Rab25在卵巢癌中的表达及其基因沉默诱导卵巢癌细胞凋亡的实验研究
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摘要
卵巢癌由于发病率不断上升,而治疗效果提高缓慢,目前已跃居为妇科第一位的致死疾病。尽管手术技巧的提高可以做到最大程度的肿瘤减灭,而铂类、紫杉醇类以及众多二线化疗药物的临床应用给化学治疗带来多重生机,但是卵巢癌患者的5年生存率仍然停滞不前。因此众多学者致力于寻求新的治疗途径和靶点。
Rab蛋白家族是Ras超家族中最大的亚家族,为小GTP结合蛋白。动物的体内体外实验都发现Rab GTPases在调节细胞间的囊泡运输的分子开关中起极其关键的作用。目前越来越多证据表明在多重人类疾病包括癌症中Rab small GTPases和它们相关调节蛋白及受动器发生改变,这些改变与众多疾病的发生发展密切相关。Rab25是近年新发现的Rab家族的蛋白,国外有研究报道Rab25的高表达参与卵巢癌的发病,与疾病的发展及不良预后密切相关。因此,有学者提出对Rab25的深入研究有望成为卵巢癌治疗的新途径。
随着分子生物学各项技术的日趋成熟,基因治疗作为一种可特异性阻断或调节致病基因表达的方法,成为卵巢癌治疗的新途径。而RNA干涉(RNAi)技术阻断基因表达具有高稳定、高效率和高特异的特点,目前为肿瘤基因治疗的新手段。
研究目的
本实验主要研究Rab25在卵巢癌组织中的表达,并观察RNA干涉技术对Rab25的抑制情况并探讨其可能的作用机制。
研究方法
第一部分Rab25在卵巢癌中的表达及意义
本实验采用免疫组织化学方法,检测了Rab25在正常卵巢组织、卵巢良性组织、卵巢恶性组织中的表达情况,分析其表达与临床分期、病理分级、组织学分型和转移的相关性。
第二部分运用RNAi特异阻断Rab25在卵巢癌中基因表达的研究及作用途径的探讨
本研究运用RNA(iRNA interference,RNA干涉)方法,构建Rab25 siRNA表达载体,借助脂质体转染卵巢癌细胞,封闭细胞中Rab25的表达,并观察其生物学行为的变化情况及发生变化的作用途径。
主要实验方法:
实验一、Rab25 siRNA转染的卵巢癌细胞株的建立
利用RNAi技术,成功构建其Rab25 siRNA表达载体pSUPER/Rab25 siRNA并进行酶切鉴定及DNA全长测序。转染A2780细胞,G418筛选稳定转染的细胞系。RT-PCR、间接免疫荧光法及Western blot分别检测Rab25的mRNA和蛋白水平的变化。
实验二、Rab25表达与卵巢癌细胞生物学行为的关系研究
在卵巢癌细胞中封闭Rab25表达后,我们观察了包括细胞增殖与凋亡,侵袭粘附以及形态学等方面的细胞生物学行为的变化情况。
1. Rab25 siRNA对卵巢癌细胞形态学的影响
以倒置显微镜及透视电镜观察贴壁状态下的各组细胞,比较细胞形态的差异,尤其是树突及胞体的大小及形态变化和典型的凋亡细胞。
2. Rab25 siRNA对卵巢癌细胞增殖与凋亡的影响
各转染组细胞及未转染正常卵巢癌细胞分别行MTT比色试验,依据测定的A490值,绘制生长曲线;流式细胞仪测定各组细胞凋亡率和细胞周期的变化;TUNEL(TdT介导的dUTP缺口末端标记)法检测卵巢癌细胞凋亡指数等。
3. Rab25 siRNA对卵巢癌细胞侵袭与粘附的影响
利用细胞基质粘附实验检测各组细胞间的粘附能力;利用transwell小室实验及划痕实验检测各组细胞的侵袭能力。
4.卵巢癌裸鼠移植瘤模型的建立及Rab25 siRNA对卵巢癌裸鼠移植瘤的作用
使用BALB/c遗传背景的雌性裸小鼠作为实验动物,4- 6周龄。于每只裸鼠背部皮下注射106 ~ 2×106个细胞,建立卵巢癌裸鼠皮下移植瘤模型。观察30-45d处死裸鼠,观察裸鼠成瘤情况。
实验三、Rab25作用途径的初步探讨
利用免疫组织化学、RT-PCR、Western blot方法检测各组细胞中Bcl-2及Bax的表达,探讨Rab25的作用途径。
研究结果
1. Rab25蛋白在卵巢交界性肿瘤与恶性肿瘤中呈高水平表达,而在卵巢良性肿瘤及正常卵巢组织中仅少量表达或无表达,差异显著(P<0.05)。Rab25的表达与卵巢癌有无转移及临床分期密切相关(P<0.05)。
2.通过双酶切鉴定和测序验证,成功克隆了Rab25基因的siRNA表达载体,并通过基因转染实现了siRNA在卵巢癌细胞内的持续表达。RT-PCR和间接免疫荧光法及Western blot结果表明,siRNA明显抑制了Rab25基因的表达;光镜观察细胞形态的结果进一步佐证了Rab25被封闭以后,细胞发生了生物学活性及形态学改变;在透视电镜下见到典型的凋亡细胞;通过MTT、流式细胞仪、TUNEL法等方法研究了Rab25 siRNA对卵巢癌细胞增殖与凋亡的影响,结果显示Rab25 siRNA可以明显抑制卵巢癌细胞的增殖,促进凋亡;通过侵袭和粘附实验结果表明Rab25 siRNA可以明显抑制卵巢癌细胞的侵袭粘附能力;成功建立裸小鼠移植瘤模型,观察30-45d后,发现转染了Rab25 siRNA的细胞体内成瘤能力显著下降。RT-PCR、免疫组织化学方法、Western blot方法从mRNA到蛋白水平检测Bcl-2和Bax表达,结果显示,与对照组相比,Bcl-2蛋白的表达水平显著降低,而Bax蛋白的表达水平明显增强。
结论
1. Rab25在卵巢癌中呈高表达,且Rab25的表达随卵巢癌分期的升高而增高。
2. Rab25的高表达参与了卵巢癌的转移。
3. Rab25 siRNA有效阻断卵巢癌细胞中Rab25 mRNA及蛋白水平的表达并影响细胞的生长增殖,促进凋亡,抑制细胞的侵袭粘附能力,抑制肿瘤生长。
4.Rab25 siRNA对卵巢癌的生长及其侵袭能力的抑制是通过启动卵巢癌细胞程序化死亡信号通路而实现的。
Ovarian cancer is a leading cause of death among gynecologic malignancies. In spite of advances in surgery and chemotherapy, its mortality rate remains relatively unchanged during recent several decade. The current optimum approach to therapy consists of cytoreductive surgery followed by combination chemotherapy. Clinical trial have established that carboplatin plus a taxane(usually paclitaxel) can be considered to be the treatment of choice for meet patients with advanced disease. Most patients will achieve a clinical complete remission with such a combination. However the median time to progression is less than 2 years, and for patients with optimal stage III disease, median survival will be approxi- mately 5 years. Therefore, new adjunctive therapy is of great importance.
Rab proteins are Ras-like small GTPases, which have crucial roles in vesicle trafficking, signal transduction, and receptor recycling which in turn regulate normal cellular activity. Recent studies have shown multiple links between Rab GTPase dysfunction and associated regulatory proteins in human diseases including cancer, and these changes are closely correlation with development of numerous diseases. The study have recently shown that Rab25, located at chromosome 1q22, is amplified at the DNA level and overexpressed at the RNA level in ovarian and breast cancer. These changes correlated with a worsened outcome in both diseases. In addition, enforced expression of Rab25 in both breast and ovarian cancer cells decreased apoptosis and increased proliferation and aggressiveness in vivo, potentially explaining the worsened prognosis. So, a better understanding of genetic alterations as well as the physiologic and pathophysiologic roles of Rab GTPases may open new opportunities for therapeutic intervention and better outcomes.
With molecular biology technology gradually mature, gene therapy as a way that specificity block or regulate virulence gene is becoming new therapeutic way of ovarian cancer. RNA interference(RNAi) technology may be a optimized means with following important features: High stability, High efficiency, High specifi- city. Hence, it may become a new means of tumor gene therapy.
Objective
To investigate the expression of Rab25 in ovarian cancer tissue and the inhibition of Rab25 gene expression by RNA interference technology, discuss the mechanism of action of Rab25 on ovarian cancer.
Methods
Part One The expression and significant of Rab25 in ovarian malignant tumor
Immunohistochemical method was employed to detect the expression of Rab25 in normal ovary, carcinoid and carcinoma ovarian tissues, then analyzed with regard to clinical stage, pathological, histology type and metastatic.
Part Two The study of inhibiting the expression of Rab25 gene in ovarian cancer by RNA interference and pathway of action.
Construction of Rab25 siRNA eukaryotic expression vectors and the confirmation on the transfected cells. The Rab25 siRNA eukaryotic expression vectors pSUPER /Rab25 siRNA was constructed by Using RNAi technology. The direction was confirmed by endonuclease digestion. Using lipidosome method, the Rab25 highly expressed human malignant ovarian cells (A2780) were transfected with the siRNA recombinant vector. Stable clones cells were obtained after G418 screening. To observe the biologic behavior of ovarian cancer cells.
一、Construction of Rab25 siRNA eukaryotic expression vectors and the confirmation on the transfected cells
According to Rab25 mRNA sequence in the Genebank,a pair of 64-nt oligonucleotides, each containing the sites of restriction endonuclease at both ends, were designed and synthesized. Oligonucleotides were annealed and ligated with linearized pSUPER by T4DNA ligase. The recombinants (named pSUPER /Rab25 siRNA ) were finally sequenced and identified by enzyme cutting and sequencing. RT-PCR assay、indirect immunofluore- scence and Western blot studies testified that the protein and mRNA level.
二、The role of Rab25 Expression in ovarian cancer cells
Knockdown the expression of Rab25 in ovarian cancer cells, we observed the change of cells proliferation and apoptosis, invasion and adhesion, morphology and so on.
1. Morphological observations of Rab25 siRNA in ovarian cancer cells Inverted microscope and electron microscope were used to observe changes in cell morphology, especially dendron, cell body and typical apoptotic cell.
2. Effect on the proliferation and apoptosis of Rab25 siRNA in ovarian cancer cells
MTT was performed to test effectdof different ovarian cancer cells, OD readings were obtained using an autoreader at 490 nm, draw Growth curve; Flow cytometry analysis was used to determine apoptosis and cell cycle of the cells; Apoptosis index of ovarian cancer cells was tested by TUNEL.
3. Effect on the invasion and adhesion of Rab25 siRNA in ovarian cancer cells
To detect the abiliry of adhesion among ovarian cancer cells by cell-adhering method; to detect the ability of invading in vitro by transwell-ECM method and wound-healing method.
4. The erecting of nude mice model of transplanted ovarian cancer celland the effect on the model of Rab25 siRNA
We erected the model on subcutaneously implanted tumor in nude mice. 106 ~ 2×106 cells were injected subcutaneously into 4- to 6-week-old athymic female nude mice. The mice were kept in pathogen-free environments and checked every 2 days for about 1-1.5 months. The date at which grossly visible tumor first appeared and the size of the tumor were recorded. The mice were killed when tumors reached 2.5 cm in diameter.
三、The initial study of mechanism of action on Rab25
To detect the expression of Bcl-2 and Bax by immunohistochemistery、RT-PCR、Western blot, and to investigate the pathway of Rab25.
Reults
1. Rab25 protein evels were high in ovarian borderline epithelial tumors and carcinoma, and it is low in normal ovary and benign tumors, the difference was significant(P<0.05). The expression of Rab25 was correlated with lymph node metastases and TNM stages. It suggested that the high level of Rab25 mignt play an important role in the carcinogenesis, development and prognosis of malignant tumors.
2. Rab25 siRNA expression vector was successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesized siRNA oligonucleotides, and achieved continuous siRNA expression in A2780 cells by gene transfection. The result of RT-PCR, indirect immunofluorescence and Western blot showed that, the efficiency of inhibition was related to target selecting. Under light microscope, they displayed large epithelioid perikaryon and stubby dendrites with occasional multidendricity as opposed to slender perikaryon and bipolar dendrites of control cells, and under electron microscope we saw typical apoptosis cells. The alteration in cell morphology suggested abnormal metabolization in Rab25 siRNA transfected cells. Clone-forming ability decreased, the proliferation of Rab25 siRNA transfected cells were inhibited compared with the control cells by using MTT assay. Compared with the controls, Rab25 siRNA transfected cells were retarded in G1 stage and the percent of cells in G1 stage increased significantly. In order to further confirm the apoptosis, flow cytometer detection was used. After the double stain of PI and annexin V FITC, more apoptosis cells could be seen in the siRNA transfected cell group. The difference of annexin V FITC positive cells between the Rab25 siRNA transfected cell groups and the control cells were significant. Statistics showed a significant growth suppression of Rab25 siRNA vector transfected cells. TUNEL showed apoptosis cells obviously. The A2780 cells that transfected Rab25 siRNA vector displayed descended invasiveness, adhesion and metastasis ability in vitro in comparison with the control cells by cell-adhering and transwell-ECM method. Nude mice model with implanted tumor were built successfully, after observeing 30-45 days, the tumors of Rab25 siRNA vector transfected cells groups was much more smaller than those of the control groups. This suggested that Rab25 siRNA suppressed tumor formation in vivo in nude mice xenografts. The results showed that compared with control groups, from mRNA level to protein level, the expression of Bcl-2 significant decreased; the expression of Bax significant increased by RT-PCR、immunohistochemistry and Western blot detection, These show that Rab25 induce cell apoptosis by regulating the level of Bcl-2/Bax.
Conclusion
1. In malignant tumors, the expression of Rab25 protein was high by using immunohistochemical methods, and the expression of Rab25 was correlated with TNM stages.
2. The high expression of Rab25 protein participated in metastasis of ovarian cancer.
3. Rab25 siRNA can interfere with the protein and mRNA expression of Rab25 in ovarian cancer cells and inhibit their growth and proliferation, enhance apoptosis, inhibit the ability of invasion and adhesion, inhibit tumor growth.
4.Rab25 siRNA can inhibit growth and invasion of ovarian cancer by induce cell apoptosis signal passway.
Rab蛋白家族是Ras超家族中最大的亚家族,为小GTP结合蛋白。动物的体内体外实验都发现Rab GTPases在调节细胞间的囊泡运输的分子开关中起极其关键的作用。目前越来越多证据表明在多重人类疾病包括癌症中Rab small GTPases和它们相关调节蛋白及受动器发生改变,这些改变与众多疾病的发生发展密切相关。Rab25是近年新发现的Rab家族的蛋白,国外有研究报道Rab25的高表达参与卵巢癌的发病,与疾病的发展及不良预后密切相关。因此,有学者提出对Rab25的深入研究有望成为卵巢癌治疗的新途径。
随着分子生物学各项技术的日趋成熟,基因治疗作为一种可特异性阻断或调节致病基因表达的方法,成为卵巢癌治疗的新途径。而RNA干涉(RNAi)技术阻断基因表达具有高稳定、高效率和高特异的特点,目前为肿瘤基因治疗的新手段。
研究目的
本实验主要研究Rab25在卵巢癌组织中的表达,并观察RNA干涉技术对Rab25的抑制情况并探讨其可能的作用机制。
研究方法
第一部分Rab25在卵巢癌中的表达及意义
本实验采用免疫组织化学方法,检测了Rab25在正常卵巢组织、卵巢良性组织、卵巢恶性组织中的表达情况,分析其表达与临床分期、病理分级、组织学分型和转移的相关性。
第二部分运用RNAi特异阻断Rab25在卵巢癌中基因表达的研究及作用途径的探讨
本研究运用RNA(iRNA interference,RNA干涉)方法,构建Rab25 siRNA表达载体,借助脂质体转染卵巢癌细胞,封闭细胞中Rab25的表达,并观察其生物学行为的变化情况及发生变化的作用途径。
主要实验方法:
实验一、Rab25 siRNA转染的卵巢癌细胞株的建立
利用RNAi技术,成功构建其Rab25 siRNA表达载体pSUPER/Rab25 siRNA并进行酶切鉴定及DNA全长测序。转染A2780细胞,G418筛选稳定转染的细胞系。RT-PCR、间接免疫荧光法及Western blot分别检测Rab25的mRNA和蛋白水平的变化。
实验二、Rab25表达与卵巢癌细胞生物学行为的关系研究
在卵巢癌细胞中封闭Rab25表达后,我们观察了包括细胞增殖与凋亡,侵袭粘附以及形态学等方面的细胞生物学行为的变化情况。
1. Rab25 siRNA对卵巢癌细胞形态学的影响
以倒置显微镜及透视电镜观察贴壁状态下的各组细胞,比较细胞形态的差异,尤其是树突及胞体的大小及形态变化和典型的凋亡细胞。
2. Rab25 siRNA对卵巢癌细胞增殖与凋亡的影响
各转染组细胞及未转染正常卵巢癌细胞分别行MTT比色试验,依据测定的A490值,绘制生长曲线;流式细胞仪测定各组细胞凋亡率和细胞周期的变化;TUNEL(TdT介导的dUTP缺口末端标记)法检测卵巢癌细胞凋亡指数等。
3. Rab25 siRNA对卵巢癌细胞侵袭与粘附的影响
利用细胞基质粘附实验检测各组细胞间的粘附能力;利用transwell小室实验及划痕实验检测各组细胞的侵袭能力。
4.卵巢癌裸鼠移植瘤模型的建立及Rab25 siRNA对卵巢癌裸鼠移植瘤的作用
使用BALB/c遗传背景的雌性裸小鼠作为实验动物,4- 6周龄。于每只裸鼠背部皮下注射106 ~ 2×106个细胞,建立卵巢癌裸鼠皮下移植瘤模型。观察30-45d处死裸鼠,观察裸鼠成瘤情况。
实验三、Rab25作用途径的初步探讨
利用免疫组织化学、RT-PCR、Western blot方法检测各组细胞中Bcl-2及Bax的表达,探讨Rab25的作用途径。
研究结果
1. Rab25蛋白在卵巢交界性肿瘤与恶性肿瘤中呈高水平表达,而在卵巢良性肿瘤及正常卵巢组织中仅少量表达或无表达,差异显著(P<0.05)。Rab25的表达与卵巢癌有无转移及临床分期密切相关(P<0.05)。
2.通过双酶切鉴定和测序验证,成功克隆了Rab25基因的siRNA表达载体,并通过基因转染实现了siRNA在卵巢癌细胞内的持续表达。RT-PCR和间接免疫荧光法及Western blot结果表明,siRNA明显抑制了Rab25基因的表达;光镜观察细胞形态的结果进一步佐证了Rab25被封闭以后,细胞发生了生物学活性及形态学改变;在透视电镜下见到典型的凋亡细胞;通过MTT、流式细胞仪、TUNEL法等方法研究了Rab25 siRNA对卵巢癌细胞增殖与凋亡的影响,结果显示Rab25 siRNA可以明显抑制卵巢癌细胞的增殖,促进凋亡;通过侵袭和粘附实验结果表明Rab25 siRNA可以明显抑制卵巢癌细胞的侵袭粘附能力;成功建立裸小鼠移植瘤模型,观察30-45d后,发现转染了Rab25 siRNA的细胞体内成瘤能力显著下降。RT-PCR、免疫组织化学方法、Western blot方法从mRNA到蛋白水平检测Bcl-2和Bax表达,结果显示,与对照组相比,Bcl-2蛋白的表达水平显著降低,而Bax蛋白的表达水平明显增强。
结论
1. Rab25在卵巢癌中呈高表达,且Rab25的表达随卵巢癌分期的升高而增高。
2. Rab25的高表达参与了卵巢癌的转移。
3. Rab25 siRNA有效阻断卵巢癌细胞中Rab25 mRNA及蛋白水平的表达并影响细胞的生长增殖,促进凋亡,抑制细胞的侵袭粘附能力,抑制肿瘤生长。
4.Rab25 siRNA对卵巢癌的生长及其侵袭能力的抑制是通过启动卵巢癌细胞程序化死亡信号通路而实现的。
Ovarian cancer is a leading cause of death among gynecologic malignancies. In spite of advances in surgery and chemotherapy, its mortality rate remains relatively unchanged during recent several decade. The current optimum approach to therapy consists of cytoreductive surgery followed by combination chemotherapy. Clinical trial have established that carboplatin plus a taxane(usually paclitaxel) can be considered to be the treatment of choice for meet patients with advanced disease. Most patients will achieve a clinical complete remission with such a combination. However the median time to progression is less than 2 years, and for patients with optimal stage III disease, median survival will be approxi- mately 5 years. Therefore, new adjunctive therapy is of great importance.
Rab proteins are Ras-like small GTPases, which have crucial roles in vesicle trafficking, signal transduction, and receptor recycling which in turn regulate normal cellular activity. Recent studies have shown multiple links between Rab GTPase dysfunction and associated regulatory proteins in human diseases including cancer, and these changes are closely correlation with development of numerous diseases. The study have recently shown that Rab25, located at chromosome 1q22, is amplified at the DNA level and overexpressed at the RNA level in ovarian and breast cancer. These changes correlated with a worsened outcome in both diseases. In addition, enforced expression of Rab25 in both breast and ovarian cancer cells decreased apoptosis and increased proliferation and aggressiveness in vivo, potentially explaining the worsened prognosis. So, a better understanding of genetic alterations as well as the physiologic and pathophysiologic roles of Rab GTPases may open new opportunities for therapeutic intervention and better outcomes.
With molecular biology technology gradually mature, gene therapy as a way that specificity block or regulate virulence gene is becoming new therapeutic way of ovarian cancer. RNA interference(RNAi) technology may be a optimized means with following important features: High stability, High efficiency, High specifi- city. Hence, it may become a new means of tumor gene therapy.
Objective
To investigate the expression of Rab25 in ovarian cancer tissue and the inhibition of Rab25 gene expression by RNA interference technology, discuss the mechanism of action of Rab25 on ovarian cancer.
Methods
Part One The expression and significant of Rab25 in ovarian malignant tumor
Immunohistochemical method was employed to detect the expression of Rab25 in normal ovary, carcinoid and carcinoma ovarian tissues, then analyzed with regard to clinical stage, pathological, histology type and metastatic.
Part Two The study of inhibiting the expression of Rab25 gene in ovarian cancer by RNA interference and pathway of action.
Construction of Rab25 siRNA eukaryotic expression vectors and the confirmation on the transfected cells. The Rab25 siRNA eukaryotic expression vectors pSUPER /Rab25 siRNA was constructed by Using RNAi technology. The direction was confirmed by endonuclease digestion. Using lipidosome method, the Rab25 highly expressed human malignant ovarian cells (A2780) were transfected with the siRNA recombinant vector. Stable clones cells were obtained after G418 screening. To observe the biologic behavior of ovarian cancer cells.
一、Construction of Rab25 siRNA eukaryotic expression vectors and the confirmation on the transfected cells
According to Rab25 mRNA sequence in the Genebank,a pair of 64-nt oligonucleotides, each containing the sites of restriction endonuclease at both ends, were designed and synthesized. Oligonucleotides were annealed and ligated with linearized pSUPER by T4DNA ligase. The recombinants (named pSUPER /Rab25 siRNA ) were finally sequenced and identified by enzyme cutting and sequencing. RT-PCR assay、indirect immunofluore- scence and Western blot studies testified that the protein and mRNA level.
二、The role of Rab25 Expression in ovarian cancer cells
Knockdown the expression of Rab25 in ovarian cancer cells, we observed the change of cells proliferation and apoptosis, invasion and adhesion, morphology and so on.
1. Morphological observations of Rab25 siRNA in ovarian cancer cells Inverted microscope and electron microscope were used to observe changes in cell morphology, especially dendron, cell body and typical apoptotic cell.
2. Effect on the proliferation and apoptosis of Rab25 siRNA in ovarian cancer cells
MTT was performed to test effectdof different ovarian cancer cells, OD readings were obtained using an autoreader at 490 nm, draw Growth curve; Flow cytometry analysis was used to determine apoptosis and cell cycle of the cells; Apoptosis index of ovarian cancer cells was tested by TUNEL.
3. Effect on the invasion and adhesion of Rab25 siRNA in ovarian cancer cells
To detect the abiliry of adhesion among ovarian cancer cells by cell-adhering method; to detect the ability of invading in vitro by transwell-ECM method and wound-healing method.
4. The erecting of nude mice model of transplanted ovarian cancer celland the effect on the model of Rab25 siRNA
We erected the model on subcutaneously implanted tumor in nude mice. 106 ~ 2×106 cells were injected subcutaneously into 4- to 6-week-old athymic female nude mice. The mice were kept in pathogen-free environments and checked every 2 days for about 1-1.5 months. The date at which grossly visible tumor first appeared and the size of the tumor were recorded. The mice were killed when tumors reached 2.5 cm in diameter.
三、The initial study of mechanism of action on Rab25
To detect the expression of Bcl-2 and Bax by immunohistochemistery、RT-PCR、Western blot, and to investigate the pathway of Rab25.
Reults
1. Rab25 protein evels were high in ovarian borderline epithelial tumors and carcinoma, and it is low in normal ovary and benign tumors, the difference was significant(P<0.05). The expression of Rab25 was correlated with lymph node metastases and TNM stages. It suggested that the high level of Rab25 mignt play an important role in the carcinogenesis, development and prognosis of malignant tumors.
2. Rab25 siRNA expression vector was successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesized siRNA oligonucleotides, and achieved continuous siRNA expression in A2780 cells by gene transfection. The result of RT-PCR, indirect immunofluorescence and Western blot showed that, the efficiency of inhibition was related to target selecting. Under light microscope, they displayed large epithelioid perikaryon and stubby dendrites with occasional multidendricity as opposed to slender perikaryon and bipolar dendrites of control cells, and under electron microscope we saw typical apoptosis cells. The alteration in cell morphology suggested abnormal metabolization in Rab25 siRNA transfected cells. Clone-forming ability decreased, the proliferation of Rab25 siRNA transfected cells were inhibited compared with the control cells by using MTT assay. Compared with the controls, Rab25 siRNA transfected cells were retarded in G1 stage and the percent of cells in G1 stage increased significantly. In order to further confirm the apoptosis, flow cytometer detection was used. After the double stain of PI and annexin V FITC, more apoptosis cells could be seen in the siRNA transfected cell group. The difference of annexin V FITC positive cells between the Rab25 siRNA transfected cell groups and the control cells were significant. Statistics showed a significant growth suppression of Rab25 siRNA vector transfected cells. TUNEL showed apoptosis cells obviously. The A2780 cells that transfected Rab25 siRNA vector displayed descended invasiveness, adhesion and metastasis ability in vitro in comparison with the control cells by cell-adhering and transwell-ECM method. Nude mice model with implanted tumor were built successfully, after observeing 30-45 days, the tumors of Rab25 siRNA vector transfected cells groups was much more smaller than those of the control groups. This suggested that Rab25 siRNA suppressed tumor formation in vivo in nude mice xenografts. The results showed that compared with control groups, from mRNA level to protein level, the expression of Bcl-2 significant decreased; the expression of Bax significant increased by RT-PCR、immunohistochemistry and Western blot detection, These show that Rab25 induce cell apoptosis by regulating the level of Bcl-2/Bax.
Conclusion
1. In malignant tumors, the expression of Rab25 protein was high by using immunohistochemical methods, and the expression of Rab25 was correlated with TNM stages.
2. The high expression of Rab25 protein participated in metastasis of ovarian cancer.
3. Rab25 siRNA can interfere with the protein and mRNA expression of Rab25 in ovarian cancer cells and inhibit their growth and proliferation, enhance apoptosis, inhibit the ability of invasion and adhesion, inhibit tumor growth.
4.Rab25 siRNA can inhibit growth and invasion of ovarian cancer by induce cell apoptosis signal passway.
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