【作者】 刘兴涛；

【导师】 张钦宪； 丁一；

【作者基本信息】 郑州大学 ， 人体解剖学与组织胚胎学， 2005， 硕士

【摘要】 胃癌是我国最常见的恶性肿瘤之一,具有发病率高、发病隐匿、易转移和死亡率高等特点。目前治疗方法主要有手术、化疗、放疗等,疗效均并不能令人满意;目前我国胃癌患者治疗后5年存活率仅为20—30%。癌基因的激活是肿瘤发病的根本原因之一,以抑制癌基因过度表达为重要内容的基因治疗成为新世纪人类攻克恶性肿瘤(包括胃癌)的重要研究方向。 RNA干扰(RNA interference,RNAi)是将双链RNA(double strand RNA, dsRNAl导入细胞内引起同源mRNA降解的一种细胞反应过程。DsRNA进入细胞后被特异性dsRNA核酸酶(Dicer)切割成多个21-25bp的短干扰RNA(short interfering RNAs,siRNAs),siRNAs与一些核酸酶结合形成RNA诱导的沉默复合体(RNA-induced silencing complex,RISC),由RISC介导同源mRNA的降解,属于转录后基因沉默(post-transcriptional gene silencing,PTGS)的一种。短发夹状RNA(short hairpin RNA,shRNA)由短的正义链、反义链(21-25bp)和连接它们的数个碱基组成,因其形似发夹而得名。shRNA在胞质内也被Dicer切割成siRNAs,发挥对靶基因的抑制作用。shRNA可自身合成双链,其化学稳定性较siRNAs更高。另可直接在体外大量合成,易于筛选有效序列,为构建RNAi重组载体奠定基础。自从1998年被发现以来,作为有力工具,RNAi技术已被用于大肠癌、食管癌、肝癌、卵巢癌等多种人类肿瘤的研究,但应用其研究胃癌少见报道。 Wnt信号通路是细胞内重要的信号传导途径,该通路的高激活状态与多种人类肿瘤密切相关。β-catenin基因编码的β-连环蛋白是该通路的关键阳性调节因子。在正常情况下,细胞内肿瘤抑制因子APC蛋白、β-连环蛋白和GSK-3β等组成蛋白复合体,β-连环蛋白可在复合体内被磷酸化降解。APC或β-catenin基因的更多还原

【Abstract】 As one of the most common malignancies in China, gastric carcinoma is characterized by high incidence 、 nonspecific appearance、 easily metastasis and high death rate .The treatments include surgery、 chemotherapy、 radiotherapy and so on, but the therapeutic effect is unsatisfactory. The 5-year survival is only 25-30%. With the development of the cancer molecular research, people discovered that oncogenes’ high expression is one of the base reasons which can facilitate carcinomatosis. To suppress the oncogenes expression is one of the important methods for cancer therapy. Gene therapies might have huge potential to cure the prevalence (including gastric carcinoma) in the new century.RNA interference (RNAi) is the process of sequence-specific posttranscriptional gene silencing (PTGS) triggered by double stranded RNA (dsRNA). The dsRNA introduced or generated in cells is subjected to digestion with a dsRNA-specific endonuclease, Dicer, into 21-25 nucleotide (nt) RNA duplexes, and the resultant duplexes, referred to as short interfering RNAs (siRNAs) duplexes, function as essential sequence specific mediators of RNAi in the RNA-induced silencing complexes (RISC). These reactions result in the cleavage of the target mRNA. As one form of RNAi, Short hairpin RNA (shRNA) is composed of both sense and antisense sequences with a few bases named as loop between them. When input into cells, shRNA can be cleavaged into siRNAs by Dicer too. ShRNA can develop to double strands itself and keep high stability in the cytoplasm. Moreover, a large of shRNA can be synthesized in vitro andcontribute to screen effective sequences to construct the recombination vector. Since discovered in 1998, RNAi had been developed as an important tool for the study of the tumor therapy. Now RNAi has been successfully used for many human tumors study, including colon cancer > oesophagus cancer^ liver cancer^ ovary cancer et al. Little research in the field of gastric cancer using this technique has been reported yet.Wnt pathway is one of the important signaling ways in cells. People believe that its high activity is associated with many malignancies. The P -catenin play a key role in Wnt pathway. In normal cells, a multiprotein complex which includes the adenomatous polyposis coli tumor suppressor protein APC, P -catenin, and the serine/threonine kinase GSK-3 P , targets beta-catenin for proteosomal destruction. The mutation of the APC and P -catenin gene often results in the accumulation of P -catenin in the cytoplasm. Over there the 3 -catenin associates with members of the TCF/lymphocyte enhancer factor family and migrates into the nucleus as a transcriptional activator to activate the transcription of a variety of target genes including c-myc n cyclin Dl and so on. Ultimately the increased P -catenin level leads to the abnormal cellular proliferation and malignancies. It is estimated that approximately 60% human tumors (including gastric cancer) exists high expression of P -catenin, which indicates that P -catenin might act as a key factor in maintaining the malignant phenotype. The specific inhibition of P -catenin may contribute to better understanding its role in the gastric cancers and cell lines.In this study, RNA interference (RNAi) was used to inhibit P -catenin expression in gastric cancer BGC-823 cells. The cellular growth was observed after the treatment. The results were used to investigate the inhibition effect of RNAi and the function of the target gene in gastric cancer. Our study would provide the laboratory evidence for the future gene therapy of gastric carcinoma using RNAi. Materials and Methods1. The culture of the gastric carcinoma BGC-823 cells: The BGC-823 cells were supplemented with 10% fetal bovine serum, lOOunits/ml penicillin andstreptomycin. The cells were maintained with 37°C and 5%CO2.2. Synthesis of the shRNA: The DNA templates were synthesized firstly, which included T7 RNA Polymerase promoter that could bind to the T7 RNA polymerase to transcript the target shRNA. The synthesized shRNAs were 49 nt and named Rin R2 #J Ro respectively. 2% gel electrophoresis was used to detect the length and concentration of the synthesized shRNAs.3. Cellular grouping: The cultured BGC-823 cells were divided into treat groups(Ri and R2) and control groups(Ci and C2 ), treated by Ri, R2> Ro and empty vector respectively. The Ci and C2 group were the experiment and negative control group respectively.4. Transfection: CodeBreaker? siRNA Transfection Reagent was used to Transfect the shRNAs into BGC-823 cells.5. The detection after transfection: RT-PCR > the MTT test and plate colony assays were used to detect the inhibition effect. The RT-PCR was used to detect the expression level of the target gene. The MTT test and plate colony assays were used to detect the cellular proliferation after transfection.6. Statistical analysis: Biostatistic analyses were done by SPSS 11.0 software package. Data from the experiment was analyzed by ANOVA. a=0.05 was the remarkable standard.Results1. ShRNAs were synthesized successfully in vitro and their lengths were 49bp.2. RT-PCR results: The P -catenin/ P -actin ratio of Ri> R2 ^ Q and C2 group were 72% > 73% ^ 90% > 93% respectively at 24h after transfection; the values of treatment groups were dramatically lower than that of the control (p < 0. 001); no significant difference was found between Ri and R2,or Q and C2 (p > 0. 05) .The expression of P -catenin at 48 and 72 h could hardly be observed; the statistic results were similar with that at 24h. The results indicated that the synthesized shRNAs could markedly inhibit the expression of P -catenin in gastric cancer BGC-823 cells.3. MTT results: The inhibition rates of Ri and R2 group after transfection were remarkably higher than the control (p<0.001). The results indicated that cancer cells growth could be inhibited by the shRNAs. The inhibition rates of Ri and R2 groups increased with time, which revealed that inhibition could keep some time. No significant difference could be found between Ri and R2,or Q and C2 (p>0.05 ) .4. Plate cloning assays results: The clone inhibition rates of Ri> R2 on the 5th day after transfection were 20%n 21%. Comparing with 2% of the control Q, the values were obviously higher (P<0.001). No significant difference was found between Ri and R2(p>0.05). The clone inhibition rates of Ri> R2 and Q were 34<%^ 31% and 3% on the 10th day respectively; 46% % 47% ^ 4% were seen on the 14th day; The statistical analysis were similar with that on the 5th day. The experiment results indicated that when 3 -catenin expression was inhibited, the proliferation of cancer cells was suppressed too.Conclusions1. ShRNA can be synthesized successfully in vitro.2. ShRNA can be used to inhibit the expression of P -catenin successfully. Moreover, the cellular proliferation was suppressed after transfection. The results reveal that P -catenin gene might be an important biology guide which probably plays a keyrole in happen and development of the gastric cancer. The results also indicate that RNAi have huge potential in the gastric cancer therapy as a new technique.3. Two shRNAs synthesized in the study can successfully lower the level of P -catenin expression and can be used to construct the recombination vector to inhibit the target gene expression for longer time.更多还原