Bladder cancer is the most common tumor of urinary system in China.With the continuous improvement of living standards,bladder cancer incidence and mortality trends among our Chinses is rising.Finding a new technology to treat this disease is imperative.Small RNA molecules are now being recognized as master regulators of gene activity. Although known initially for their gene-silencing functions, recent discoveries have revealed that small RNAs can also activate gene expression.Some studies recently demonstrated that gene transcription can be stimulated by transfecting cells with small double-stranded RNA (dsRNA) constructs designed to target gene promoter sequences.Thus,these small activating RNAs (saRNAs) result in RNA-induced gene activation (RNAa). These studies established that the transcription of a number of genes is enhanced by saRNAs, including those encoding E-cadherin, p21,vascular endothelial growth factor (VEGF) and progesterone receptor(PR).For the past few years, researchers have been stalled by the challenge of delivering small RNA molecules in vivo due to its instability and nuclease easily degradation.The chemical modifications can protect the small double-stranded RNA against degradation by nucleases, minimize the off-target effects, increase the potency of the knockdown, and avoid the triggering of immune responses that normally protect against viruses.Ribose modification is the most common mode of chemical modifications. The study includes were first screen the robise modified saRNAs whose effect is not weakened, and to assess the in vitro serum stability.To evaluate the tumor inhibition effect of robise modified saRNAs by xenograft model of bladder cancer and screen the best way to modify saRNAs.Further to evaluate the drug metabolism characteristics of robise modified saRNAs compare to non-modified saRNAs.These kinds of design in saRNAs molecules will improved their physicochemical and pharmaceutical properties and shown great expectation to overcome the barriers to RNAa based therapies.

膀胱移行细胞癌是我国泌尿系统最常见的恶性肿瘤，利用新手段新方法进行膀胱癌治疗的研究十分必要。本人所参加的研究组自2007 年初起一直致力于RNA 诱导的基因激活机制及其在泌尿系肿瘤治疗中应用的研究。本研究的目标是核糖修饰小激活RNA 分子（saRNAs）全身给药治疗膀胱癌的体内方案。研究内容包括首先筛选出不影响激活效应的核糖修饰saRNAs,并评估其体外血清稳定性；应用体内试验评价核糖修饰saRNAs 对裸鼠异种移植膀胱癌模型的肿瘤生长及转移抑制效果，筛选出抗肿瘤作用最强的核糖修饰saRNAs 分子；进一步评估该核糖修饰saRNAs 相对于未修饰saRNAs 在体内的药物代谢学特点。本研究将为RNAs 激活技术的进一步临床研究提供理论基础。

本基金项目基于小RNA激活理论，对已经证实的具有激活功效的两条saRNA dsP21-322 和dsEcad-215进行核糖修饰，探讨不同核糖修饰对saRNAs 激活功效的影响，从中筛选出能够加强saRNA激活效能的核糖修饰方法。通过体内实验，证明该核糖修饰saRNA对膀胱肿瘤生长的抑制作用。针对dsP21-322，我们设计了5对不同核糖修饰saRNAs 分子，分别为正义链2’-OMe修饰，反义链2’-OMe修饰，正义链2’-F修饰，反义链2’-F修饰，双链2’-F修饰，研究结果发现，反义链全嘧啶2’F修饰dsP21-322-2’F相对于未修饰dsP21-322，能够增强其对p21的激活效应，并且能增强其对膀胱癌细胞的抑制作用，并能增强其在血清中的稳定性，而其他4种核糖修饰与未修饰dsP21-322相比，p21的激活效应均有不同程度的降低。针对dsEcad-215，我们设计了同样的5种核糖修饰，但研究结果显示，所有的核糖修饰与未修饰dsEcad-215相比，E-cadherin的表达水平均有不同程度的降低。通过建立裸鼠皮下异种移植瘤的模型，探讨了dsP21-322-2’F 的体内抗肿瘤作用。我们的研究发现，与未修饰dsP21-322相比，dsP21-322-2’F 对膀胱移植瘤的生长抑制作用明显增强，在肿瘤组织中P21的mRNA和蛋白表达量均明显升高，肿瘤组织中微血管密度降低，凋亡相关Bcl-2家族蛋白发生变化，其中Bcl-2、Bcl-xL表达降低，Bad表达升高，提示dsP21-322-2’F抑制肿瘤生长可能与抑制肿瘤血管生成、诱导肿瘤细胞凋亡相关。本研究证明反义链2’-F全嘧啶修饰对于saRNAs是一种可行有效的修饰方法，为今后saRNA的核糖修饰提供参考指导；也为小RNA激活技术对于膀胱恶性肿瘤的临床治疗提供了理论基础。