摘要
研究背景：
卵巢癌是妇科恶性肿瘤中死亡率最高的恶性肿瘤,在年龄介于55-74岁妇女所患的恶性肿瘤中,卵巢癌是第五位常见导致死亡的癌症。大多数卵巢癌患者被确诊时已属晚期,卵巢癌的治疗通常先进行手术治疗,然后再采用以铂类及紫杉烷为基础的联合化疗。晚期卵巢癌患者在经过理想的肿瘤细胞减灭术后,使其残余病灶的直径小于1厘米,再给予手术后的后续化疗,其中位生存时间也就刚刚超过4年。由于对化疗耐药卵巢癌细胞克隆的生长,几乎所有患者最终会出现卵巢癌的复发并随着疾病的进展而导致死亡。因此,发现新颖有效的治疗方法对卵巢癌的治疗是绝对有必要的。
血管内皮生长因子(VEGF)是一种强烈的促血管生长因子,它能增加血管内皮细胞的通透性,刺激血管内皮细胞的分裂及游走。VEGF已被认为在肿瘤的生长及侵袭方面起着重要作用,它已成为肿瘤治疗方面一个新出现的靶点。以往的研究已经证实卵巢癌细胞表达VEGF,近来研究显示卵巢癌患者血清中VEGF水平呈现显著地升高。VEGF参与了卵巢癌的进展和腹水形成,是一个重要的与卵巢癌浸润,转移和腹水形成相关的生长因子。已有报道把VEGF作为一种评估卵巢肿瘤标志物及预测卵巢癌患者预后的预测因子。
近来研究已经表明,在哺乳动物中小干扰RNA(siRNA)能通过对生物学上保守结构的RNA进行干扰,从而抑制相应靶基因表达。RNA干扰具有高度序列特异性,能使靶蛋白表达关闭。许多研究已经证实RNA干扰能阻断多种类型哺乳动物病毒复制,抑制癌肿生长。RNA干扰已经被开发成为反向遗传学的一个强有力的工具,并且已显示出广阔的治疗学上应用前景。
本研究中,我们对卵巢癌细胞系CaoV3通过siRNA对其VEGF的表达进行了基因沉默,并评估VEGF表达下调对细胞增殖、细胞调亡及浸润相关蛋白表达的影响。期望能为靶向VEGF的卵巢癌基因治疗提供体外实验依据。
第一部分VEGF靶向siRNA制备与转染及干扰效果检测
目的：
制备VEGF靶向siRNA并转染至CaoV3,明确VEGF靶向siRNA是否能特异及有效地沉默CaoV3细胞VEGF基因表达。
材料与方法：
细胞培养
人上皮性卵巢癌细胞系CaoV3购自ATCC公司(美国组织培养库)。细胞放置于含有10%胎牛血清加链霉素／青霉素的RPMI-1640培养液中培养,保持培养箱的温度在37℃,CO2浓度为5%,95%空气湿度。
siRNA制备与转染
VEGF特异性siRNA片段试剂盒BLOCK-iTTM Complete Dicer RNAi购自于美国Invitrogen公司,按制造商的说明制备siRNA。VEGF基因的引物为5'-GAA CTT TCT GCT GTC TTG G-3'(上游引物),和5'-TTT TCT TGT CTT GCT CTA TCT-3'(下游引物)；LacZ引物序列5'-ACCAGAAGCGGRGCCGGAAA-3'(上游引物),5'-CCACAGCGGATGGTTCGGAT-3'(下游引物)。将细胞置于含有30ng siRNA和5μl脂质体的2000转染试剂进行转染,经过48小时转染后,细胞被收获为下一个实验作准备。本实验中lacZ d-siRNA作为RNA干扰阴性对照组,不加d-siRNA作为RNA干扰空白对照组。定量逆转录多聚酶链反应(qRT-PCR)
当VEGF-siRNA进行48小时转染后,用实时荧光定量qRT-PCR对VEGF mRNA的表达进行检测,总RNA由购自美国公司的TRIzol试剂提取后,加入RNA酶游离胰脱氧核糖核酸酶Ⅰ,在37℃下孵育30分钟,然后加入20μl含有反转录酶和随机六聚物引物的反应混合物进行逆转录。
VEGF的引物与"siRNA制备与转染”中使用的引物相同,用Beta-actin作为内参,其扩增引物为：5'-GTG GAC ATC CGC AAA GAC-3'(上游引物)和5'-AAA GGG TGT AAC GCA ACT AA-3(下游引物)’。qRT-PCR采用Applied Biosystems 7900HT system with the SYBR(?) Premix Ex TaqTM kit (TaKaRa),按照制造商的说明书进行。
蛋白质印迹(Western Blot)
用于Western Blot检测VEGF蛋白的一抗为鼠抗人-VEGF单克隆抗体,(Santa Cruz,美国,sc-7269,1：500稀释),等量提取的蛋白装载到SDS/PAGE胶上并被分离后,再转移到PVDF膜上,然后用含有5%脱脂奶粉的TBST液封闭1小时,接着再用一抗在常温下孵育2小时,膜再用TBST液洗涤4次,然后再用抗鼠IgG-HRP(DAKO)在常温下孵育1小时,最后蛋白用ECL体系进行检测。蛋白相对表达量=待测蛋白的光密度值／内参的光密度值。
结果：
靶向siRNA下调CaoV3细胞株VEGF的表达
VEGF靶向siRNA与lacZ d-siRNA(阴性对照)瞬时转染CaoV3细胞48小时后,VEGF mRNA表达水平在空白对照、阴性对照与VEGF d-siRNA组分别为1.64±0.07,1.61±0.04和,0.48±0.02, VEGF靶向siRNA对VEGF mRNA表达的抑制率为70.02%(P=0.000),然而lacZ d-siRNA转染至CaoV3细胞对其VEGF mRNA的表达没有影响(P>0.05)。Western-blot结果进一步证实了这种表达减少。VEGF d-siRNA组CaoV3细胞VEGF蛋白的表达水平(0.31±0.07)与空白对照(0.82±0.08)、阴性对照组(0.81±0.09)相比显示了显著的减少,VEGF靶向siRNA对VEGF蛋白表达的抑制率为62.21%(P=0.000)。这些数据显示siRNA能在：mRNA及蛋白水平强力抑制CaoV3细胞VEGF的表达。
结论：
VEGF靶向siRNA在体外能特异性及有效地抑制CaoV3细胞VEGF mRNA及蛋白的表达。
第二部分siRNA靶向干扰VEGF对卵巢癌细胞增殖、凋亡及侵袭力的影响研究
目的：
明确siRNA靶向干扰VEGF能有效地抑制CaoV3细胞增殖,诱导细胞凋亡,降低细胞侵袭潜能。
材料与方法：
细胞培养
人上皮性卵巢癌细胞系CaoV3购自ATCC公司(美国组织培养库)。细胞放置于含有10%胎牛血清加链霉素／青霉素的1640培养液中培养,保持培养箱的温度在37℃,CO2浓度为5%,95%.空气湿度。
siRNA制备与转染
VEGF特异性siRNA片段试剂盒BLOCK-iTTM Complete Dicer RNAi购自于美国Invitrogen公司,按制造商的说明制备siRNA。VEGF基因的引物为5'-GAA CTT TCT GCT GTC TTG G-3'(上游引物),和5'-TTT TCT TGT CTT GCT CTA TCT-3'(下游引物)；LacZ引物序列5'-ACCAGAAGCGGRGCCGGAAA-3'(上游引物),5'-CCACAGCGGATGGTTCGGAT-3'(下游引物)。将细胞置于含有30ng siRNA和5μl脂质体的2000转染试剂进行转染,经过48小时转染后,细胞被收获为下一个实验作准备。本实验中lacZ d-siRNA作为RNA干扰阴性对照组,不加d-siRNA作为RNA干扰空白对照组。
细胞增殖测定
细胞增殖测定使用溴标法试剂盒(Roche Diagnostics),按照制造商的说明进行测定。简单地说,BrdU溶液加入到培养基中,2小时后弃去培养基,细胞用FixDenat溶液在常温下固定30分钟,然后弃去FixDenat液,细胞用抗-BrdU工作液孵育30分钟,最后免疫复合物由随后的底物反应探得。
凋亡检测
caspase-3的活性用CaspACETM Assay System kit(Colorimetric, Promega)测定,按照制造商的说明操作。将细胞置于98μl反应混合液中,再向其内加入半胱天冬酶底物。在常温下孵育4小时,吸光度由分光光度计在405nm波长处测定,然后计算caspase-3的比活性。再用TUNEL分析法对CaoV3凋亡情况进行了形态学上的测定,采用原位细胞死亡探测试剂盒,POD(Roche, Germany),按照制造商的说明进行测定。
蛋白质印迹(Western Blot)
用于Western Blot检测的一抗均为鼠抗人单克隆抗体,分别为抗-Survivin (Santa Cruz,美国,sc-17779,1：500稀释),抗-MMP2 (Santa Cruz,美国,sc-58386,1：1000稀释),抗-MMP9 (Santa Cruz,美国,sc-21733,1：1000稀释).等量提取的蛋白装载到SDS/PAGE胶上并被分离后,再转移到PVDF膜上,然后用含有5%脱脂奶粉的TBST液封闭1小时,接着再用一抗在常温下孵育2小时,膜再用TBST液洗涤4次,然后再用抗鼠IgG-HRP(DAKO)在常温下孵育1小时,最后蛋白用ECL体系进行检测。蛋白相对表达量=待测蛋白的光密度值／内参的光密度值。
结果：
1. VEGF靶向siRNA抑制CaoV3细胞增殖
为了确定VEGF siRNA是否能影响CaoV3细胞的增殖,用溴标法测定细胞活性。VEGF siRNA组CaoV3细胞的增殖(0.67±0.30)与空白对照、阴性对照组相比(1.85±0.43与1.79±0.37)显著地减少了,细胞增殖抑制了62.56%(P=0.000)。在空白对照组与阴性对照组之间CaoV3细胞的增殖没有差异(P>0.05),因此VEGF表达的下调能抑制CaoV3细胞的增殖。
2. VEGF靶向siRNA诱导CaoV3细胞凋亡
检测caspase-3的酶活性来评估VEGF siRNA对CaoV3细胞凋亡的影响。结果VEGF siRNA组caspase-3的活性(1.83±0.11)与阴性对照组相比(1.04±0.09)显著地增加了,caspase-3的活性增加了75.96%(P=0.000)；而阴性对照组(1.04±0.09)与空白对照组(1.00±0.00)之间caspase-3的活性无差异(P>0.05)。
为了检测VEGF siRNA在体外是否能诱导人卵巢癌细胞系CaoV3细胞的凋亡,细胞固定后用DNA末端标记法(TUNEL法)分析细胞的凋亡。用VEGF siRNA转染48h后,几乎所有的细胞从培养皿上消失了,留下来的少数细胞几乎100%凋亡阳性,然而,空白对照、阴性对照组细胞数量稳定地增长,并且凋亡细胞小于5%。
3. VEGF靶向siRNA下调CaoV3细胞survivin、MMP2与MMP9蛋白的表达
为了研究VEGF siRNA能否抑制CaoV3细胞表达survivin、MMP2与MMP9蛋白,用Western-blot法检测这些蛋白的表达水平,结果阴性对照组survivin、MMP2与MMP9蛋白的表达没有改变,但’VEGF siRNA组CaoV3细胞这三种蛋白的表达显著减少(P均=0.000)。
结论：
siRNA靶向干扰VEGF能有效抑制CaoV3细胞增殖,诱导细胞凋亡,降低细胞侵袭潜能。
Background
Ovarian carcinoma has the highest mortality rate among all the gynecologic malignancies. It represents the fifth most common cancer, resulting in mortality in women between 55 and 74 years of age. Most commonly diagnosed in advanced stages, it is treated initially with surgery followed by a combination of platinum and taxane based chemotherapy. Patients with advanced disease undergoing optimal debulking surgery to less than a centimeter residual have a median survival of just over 4 years after adjuvant chemotherapy. Due to the development of chemotherapy resistant clones, almost all such women ultimately develop recurrent ovarian cancer and die of progressive disease. Thus, finding novel effective strategies are desperately needed in this disease.
Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that stimulates permeability, cell division, and migration of vascular endothelial cells. VEGF, an emerging target in tumor therapy, has been considered to play an important role in tumor growth and invasion. In ovarian carcinoma, VEGF expression was detected previously. It has been reported recently that serum levels of VEGF are significantly elevated in patients with ovarian epithelial cancers. VEGF is an important growth factor associated with progression of ovarian cancer and accumulating of ascetic fluid and plays an important role in invasion, metastasis and ascetic fluid accumulating in ovarian cancer. VEGF has been reported as a marker for assessing ovarian tumor and as a predictor for the outcome of ovarian carcinoma.
Recently, small interfering RNA (siRNA) has been shown to inhibit the expression of a corresponding target gene in mammals via the biologically conserved mechanism of RNA interference (RNAi). RNAi is a highly sequence specific method and allows to turn off the expression of a target protein. Many studies have demonstrated that RNAi can block replication of various types of mammalian viruses, and inhibit growth of carcinoma. RNAi is now being exploited as a powerful tool for reverse genetics, and shows great promise for therapeutic applications.
In this study, we down-regulated VEGF expression by siRNA in a human epithelial ovarian carcinoma cell line CaoV3 with relatively high VEGF expression, and evaluated the effect of decreased VEGF expression on cell proliferation, cell apoptosis and invasion related protein expression. We expect that our study could provide experiment evidence for gene therapy of ovarian carcinoma on targeting VEGF in vitro.
PART I Preparation and transfection of VEGF siRNA and detection of its interference effect
Objective:
siRNA against VEGF was prepared and transfected to ovarian carcinoma cell line CaoV3, in order to identify siRNA targeting VEGF can block VEGF mRNA and protein expression in CaoV3 efficiently and specifically.
Materials and Methods:
Cell culture
Human epithelial ovarian carcinoma cell line CaoV3 was purchased from ATCC (American Type Culture Collection, USA), and maintained in the RPMI-1640 medium containing 10% fetal bovine serum supplemented with streptomycin/penicillin in an incubator at 37℃with 5% CO2 and 95% air.
siRNA preparation and transfection
The VEGF specific siRNA fragments were obtained by BLOCK-iTTM Complete Dicer RNAi Kit (Invitrogen, USA), following the manufacture's instructions. The primers for VEGF gene are 5'-GAA CTT TCT GCT GTC TTG G-3'(Forward) and 5'-TTT TCT TGT CTT GCT CTA TCT-3'(Reverse), the primers for LacZ gene are 5'-ACCAGAAGCGGRGCCGGAAA-3'(Forward) and 5'-CCACAGCGGATGGTTCGGAT-3'(Reverse). Cells were transfected with 30ng siRNA and 5μ1 Lipofectamine 2000 (Invitrogen, USA). After transfected for 48 h, cells were harvested for the next experiments. lacZ d-siRNA (negative control), no d-siRNA (blank control).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
After transfected with VEGF-siRNA 48h, VEGF mRNA expression was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted by TRIzol Reagent (Invitrogen, USA), treated with RNase free DNase I for 30 min at 37℃, and retro-transcribed in 20μ1 of reaction mixture using M-MuLV reverse transcriptase and random hexamer primers.
The primers of VEGF were same as used in "siRNA preparation and transfection" part. Beta-actin was used as an internal standard and amplified with primers:5'-GTG GAC ATC CGC AAA GAC-3'(Forward) and 5'-AAA GGG TGT AAC GCA ACT AA-3'. qRT-PCR was performed by Applied Biosystems 7900HT system with the SYBR(?) Premix Ex TaqTM kit (TaKaRa) following the manufacture's protocols. All reactions were run in triplicate. VEGF mRNA expression was measured by the 2-△Ct method.△Ct value was obtained by substracting beta-actin Ct value from VEGF Ct value.
Western blot
The primary antibodies used in Western blot were anti-VEGF (Santa Cruz, USA, sc-7269,1:500 dilution), Equal extracted protein was loaded and separated by SDS/PAGE gels, then transferred to PVDF membrane. Membrane was blocked with 5% non-fat dry milk in TBST for 1h, and then incubated with the primary antibodies for 2h at RT. Membrane was rinsed four times with TBST, and then incubated with the secondary anti-mouse IgG-HRP (DAKO) for 1h at RT. Protein was visualized with the ECL system (Santa Cruz,USA). Individual signal quantitation was determined as IOD (integrated optical density) data. The protein expression was relative to the IOD of beta-Actin.
Results:
Down-regulation of VEGF by siRNA
The VEGF d-siRNA and lacZ d-siRNA (negative control) were transiently transfected into CaoV3 cells for 48 h. The VEGF mRNA level in blank control (no d-siRNA), negative control and VEGF d-siRNA group were 1.64±0.07,1.61±0.04 and 0.48±0.02, respectively. The inhibition ratio was 70.02% by VEGF siRNA (P =0.000), while transfection with the lacZ d-siRNA had no effect on the expression of VEGF mRNA in CaoV3 cells (P> 0.05). This reduction was further confirmed by Western blot. VEGF protein expression in VEGF-siRNA transfected CaoV3 cells (0.31±0.07) showed a marked reduction, compared with the blank and negative control (0.82±0.08 and 0.81±0.09). The inhibition ratio was 62.21% by VEGF siRNA. These data indicated that VEGF expression can be strongly inhibited both on mRNA and protein levels by siRNA.
Conclusion:
Our study implied that siRNA targeting VEGF can block VEGF mRNA and protein expression in CaoV3 efficiently and specifically.
PART II Proliferation suppression, apoptosis and invasiveness of ovarian carcinoma cells induced by siRNA against VEGF
Objective:
To explore whether the siRNA targeting VEGF can inhibit cell proliferation, induce cell apoptosis, and decrease the cell invasive potential of CaoV3.
Materials and Methods:
Cell culture
Human epithelial ovarian carcinoma cell line CaoV3 was purchased from ATCC (American Type Culture Collection, USA), and maintained in the RPMI-1640 medium containing 10% fetal bovine serum supplemented with streptomycin/penicillin in an incubator at 37℃with 5% CO2 and 95% air.
siRNA preparation and transfection
The VEGF specific siRNA fragments were obtained by BLOCK-iTTM Complete Dicer RNAi Kit (Invitrogen, USA), following the manufacture's instructions. The primers for VEGF gene are 5'-GAA CTT TCT GCT GTC TTG G-3'(Forward) and 5'-TTT TCT TGT CTT GCT CTA TCT-3'(Reverse), the primers for LacZ gene are 5'-ACCAGAAGCGGRGCCGGAAA-3'(Forward) and 5'-CCACAGCGGATGGTTCGGAT-3'(Reverse). Cells were transfected with 30ng siRNA and 5μl Lipofectamine 2000 (Invitrogen, USA). After transfected for 48 h, cells were harvested for the next experiments. lacZ d-siRNA (negative control),,blank control (no d-siRNA).
Proliferation detection
Cell proliferation was studied using a colorimetric bromodeoxyuridine (BrdU) kit (Roche Diagnostics) according to the manufacturer's instructions. Briefly, BrdU solution was added into the medium. The medium was discarded after 2 h, and cells were fixed with FixDenat solution for 30 min at RT. After removing FixDenat, cells were incubated with anti-BrdU working solution for 30 min. The immune complexes were detected by the subsequent substrate reaction.
Apoptosis detection
Caspase-3 activity was detected by the CaspACETM Assay System kit (Colorimetric, Promega) according to manufacturer's instructions. Cells were resuspended in 98μ1 of reaction mixtures and mixed with 2μ1 of caspase substrate. After incubation at RT, absorbance was measured in the well at 405nm by spectrophotometer, and then the caspase-3 specific activity was calculated.
For the detection of apoptotic cells with nuclear fragmentation, a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) method was performed by in situ cell death detection kit, POD (Roche, Germany) following manufacturer's instructions.
Western blot
The primary antibodies used in Western blot were anti-Survivin (Santa Cruz, USA, sc-17779,1:500 dilution), anti-MMP2 (Santa Cruz, USA, sc-58386,1:1000 dilution), anti-MMP9 (Santa Cruz, USA, sc-21733,1:1000 dilution). Equal extracted protein was loaded and separated by SDS/PAGE gels, then transferred to PVDF membrane. Membrane was blocked with 5% non-fat dry milk in TBST for 1h, and then incubated with the primary antibodies for 2h at RT. Membrane was rinsed four times with TBST, and then incubated with the secondary anti-mouse IgG-HRP (DAKO) for 1h at RT. Protein was visualized with the ECL system (Santa Cruz,USA). Individual signal quantitation was determined as IOD (integrated optical density) data. The protein expression was relative to the IOD of beta-Actin.
Results:
Proliferation inhibition by VEGF siRNA
To determine whether VEGF siRNA affects cell proliferation in CaoV3 cells, the cell viability was determined by the colorimetric BrdU assay. The cell proliferation was reduced significantly in VEGF siRNA transfected cells (0.67±0.30), compared with the blank and negative control (1.85±0.43 and 1.79±0.37), with a 62.56% inhibition of cell proliferation. (P=0.000) There was no difference between the blank control and the negative control CaoV3 cells(P>0.05). Thus, down-regulation of VEGF expression could inhibit CaoV3 cell proliferation.
Apoptosis induced by VEGF siRNA
The enzymatic activity of caspase-3 was detected to evaluate the effect of VEGF siRNA on cell apoptosis in CaoV3 cells. The caspase-3 activity was increased from 1.04±0.09 in the negative control to 1.83±0.11 in the VEGF siRNA transfected group. The caspase-3 activity increased 75.96%(P< 0.05). There was no difference between the blank control (1.00±0.00) and the negative control (1.04±0.09)CaoV3 cells(P >0.05).
To examine whether VEGF siRNA can induce apoptosis in human ovarian carcinoma cell line CaoV3 in vitro, cells were fixed and analyzed for apoptosis by TUNEL assay. After transfected with VEGF-siRNA 48h, almost all cells disappeared from the plates and of the few remaining ones nearly 100% were TUNEL-positive, whereas the blank control and negative control steadily increased in amount of cells and displayed less than 5% of the cells were TUNEL-positive
Down-regulation of survivin, MMP2, and MMP9 protein expression by VEGF siRNA
To investigate whether VEGF siRNA inhibited these protein expressions, the protein level was determined by Western blot. The negative control did not change survivin, MMP2, and MMP9 protein expression, but transfecting VEGF d-siRNA in CaoV3 cells markedly reduced the levels of all three protein (P=0.000,respectively).
Conclusion:
Our study implied that siRNA targeting VEGF can inhibit cell proliferation, induce cell apoptosis, and decrease the cell invasive potential of CaoV3.
引文
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