RNA联合干扰STAT3及Survivin对膀胱癌细胞的抑制作用研究

发布时间：2018-08-22 20:47

【摘要】：研究背景：膀胱癌是指发生在膀胱粘膜上的恶性肿瘤。在西方，膀胱癌发病率仅次于前列腺癌，居第2位；在我国，膀胱癌是十大常见肿瘤之一，是泌尿系统发病率和死亡率双第一的恶性肿瘤。膀胱癌可发生于任何年龄，甚至于儿童，2012年全国肿瘤登记地区膀胱癌的发病率为6.61/10万，列恶性肿瘤发病率的第9位。男性发病率为女性的3～4倍，2013年男性膀胱癌发病率为10/10万，列恶性肿瘤的第六位。膀胱癌具有多灶性、多基因参与、多阶段异质性生长、高发病率、高复发率等恶性生物学特性，治疗难度大，膀胱癌目前的治疗方法主要包括手术、放射性治疗和术后膀胱腔内灌注抗癌药物，该方法对患者损伤大，病人生活质量严重下降，而且该方法无法解决高复发率（60%以上）和高耐药性的难题。在这种情况下，小干扰RNA基因靶向疗法引起了科研人员的极大关注。 RNA干扰(RNA interference)是一种转录后的基因沉默，RNA经切割后，触发某种转录后监控程序，识别有同源序列的mRNA，对其产生特异性切割，从而阻断其翻译功能，该方法能够从基因水平上抑制癌基因的过度表达，却不干扰正常细胞的生理活动。利用siRNA进行特异性基因抑制己被证实在病毒感染性疾病及肿瘤治疗中具有极大的潜力，是极有希望根治膀胱癌的最新方法。基因治疗的关键是靶点，我们选择了STAT3和Survivin作为实验对象。 STAT3是肿瘤多条信号传导通路的连接点与调控环节，它在多种肿瘤中持续性激活且高量表达，并全程参与了肿瘤的侵袭转移、血 III管发生、凋亡抵抗和免疫逃避等过程，是已知的最重要的癌基因之一。 Survivin是凋亡抑制蛋白家族的新成员，它通过多个途径抑制效应性蛋白酶，从而促进转化细胞的异常增殖。Survivin在所有常见恶性肿瘤中表达，在正常组织中无表达。作为一种广泛的肿瘤抗原，，Survivin可以作为抗肿瘤免疫治疗的一个广泛适用的靶基因。目的：有越来越多的证据表明，信号转导和转录激活因子3（STAT3）和凋亡抑制基因Survivin在膀胱癌的发生发展中起到了重要作用，但是沉默这两种基因对膀胱癌细胞T24的影响是未知的。因此，我们观察在体内、体外沉默STAT3和Survivin基因后，T24细胞的生长情况，为膀胱癌基因治疗的提供理论依据和靶点。方法： 1网络在线设计两组小干扰RNA，分别靶向抑制STAT3、Survivin两种基因，生物信息学方法验证siRNA的特异性和有效性； 2采用Invitrogen试剂盒制备表达载体，瞬时转染T24细胞； 3用实时荧光定量PCR检测转染前后STAT3和Survivin的基因表达情况；Western blot比较转染前后表达水平的差异；MTT实验测定细胞的增殖及细胞活性； 4将两条干扰效果最好的siRNA串联建立复合干扰RNA，用上述方法检验干扰效果； 5建立裸鼠膀胱癌移植瘤模型，测定干扰对肿瘤体积和重量的影响，以分析siRNA的体内干扰效果。数据均用SPSS软件分析v17.0（IBM，阿蒙克，NY，USA）。两组间比较使用t检验。采用单向方差分析确定多组间统计学意义（方差分析）。数据均值±标准偏差（SD）。P＜0.05被认为有统计学意义。结果： 1.成功构建并合成了四个STAT3siRNA和四个Survivin siRNA。 2.四个siRNA中的三个显著降低了STAT3的表达，其中STAT3-3是最有效的STAT3抑制剂。所有的Survivin siRNA有效的下调Survivin表达，核酸与蛋白含量检测结果表明，Survivin-4是最有效的Survivin抑制剂。而且，质粒STAT3-3和Survivin-4分别能显著降低STAT3和Survivin的表达，两者串联的复合干扰RNA也能显著降低STAT3和Survivin的表达，三者在核酸和细胞水平上的干扰效果没有显著差异。 3.体内检测结果表明，上述质粒干扰显著降低裸鼠膀胱癌移植瘤模型的肿瘤体积和重量，差异有统计学意义。结论：研究结果表明，我们设计的siRNA能有效抑制STAT3和Survivin基因在T24细胞中的表达，从而抑制膀胱癌细胞T24的增殖。将两种siRNA串联在同一质粒中，抑制效果同样明显，为基因药物的研制探索了新的途径。同时抑制这两种基因不能显著提高在T24细胞中的抗癌效果，这表明它们可能在T24细胞同一信号传导通路发挥作用。这些结果为研究膀胱癌的发生发展提供了宝贵的材料，也为膀胱癌临床基因治疗指明了靶向目标。
[Abstract]:Research background:
Bladder cancer is a malignant tumor that occurs on the bladder mucosa. In the west, the incidence of bladder cancer is second only to prostate cancer, ranking second; in our country, bladder cancer is one of the ten common tumors, is the first malignant tumor in the incidence and mortality of the urinary system. Bladder cancer can occur at any age, even in children, in 2012 the national cancer. The incidence of bladder cancer was 6.61/100,000, ranking ninth in the incidence of malignant tumors. Male incidence was 3-4 times that of females. Male incidence of bladder cancer was 10/100,000 in 2013, ranking sixth in the incidence of malignant tumors.
Bladder cancer has many malignant biological characteristics, such as multi-focal, multi-gene participation, multi-stage heterogeneous growth, high incidence, high recurrence rate, and so on. The treatment of bladder cancer is very difficult. Current treatment methods mainly include surgery, radiotherapy and postoperative intravesical instillation of anticancer drugs. This method is harmful to patients, and the quality of life of patients is seriously reduced. In this case, small interfering RNA gene targeting therapy has attracted great attention of researchers.
RNA interference is a kind of gene silencing after transcription. After RNA is cut, it triggers a post-transcriptional monitoring program, identifies the mRNA with homologous sequence and produces specific cleavage to it, thus blocking its translation function. This method can inhibit the overexpression of oncogenes at the gene level, but does not interfere with the physiological activities of normal cells. Specific gene suppression with siRNA has been demonstrated to have great potential in the treatment of viral infectious diseases and tumors, and is a promising new method for radical treatment of bladder cancer.
The key of gene therapy is target. We chose STAT3 and Survivin as experimental objects.
STAT3 is the junction and regulation link of multiple signal transduction pathways in tumors. It is persistently activated and overexpressed in various tumors and participates in the invasion and metastasis of tumors.
III tube formation, apoptosis resistance and immune escape are one of the most important oncogenes known.
Survivin is a new member of the inhibitor of apoptosis protein family. It inhibits effector proteases through multiple pathways and promotes the abnormal proliferation of transformed cells. Survivin is expressed in all common malignant tumors but not in normal tissues. As a wide range of tumor antigens, Survivin can be used as a wide range of anti-tumor immunotherapy. Suitable target genes.
Objective:
There is increasing evidence that signal transduction and activator of transcription 3 (STAT3) and apoptosis suppressor gene Survivin play an important role in the development of bladder cancer, but the effect of silencing these two genes on bladder cancer cell T24 is unknown. The growth condition provides theoretical basis and target for gene therapy of bladder cancer.
Method:
1. Two groups of small interfering RNA were designed on-line, targeting STAT3 and Survivin, respectively. Bioinformatics methods were used to verify the specificity and effectiveness of siRNA.
2 the expression vector was prepared by Invitrogen kit and transiently transfected into T24 cells.
The expression of STAT 3 and Survivin was detected by real-time fluorescence quantitative PCR, the expression of STAT 3 and Survivin was compared by Western blot, and the proliferation and cell viability were measured by MTT assay.
4 establish two interference siRNA with the best interference in series to establish Compound Jamming RNA.
5 To establish a nude mouse model of bladder cancer xenograft and determine the effect of interference on tumor volume and weight in order to analyze the effect of siRNA interference in vivo.
The data were analyzed with SPSS software v17.0 (IBM, Amund, NY, USA). The t test was used for comparison between the two groups. The one-way ANOVA was used to determine the statistical significance among the groups (ANOVA). The data mean (+) standard deviation (SD). P < 0.05 was considered statistically significant.
Result:
1. four STAT3siRNA and four Survivin siRNA. were successfully constructed and synthesized.
2. Three of the four siRNA significantly reduced the expression of STAT3, of which STAT3-3 was the most effective STAT3 inhibitor. All of the survivivin siRNA effectively down-regulated the expression of Survivin. Nucleic acid and protein assays showed that Survivin-4 was the most effective Survivin inhibitor. Moreover, plasmid STAT3-3 and Survivin-4 significantly decreased the expression of STAT3 and Surivin-4, respectively. The expression of vivin and the expression of STAT3 and Survivin were also significantly decreased by the combined interfering RNA in series. There was no significant difference between the interfering effect of the three on nucleic acid and cell level.
3. The results of in vivo detection showed that the above plasmid interference significantly reduced the tumor volume and weight of nude mice bladder cancer xenograft tumor model, the difference was statistically significant.
Conclusion:
The results showed that the siRNA designed by us could effectively inhibit the expression of STAT3 and Survivin genes in T24 cells and thus inhibit the proliferation of bladder cancer cell line T24. The inhibition effect of two siRNAs in the same plasmid was also obvious, which explored a new way for the development of gene drugs. These results provide valuable materials for the study of the genesis and development of bladder cancer and point out the target of gene therapy for bladder cancer.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.14

【相似文献】