摘要
背景：
前列腺癌是全世界男性面临的重要的公共卫生问题。前列腺癌由激素依赖性进展为去势抵抗性,是导致肿瘤治疗失败、患者死亡的重要原因。小RNA诱导的基因激活是分子生物学领域新兴的话题,一系列研究显示特殊设计的小RNA能够在转录水平上调靶基因的表达。该方法为去势抵抗性前列腺癌(CRPC)的基因靶向治疗提供了全新的策略。
目的：
利用小激活RNA (saRNA)上调CRPC中缺陷基因INTS6的表达,在体内和体外两个水平研究INTS6对CRPC的生物学作用及机制。
方法：
1.利用免疫组化方法检测组织芯片中28对前列腺癌和瘤旁组织INTS6蛋白的表达情况。
2.针对INTS6基因启动子区,按照一定的原则设计saRNA.利用定量PCR、蛋白印迹以及细胞活力测定等方法,在CRPC细胞和正常上皮来源的细胞中挑选既能够上调INTS6表达,又对肿瘤细胞的活力具有一定专一性抑制作用的小RNA,并对RNA激活的时间、剂量、表遗传学和双链选择等特征进行鉴定。
3.利用流式细胞技术和Transewell实验,检测saRNA处理后CRPC细胞周期、凋亡、迁移和侵袭能力的变化。通过定量PCR、蛋白印迹和细胞免疫荧光等技术手段,评价saRNA诱导INTS6基因激活对Wnt信号通路的影响。
4.构建裸鼠的皮下荷PC3肿瘤模型,瘤内注射saRNA,观察在体水平saRNA对CRPC的抑制作用。
结果：
1.与瘤旁组织相比,前列腺癌组织中INTS6蛋白表达水平下降。
2.设计并筛选得到INTS6saRNA-915,该小RNA能够在转录水平上调CRPC细胞中INTS6基因的表达,并且表现出一定的肿瘤特异性抑制作用。saRNA对靶基因的激活作用出现于处理后第2天,在第4--5天达到高峰,20nM的处理浓度激活效果较强。saRNA作用后,靶基因启动子区出现相应的表遗传学修饰改变。saRNA任何一条单链结构或功能的丧失都会影响其对INTS6基因的激活作用。
3. saRNA作用于CRPC细胞后,抑制细胞的增殖能力,诱导细胞发生G0/G1期周期阻滞,同时抑制细胞的迁移和侵袭能力。saRNA在发挥上述作用的同时,细胞中P-catenin、cyclin D1、Fra-1和MET等Wrnt关键信号蛋白受到抑制,这种抑制作用依赖INTS6的正常表达。
4.成功构建裸鼠的皮下荷PC3肿瘤模型。瘤内给予脂质体包裹的INTS6saRNA,观察到移植瘤的生长受到明显抑制,同时检测到瘤组织中INTS6蛋白表达升高,增殖相关蛋白Ki67和PCNA表达下调。
结论：
针对前列腺癌中缺陷的抑癌基因基INTS6设计并导入激活RNA能够有效上调基因的表达,同时抑制肿瘤的增殖和运动能力。该方法为去势抵抗性前列腺癌的基因靶向治疗提供了新的实验依据。
Background
Prostate cancer becomes one of the most important public-health issue for male population in modern world. The progression of hormone dependent prostate cancer into castration resistant prostate cancer (CRPC) is the main cause of the treatment failure and the cancer related patient death. Small RNA induced gene activation is a notable topic in molecular biology field recently. A serial of researches demonstrated that specially designed small RNAs were potent to activate the targeted gene transcriptionally. This technique provides a notable strategy for the treatment of CRPC.
Objectives To activate the inefficient gene of INTS6in CRPC by small activating RNA(saRNA), and to study the function of INTS6in CRPC both in vitro and in vivo.
Method
1. INTS6protein level was assessed in28paired prostate cancer and adjacent prostate tissue groups by immunohistochemical staining.
2. SaRNAs, which target the INTS6promoter region, were designed according to the recent reports and our experience. Quantitative PCR, protein blot assay and cell viability assessment were used to identify the potency and specialty of designed saRNA in activating INTS6gene in CRPC cells and normal epithelial cells. The appropriate course and dose, the epigenetic modulation and the strand selection of saRNA were also assessed.
3. The cell cycle distribution, apoptosis, migration and invasion ability of CRPC cells after saRNA treatment were assessed by flow cytometry and transwell exam. The effect of saRNA induced INTS6activation in Wnt signal pathway was also assessed by quantitative PCR, protein blot assay and cyto-immunoflourescence assay.
4. Intratumoral injection of saRNA was performed on nude mice with PC3xenograft, and the tumor growth was assessed.
Results
1. The protein level of INTS6decreased in prostate cancer, when compared with adjacent prostate tissue.
2. One of designed saRNAs, INTS6saRNA-915, up-regulated the expression of INTS6in CRPC cells on the transcriptional level, with the inhibition ability specialized on cancer cells. The activation by saRNA appeared at the2nd day after treatment, and achieved its maximum effect around the4th to5th day. The treatment with the dose of20nM showed the strongest targeted gene activation. Epigenetic modulation was detected on the targeted promoter region after asRNA treatment. Structural or functional loss of either single strand would diminish the potency of targeted gene activation by saRNA.
3. Decrease of cell proliferation, migration and invasion ability was observed in saRNA treated CRPC cells, with a significant G0/G1phase arrest. The suppression of Wnt signal, such as β-catenin, cyclin D1, Fra-1and MET, were also observed in saRNA treated cells. This kind of suppression probably depended on the normal expression of INTS6.
4. Subcutaneous PC3xenograft was successfully establish in nude mice. Tumor growth was suppressed in INTS6saRNA treating group. Up-regulation of INTS6and down-regulation of Ki67and PCNA (proliferation related proteins) were detected in saRNA treating group.
Conclusions
Design and import of saRNA, which targets the inefficient gene INTS6in prostate cancer, is an effective way to active the gene expression and to inhibit the tumor proliferation ability and motility. All these evidences demonstrate a potential technique for the treatment of castration resistant prostate cancer.
引文
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