Twist基因在膀胱癌中表达与RNA干扰的实验研究
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摘要
膀胱肿瘤是泌尿外科最常见的恶性肿瘤,其临床特点主要是高发病率、高复发率和低度恶性。细胞凋亡是机体重要的生理过程,也是机体维持细胞群体数量稳定的重要机制。肿瘤是一类细胞周期失控性疾病,细胞凋亡和增殖失去平衡是肿瘤产生和发展的主要原因之一,也是肿瘤细胞的基本特征。Twist是一个在进化过程中高度保守的转录因子,Twist蛋白被认为是一种癌蛋白,能影响许多肿瘤细胞的凋亡、侵袭和转移。RNA干扰是一项新兴的高效、特异、安全、可操作性强的基因阻断技术。它的发现改变了人们对细胞基因调控的传统理解,RNA干扰技术在对基因功能研究、信号转导基因上下游关系确认以及抗病毒、抗肿瘤治疗中有着广阔的应用前景。本研究应用免疫组化法探讨了Twist在膀胱癌组织中的表达与临床病理特征之间的关系,成功构建了靶向Twist基因的shRNA质粒表达载体,并筛选出其中抑制效果最为明显的shRNA质粒表达载体。用RNAi技术沉默膀胱癌T24细胞Twist基因的表达,观察其对人膀胱癌细胞生物学行为的影响。
实验结果提示Twist在人膀胱癌组织中高表达,并与肿瘤的病理分级、临床分期有显著相关性。Twist是一个膀胱癌临床辅助诊断的分子标志物,并极可能是膀胱癌的一个潜在的治疗靶点,具有广阔的临床应用价值。人膀胱癌细胞系T24中Twist高表达,可以用作Twist基因沉默研究的靶细胞。RNAi沉默Twist基因后,明显地改变了人膀胱癌细胞生物学行为,表现细胞增殖能力降低,细胞凋亡的增加,细胞迁移速度明显减低。Twist基因干扰还可以明显提高膀胱癌T24细胞对化疗药物的敏感性,这将有助于减少化疗药物的毒副作用,增加化疗药物的疗效,对减少膀胱癌的复发起到重要作用。
本研究提示:针对Twist基因及其表达产物的检测,将有助于判断膀胱癌的恶性程度,指导膀胱癌治疗;RNAi技术可以做为沉默膀胱癌中Twist基因的有效方法;而靶向Twist的基因治疗,可能为膀胱癌的治疗提供新的策略。
Bladder cancer is the most common tumors of genitourinary tract. The most important clinical character of it is high incidence rate ,high recurrence rate ,and low malignancy. In recent years, molecule mechanisms about bladder cancer is always the hot point, such as gene mutation , protooncogene activation , antioncogene inactivation , apoptosis disequilibrium and so on.The tumor is the result of unlimited cell proliferation, and cell growth is determined by genes.The tumor genes and the tumor suppressor genes exist in the human body, the functions of these two genes are different.The occurrence of tumor is due to increase tumor gene function and inhibit tumor suppressor gene function.A variety of external factors such as radiation, carcinogenic chemicals and viruses induced tumor through the changes of gene.Bladder cancer biological characteristics of malignant include multifocal, multi-gene involvement and heterogeneous multi-stage growth.At present, the treatments of bladder cancer include bladder surgery and anti-cancer drug instillation after surgery.However, easy to relapse after surgery and the height of the chemotherapy drug resistance have been the focus of the study and clinical issues.After nearly 20 years of development,gene therapy has been considered to be following the surgery, chemotherapy and other conventional treatment a very promising way to cure bladder cancer.
Twist gene is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Twist could activate many kinds of tumor related genes, and it has close correlation with tumor generation, development and prognosis. Epithelial mesenchymal transformation( EMT) refers to the epithelial cells into mesenchymal cells in a specific physiological and pathological circumstances.EMT plays important roles in embryonic development, chronic inflammation, tumor progression and a variety of fibrotic diseases.Twist is a important transcription factor in the process of EMT .Twist protein is considered to be a cancer protein, can affect the number of tumor cells apoptosis, invasion and metastasis.Twist maybe a new target in gene therapy.
The present study observed the Twist protein expression in human bladder cancer tissues using immunohistochemical methods and analyzed the correlation between Twist protein expression and clinical pathological characters. Using RNA interference technique silencing Twist gene, the effects and its relative mechanism of Twist shRNA on the human bladder cancer cell line T24 cells was observed. The present study was divided into three parts. The follows are:
1 Expressions of Twist gene in bladder carcinoma and its clinicopathological significance
Objective: To investigate the expressions of Twist gene in bladder carcinoma and its clinicopathological significance. Methods: By immunohistochemical assay (SP), the expression of Twist gene was done in 5 cases of normal tissues and 95 cases of bladder carcinoma tissues. The results were analyzed by corresponding statistica methods. Results: Twist gene showed higher expression in bladder carcinoma. The rate of positive expression was 87.4% , while the positive rate was 20% in nomal tissues. The difference was of significance between the two tissues (P<0.05). The expressions of Twist gene was correlated with WHO pathology classification and TNM stage (P<0.05), and was incorrelated with age and sex. Conclusions: The expression of Twist is up-regulated in bladder carcinoma tissues , and it plays an important role in progression of tumor.
2 Construction and screening of plasmid expression vectors encoding the short hairpin RNA targeting Twist gene
Objective: To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting Twist mRNA. Methods: Two plasmid expression vectors coding for shRNA targeting 777 and 845 of Twist gene sequence and a control vector containing random DNA fragment were constructed. The recombinant plasmids were identified by PCR, and then transfected separately into bladder cancer cell line-T24. The Twist gene silencing effect was detected by RT-PCR and Western blotting. Results: The expected band was amplified from the plasmids coding for shRNA by PCR. Transfection of T24 cells expressing Twist gene with the shRNA plasmids resulted in a inhibition of Twist mRNA and protein expressions by 68% and 76%, respectively. Conclusion: The plasmid expression vectors coding for shRNA targeting Twist mRNA have been constructed successfully,of which pGenesil-shRNA1 most effectively silences Twist gene in T24 cells.
3 Effect of silencing Twist gene expression by RNA interference on proliferation of human bladder carcinoma T24 cell line
Objective: To investigate whether Twist gene downregulation by RNA interference (RNAi) leads to inhibition of proliferation and arrest of cell cycle in human bladder carcinoma T24 cell line. Methods: The shRNA expression plasmid targeting to Twist gene was constructed and transfected into bladder cancer T24 cell line with Lipofectamin2000. RT-PCR and Western Blot was used to monitor the validity of specific shRNA in downregulation of Twist expression. Then the MTT assay was performed for detecting cell proliferation and PI flow cytometric analysis for cell cycle. Results: The specific Twist shRNA was confirmed to be efficient in silencing Twist expression. Twist gene downregulation by RNAi inhibited cell proliferation remarkably( P < 0. 05) and arrested cell cycle at G1 phase significantly( P < 0. 05). There was an increase of cell number at G1 phase [ (74.34±3.24) % vs (48. 17±1.62) % , P < 0. 05 ] and a decrease of S phase [ (11.58±1.02) % vs (33.71±3.54) % , P < 0. 05 ] in T24 cells treated with Twist shRNA compared with untreated T24 cells. Conclusions: Twist specific shRNA expression vector could specifically and efficiently inhibit the expression of Twist gene to regulate cell cycle and inhibit cell proliferation in human bladder carcinoma T24 cell line.
In summary, Twist gene expression products might be the auxiliary diagnosis way in assessing bladder cancer malignance and invasion ability, which might direct the bladder cancer operation and further treatment. The present study showed that Twist shRNA plasmids could silence Twist gene effectively and RNAi gene therapy targeting Twist might offer the new direction for treatment bladder cancer.
实验结果提示Twist在人膀胱癌组织中高表达,并与肿瘤的病理分级、临床分期有显著相关性。Twist是一个膀胱癌临床辅助诊断的分子标志物,并极可能是膀胱癌的一个潜在的治疗靶点,具有广阔的临床应用价值。人膀胱癌细胞系T24中Twist高表达,可以用作Twist基因沉默研究的靶细胞。RNAi沉默Twist基因后,明显地改变了人膀胱癌细胞生物学行为,表现细胞增殖能力降低,细胞凋亡的增加,细胞迁移速度明显减低。Twist基因干扰还可以明显提高膀胱癌T24细胞对化疗药物的敏感性,这将有助于减少化疗药物的毒副作用,增加化疗药物的疗效,对减少膀胱癌的复发起到重要作用。
本研究提示:针对Twist基因及其表达产物的检测,将有助于判断膀胱癌的恶性程度,指导膀胱癌治疗;RNAi技术可以做为沉默膀胱癌中Twist基因的有效方法;而靶向Twist的基因治疗,可能为膀胱癌的治疗提供新的策略。
Bladder cancer is the most common tumors of genitourinary tract. The most important clinical character of it is high incidence rate ,high recurrence rate ,and low malignancy. In recent years, molecule mechanisms about bladder cancer is always the hot point, such as gene mutation , protooncogene activation , antioncogene inactivation , apoptosis disequilibrium and so on.The tumor is the result of unlimited cell proliferation, and cell growth is determined by genes.The tumor genes and the tumor suppressor genes exist in the human body, the functions of these two genes are different.The occurrence of tumor is due to increase tumor gene function and inhibit tumor suppressor gene function.A variety of external factors such as radiation, carcinogenic chemicals and viruses induced tumor through the changes of gene.Bladder cancer biological characteristics of malignant include multifocal, multi-gene involvement and heterogeneous multi-stage growth.At present, the treatments of bladder cancer include bladder surgery and anti-cancer drug instillation after surgery.However, easy to relapse after surgery and the height of the chemotherapy drug resistance have been the focus of the study and clinical issues.After nearly 20 years of development,gene therapy has been considered to be following the surgery, chemotherapy and other conventional treatment a very promising way to cure bladder cancer.
Twist gene is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Twist could activate many kinds of tumor related genes, and it has close correlation with tumor generation, development and prognosis. Epithelial mesenchymal transformation( EMT) refers to the epithelial cells into mesenchymal cells in a specific physiological and pathological circumstances.EMT plays important roles in embryonic development, chronic inflammation, tumor progression and a variety of fibrotic diseases.Twist is a important transcription factor in the process of EMT .Twist protein is considered to be a cancer protein, can affect the number of tumor cells apoptosis, invasion and metastasis.Twist maybe a new target in gene therapy.
The present study observed the Twist protein expression in human bladder cancer tissues using immunohistochemical methods and analyzed the correlation between Twist protein expression and clinical pathological characters. Using RNA interference technique silencing Twist gene, the effects and its relative mechanism of Twist shRNA on the human bladder cancer cell line T24 cells was observed. The present study was divided into three parts. The follows are:
1 Expressions of Twist gene in bladder carcinoma and its clinicopathological significance
Objective: To investigate the expressions of Twist gene in bladder carcinoma and its clinicopathological significance. Methods: By immunohistochemical assay (SP), the expression of Twist gene was done in 5 cases of normal tissues and 95 cases of bladder carcinoma tissues. The results were analyzed by corresponding statistica methods. Results: Twist gene showed higher expression in bladder carcinoma. The rate of positive expression was 87.4% , while the positive rate was 20% in nomal tissues. The difference was of significance between the two tissues (P<0.05). The expressions of Twist gene was correlated with WHO pathology classification and TNM stage (P<0.05), and was incorrelated with age and sex. Conclusions: The expression of Twist is up-regulated in bladder carcinoma tissues , and it plays an important role in progression of tumor.
2 Construction and screening of plasmid expression vectors encoding the short hairpin RNA targeting Twist gene
Objective: To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting Twist mRNA. Methods: Two plasmid expression vectors coding for shRNA targeting 777 and 845 of Twist gene sequence and a control vector containing random DNA fragment were constructed. The recombinant plasmids were identified by PCR, and then transfected separately into bladder cancer cell line-T24. The Twist gene silencing effect was detected by RT-PCR and Western blotting. Results: The expected band was amplified from the plasmids coding for shRNA by PCR. Transfection of T24 cells expressing Twist gene with the shRNA plasmids resulted in a inhibition of Twist mRNA and protein expressions by 68% and 76%, respectively. Conclusion: The plasmid expression vectors coding for shRNA targeting Twist mRNA have been constructed successfully,of which pGenesil-shRNA1 most effectively silences Twist gene in T24 cells.
3 Effect of silencing Twist gene expression by RNA interference on proliferation of human bladder carcinoma T24 cell line
Objective: To investigate whether Twist gene downregulation by RNA interference (RNAi) leads to inhibition of proliferation and arrest of cell cycle in human bladder carcinoma T24 cell line. Methods: The shRNA expression plasmid targeting to Twist gene was constructed and transfected into bladder cancer T24 cell line with Lipofectamin2000. RT-PCR and Western Blot was used to monitor the validity of specific shRNA in downregulation of Twist expression. Then the MTT assay was performed for detecting cell proliferation and PI flow cytometric analysis for cell cycle. Results: The specific Twist shRNA was confirmed to be efficient in silencing Twist expression. Twist gene downregulation by RNAi inhibited cell proliferation remarkably( P < 0. 05) and arrested cell cycle at G1 phase significantly( P < 0. 05). There was an increase of cell number at G1 phase [ (74.34±3.24) % vs (48. 17±1.62) % , P < 0. 05 ] and a decrease of S phase [ (11.58±1.02) % vs (33.71±3.54) % , P < 0. 05 ] in T24 cells treated with Twist shRNA compared with untreated T24 cells. Conclusions: Twist specific shRNA expression vector could specifically and efficiently inhibit the expression of Twist gene to regulate cell cycle and inhibit cell proliferation in human bladder carcinoma T24 cell line.
In summary, Twist gene expression products might be the auxiliary diagnosis way in assessing bladder cancer malignance and invasion ability, which might direct the bladder cancer operation and further treatment. The present study showed that Twist shRNA plasmids could silence Twist gene effectively and RNAi gene therapy targeting Twist might offer the new direction for treatment bladder cancer.
引文
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