肿瘤抑素基因表达与肾癌关系以及鸟氨酸脱羧酶基因表达对肿瘤抑素表达调控作用的研究
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摘要
肿瘤是一类常见病、多发病,其中恶性肿瘤是目前危害人类健康最严重的一类疾病。在我国,随着人口老龄化,肿瘤的发病率和死亡率呈上升趋势。近年来,虽然肿瘤治疗已取得一些突破性进展,但由于肿瘤发病因素的多样性和肿瘤细胞的复杂性,肿瘤治疗仍是全球面临的一项难题。1971年Folkman提出“肿瘤的生长和转移依赖于新生血管的形成”,随后肿瘤抗血管生成疗法成为研究热点。
肿瘤抑素(tumstatin)是继血管抑素(angiostatin)和内皮抑素(endostatin)之后新发现的基底膜来源的肿瘤血管生成抑制因子；来源于Ⅳ型胶原α3链的C末端,由244个氨基酸组成,分子量为28kD。肿瘤抑素不但能够抑制血管内皮细胞增殖,诱导内皮细胞凋亡,进而抑制血管生成,从而抑制肿瘤生长；而且能够直接抑制肿瘤细胞增生,促进肿瘤细胞凋亡。在几种内源性血管生成抑制剂(angiostatin,endostatin,canstatin,restin,tumstatin)中,肿瘤抑素具有最强的抗血管生成效应,呈现良好的应用前景。
肾癌是最常见的一种肾实质肿瘤,其发病率在泌尿系肿瘤中占第二位,病因尚不清楚。目前肾癌的根治首选手术治疗,术后五年存活率为40%：而放疗和化疗只能作为辅助治疗方法,不能使肿瘤彻底控制。这些传统的治疗方法在治疗肿瘤的同时也损害机体正常的组织细胞,而肿瘤抗血管生成疗法只抑制病理性的肿瘤血管生成,不影响机体正常的血管生成,有望成为治疗肿瘤的有力武器之一。肿瘤抑素主要分布于肾小球基底膜,肺泡血管基底膜等,其与肾癌的关系尚未见报道。本文从组织和细胞水平研究了肿瘤抑素基因表达与肾癌的关系,并构建了肿瘤抑素真核表达载体以探讨其用于基因治疗的可行性。
鸟氨酸脱羧酶(ornithine decarboxylase, ODC)能催化鸟氨酸脱羧生成腐胺,是多胺合成途径中的第一个限速酶。现已发现在多种人类癌细胞和组织中ODC含量及活性明显升高,其与细胞转化、肿瘤侵袭和血管生成有关。为验证ODC过表达是否通过影响肿瘤抑素的表达来调控血管生成,本文首先检测了ODC和肿瘤抑素在肾组织和细胞中表达的相关性,然后筛选出含ODC过表达载体的HEK293细胞株,从mRNA、蛋白质、细胞增殖、启动子水平检测了ODC对肿瘤抑素表达的调控作用。研究结果表明ODC过表达能够抑制肿瘤抑素的表达,提示肿瘤组织中ODC过表达抑制肿瘤抑素的表达,是其促进血管生成的机制之
本研究过程中需要肿瘤抑素抗体,但课题开始时肿瘤抑素抗体尚未商品化,故本文首先构建了肿瘤抑素原核表达载体,在大肠杆菌中进行了高效表达,表达的包涵体蛋白经变性、纯化、复性后免疫小鼠,制备鼠抗人肿瘤抑素多克隆抗体,经ELISA和western blot验证其能够与肿瘤抑素特异性结合。这为本文的完成提供了实验材料,为后续研究奠定了基础。
第一部分人肿瘤抑素原核表达载体的构建、表达、纯化及其多克隆抗体的制备
[目的]
构建人肿瘤抑素基因原核表达载体,诱导表达后纯化获得重组人肿瘤抑素融合蛋白,制备鼠抗人肿瘤抑素抗体,为后续研究奠定基础。
[方法]
1.RT-PCR扩增肿瘤抑素的编码序列,TA克隆后NcoI、XhoI双酶切回收目的片段,插入到原核表达载体pTriEx-4中,构建重组表达质粒pTriEx-tumstatin。
2.将pTriEx-tumstatin表达质粒转化大肠杆菌E.coli JM109(DE3), IPTG诱导肿瘤抑素融合蛋白表达,SDS-PAGE电泳鉴定表达产物。
3.根据亲和层析原理,采用Ni SepharoseTM 6 Fast Flow纯化出重组人肿瘤抑素蛋白,透析复性,PEG8000浓缩,SDS-PAGE及western blot鉴定纯化产物。
4.纯化的肿瘤抑素融合蛋白与免疫佐剂混合,作为免疫原,免疫小鼠,取血清制备鼠抗人肿瘤抑素多抗；ELISA及western blot测定抗血清的效价及特异性。
[结果]
1.RT-PCR扩增出肿瘤抑素编码序列(735bp)。
2.重组表达质粒pTriEx-tumstatin构建成功,酶切鉴定及DNA序列分析证明该质粒含有肿瘤抑素全部编码序列,开放阅读框架正确。
3. pTriEx-tumstatin转化入JM109(DE3)诱导表达,表达产物经纯化、复性、浓缩,SDS-PAGE及western blot鉴定,分子量约为30kD,与预期结果相符。
4.制备出鼠抗人肿瘤抑素多克隆抗体,并测定了其效价及特异性。
[结论]
成功构建了含有人肿瘤抑素基因编码序列的原核表达载体pTriEx-tumstatin,并在E.coli JM109(DE3)中高效表达,经Ni SepharoseTM 6 Fast Flow纯化得到肿瘤抑素融合蛋白；以其为免疫原制备鼠抗人肿瘤抑素多抗,为后续的研究工作奠定了基础。
第二部分肿瘤抑素基因表达与肾癌关系的研究
[目的]
检测肿瘤抑素在肾癌组织和细胞中的表达水平；构建人肿瘤抑素基因真核表达载体,观察其对血管内皮细胞及肾癌细胞增殖的作用。
[方法]
1.半定量RT-PCR和western blot分别检测肾癌标本中肿瘤抑素mRNA和蛋白的表达情况。
2.半定量RT-PCR和western blot检测肾癌细胞(ACHN)和正常肾细胞(HEK293)中肿瘤抑素mRNA和蛋白的表达差异。
3.构建含肿瘤抑素基因的真核表达质粒(pcDNA-tumstatin),酶切鉴定并测序分析。
4.以脂质体LipofectamineTM 2000介导重组质粒pcDNA-tumstatin转染血管内皮细胞ECV304和人肾癌细胞ACHN,RT-PCR及western blot鉴定其在这两种细胞中的表达情况。
5.用CCK-8细胞活性检测法检测pcDNA-tumstatin转染后对ECV304和ACHN细胞增殖的影响。
6.流式细胞术检测pcDNA-tumstatin转染ECV304细胞后对细胞周期分布的影响,western blot检测肿瘤抑素基因表达对细胞周期调节蛋白cyclin D1表达的影响。
[结果]
1.肾组织标本中肿瘤抑素mRNA和蛋白的表达丰度以均数±标准差表示,结果显示：肾癌组织中肿瘤抑素mRNA和蛋白的表达量明显低于正常肾组织中的表达量(P<0.05)。
2.RT-PCR和western blot结果显示：肾癌细胞ACHN中肿瘤抑素mRNA和蛋白的表达量明显低于胚肾HEK293细胞中的表达量(P<0.05)。
3.经限制性内切酶酶切鉴定,pcDNA-tumstatin中肿瘤抑素基因片段大小符合；DNA测序显示序列及插入方向正确。
4.RT-PCR及western blot结果显示pcDNA-tumstatin能够在血管内皮细胞ECV304和肾癌细胞ACHN中表达。
5. pcDNA-tumstatin转染后可明显抑制血管内皮细胞ECV304的增殖,而对肾癌细胞ACHN的增殖无影响。
6.流式细胞术结果显示pcDNA-tumstatin转染的ECV304细胞周期阻滞在G1期；western blot结果显示肿瘤抑素基因表达抑制G1期主要的细胞周期蛋白cyclin D1的表达。
[结论]
肾癌组织和细胞中肿瘤抑素mRNA和蛋白表达下调,表明肿瘤抑素的表达变化可能与肾癌的进展有关。真核表达载体pcDNA3.1(+)介导的肿瘤抑素过表达可特异性抑制血管内皮细胞增殖,诱导其细胞周期阻滞于G1期,并且该阻滞作用与细胞周期蛋白cyclin D1表达下调有关,初步证实肿瘤抑素可作为肾癌治疗的治疗剂,并通过基因治疗手段来持续给药。
第三部分鸟氨酸脱羧酶基因表达对肿瘤抑素表达调控作用的研究
[目的]
构建鸟氨酸脱羧酶过表达载体pcDNA-ODC;建立ODC过表达的HEK293细胞株；研究ODC基因表达对肿瘤抑素表达的调控作用。
[方法]
1.半定量RT-PCR检测肾癌组织中ODC和肿瘤抑素的表达情况。
2.半定量RT-PCR和western blot检测癌细胞(肾癌细胞ACHN和肺癌细胞A549)中ODC和肿瘤抑素的表达情况。
3.构建含ODC基因的过表达质粒pcDNA-ODC,酶切鉴定并测序分析。
4.重新转化与ODC基因翻译起始位点互补的ODC反义真核表达质粒pcDNA-ODCr,酶切鉴定并测序分析。
5.以脂质体LipofectamineTM 2000介导pcDNA3.1质粒和pcDNA-ODC重组质粒分别转染人胚肾细胞HEK293,利用G418进行筛选,3-4周后,挑取单克隆、消化接种后继续培养建立稳转细胞株,并以western blot鉴定。
6.实验分为5组,分别为：PBS组、pcDNA3.1空载体稳转组、pcDNA-ODC稳转组、pcDNA-ODC与pcDNA-ODCr共转染组和腐胺处理组。分别提取各组HEK293细胞总蛋白,western blot检测ODC和肿瘤抑素蛋白的表达情况。
7.分别提取上述五组细胞总RNA,半定量RT-PCR检测ODC和肿瘤抑素mRNA的表达情况。
8.构建肿瘤抑素启动子的荧光素酶报告基因质粒(pGL-tumstatin2.2kb和pGL-tumstatin0.5kb),利用脂质体将pGL-tumstatin2.2kb质粒与内参照质粒pRL-TK共转染上述五组细胞,荧光素酶报告基因分析检测启动子活性。
9.获取上述五组细胞的条件培养基,作用于血管内皮细胞ECV304,CCK-8检测其对血管内皮细胞增殖的影响。
[结果]
1.半定量RT-PCR结果显示：38例肾癌标本中,有32例ODC mRNA是过表达的；在这32例ODC过表达的标本中有24例肿瘤抑素mRNA的表达量低于正常组织中的表达量。统计学分析ODC过表达与肿瘤抑素表达下调有关。
2.半定量RT-PCR和western blot结果显示：肾癌细胞ACHN、肺癌细胞A549中ODC mRNA和蛋白的表达量明显高于相应的正常细胞(人胚肾细胞HEK293和人胚肺细胞HELF),而肿瘤抑素mRNA和蛋白的表达量明显低于相应的正常细胞。统计学分析二者存在相关性。
3.经限制性内切酶和DNA测序鉴定,pcDNA-ODC质粒中,ODC基因片段大小符合,插入方向和序列正确。
4.经酶切鉴定和测序分析,所转化的pcDNA-ODCr质粒,目的片段基因序列及插入方向正确,说明所转化的pcDNA-ODCr质粒是携带与ODC翻译起始位点互补的基因序列的ODC反义真核表达质粒。
5.RT-PCR和western blot结果显示：筛选到的含pcDNA-ODC质粒的细胞能够高水平表达ODC。
6.RT-PCR和western blot结果显示：相对于空白对照组和空载体组,pcDNA-ODC稳转组中ODC mRNA和蛋白的表达量明显升高,在转染pcDNA-ODCr的pcDNA-ODC稳转细胞组中ODC mRNA和蛋白的表达量接近空白对照组,腐胺处理组中ODC表达无明显变化；而肿瘤抑素mRNA和蛋白的表达量在pcDNA-ODC稳转组和腐胺处理组中明显下降,转染pcDNA-ODCr的pcDNA-ODC稳转细胞组中肿瘤抑素mRNA和蛋白的表达量恢复至正常水平。RT-PCR结果显示：各处理组中血管内皮生长因子VEGF mRNA的表达量保持不变。
7.构建了肿瘤抑素基本启动子(2149bp)和已报道的共用启动子(530bp)的荧光素酶报告质粒(pGL-tumstatin2.2kb和pGL-tumstatin0.5kb),荧光素酶报告基因分析结果显示两者都具有启动子活性,其中pGL-tumstatin2.2kb活性明显,故选择该报告质粒进行下一步实验。结果显示ODC过表达和外源性腐胺的给予可抑制肿瘤抑素启动子活性,抑制率分别为45.8%、45.2%。
8.培养的血管内皮细胞ECV304中加入从各处理组获得的条件培养基继续培养18h后用CCK-8检测,结果显示：与PBS处理组相比,来自pcDNA-ODC稳转组和腐胺处理组的条件培养基能够诱导1.41±0.07、146±0.07倍的血管内皮细胞增殖；而来自空载体稳转组和pcDNA-ODCr转染的pcDNA-ODC稳转细胞组的条件培养基对血管内皮细胞增殖无明显影响。
[结论]
成功构建了ODC过表达的真核表达质粒pcDNA-ODC,并筛选出能够高表达ODC的人胚肾HEK293细胞株；ODC基因过表达和外源性腐胺的增加能够抑制肿瘤抑素基因的表达,从而使血管内皮细胞增殖加快,提示ODC过表达可能通过抑制肿瘤抑素表达而促进血管生成。
全文摘要总结
本课题成功构建了肿瘤抑素的原核表达载体pTriEx-tumstatin,并在大肠杆菌E.coli JM109(DE3)中高效表达了带有His.tag的融合蛋白,其纯化后作为免疫原,制备出鼠抗人肿瘤抑素多抗；利用该抗体研究发现肾癌组织和细胞中肿瘤抑素的表达量明显低于其在正常肾组织和细胞中的表达量,提示肿瘤抑素基因表达下调与肾癌的进展有关。利用表达肿瘤抑素的真核表达载体pcDNA-tumstatin,体外证明其能够特异性抑制血管内皮细胞增殖,从而抑制肿瘤生长。进一步分子机理研究证明,肿瘤抑素过表达可能是通过抑制细胞周期蛋白cyclin D1基因表达而使血管内皮细胞增殖停滞于G1期,为肾癌基因治疗研究提供了理论依据。
鸟氨酸脱羧酶(ODC)是多胺合成途径中的第一个限速酶。ODC过表达能够促进血管生成。肾癌组织和细胞中ODC的表达升高,而肿瘤抑素的表达下降,者有相关性。利用含ODC过表达载体的稳转细胞,进一步研究证明ODC基因过表达和腐胺水平增加能够抑制肿瘤抑素表达,从而使血管内皮细胞增殖加快,提示ODC过表达可能通过抑制肿瘤抑素表达而调控血管生成。
Tumor is one of the most common and frequently encountered diseases. In particular, malignant tumor is the leading cause of influencing healthy. Nowadays, with the population aging, the morbidity and mortality of tumor continue to show an upward tendency in China. Despite having some progress, searching for highly sensitive, specific and efficient treatment for tumor remains difficult, because of the diversity of etiological factor and complexity of tumor cells. In 1971, Folkman proposed that tumor growth and progression relied on neovasculature. And then anti-agiogenic therapy became a hot research subject.
Tumstatin is a novel endogenous inhibitor of angiogenesis, the same as angiostatin and endostatin, liberated from basement membrane. It is the bioactive NC I domain (28KD) of ColⅣα3, composed of 244 amino acids. Tumstatin can specifically suppress proliferation of endothelial cells (EC), induce apoptosis of EC and inhibit pathological angiogenesis and tumor growth. Among the type of endogenous angiogenesis inhibitors discovered recently such as arresten, endostatin, castatin; tumstatin is the most powerful inhibitor. Therefore, it is regarded as a promising therapeutic candidate in the control of tumor angiogenesis and growth.
Renal carcinoma is one of the most common malignant tumors originating from renal parenchyma with high and growing incidence. The definite etiopathogenesis of renal cancer is unkown. At present, early detection, diagnosis and treatment with surgery (radical or partial nephrectomy) have yielded positive results. However, overall five year survival rate for this disease is around 40%. Radiotherapy and chemotherapy, adjunctive therapies for cancer treatment, can't cure tumor completely. And these traditionary therapies may also damage the normal tissues and cells. Nevertheless, anti-agiogenic therapy inhibits pathological angiogenesis specifically, while angiogenesis associated with development and tissue repair are unaffected. So, it becomes the potent treatment for cancer with good prospect. The distribution of tumstatin is limited to glomerular basement membrane, alveolar capillary basement membrane and the vascular basement membrane of several organs. So far, there is no information on the relationship between tumstatin and renal carcinoma. Our article revealed the tumstatin gene expression in renal cancer with mRNA and protein level to observe the relatioship between tumstatin gene expression and the pathogenesis of renal cancinoma. Moreover, we constructed a eukaryotic expression plasmid containing tumstatin gene to explore the feasibility for the utilization of gene therapy.
Ornithine decarboxylase (ODC) is the first rate-limiting enzyme of polyamine biosynthesis, catalyzing the decarboxylation of ornithine to produce putrescine. ODC, overexpressed in various cancers, is associated with cell transformation, tumor invasion and angiogenesis. In order to study whether ODC overexpression facilitate angiogenesis by inhibiting tumstatin expression, we first analyzed the relationship between ODC expression levels and tumstatin production in renal tissues and cells, and then established the ODC-overexpression HEK293 cells to examine the effect of ODC overexpression on tumstatin expression and vascular endothelial cell proliferation. Our results showed that ODC overexpression can supress the expression of tumstatin, which indicated that ODC promoting tumor angiogenesis by suppressing tumstatin expression in many cancers.
In this study, we required anti-tumstatin antibody for sequential experiment, but there is no commercial antibody of tumstatin then. Therefore, we constructed the human tumstatin expression plasmid, purified and refolded the expressed fusion protein which was used to immunize the Balb/c mouse as the immunogen, and then prepared an anti-tumstatin polyclonal antibody. ELISA and western blot showed that the prepared antibody could combine the tumstatin protein specifically. The preparation of the polyclonal antibody facilitated the following study.
PART ONE PROKARYOTIC EXPRESSION OF HUMAN TUMSTATIN GENE AND PREPARATION OF POLYCLONAL ANTI-TUMSTATIN ANTIBODY
Objective:
To construct the human tumstatin prokaryotic expression plasmid, purified the recombinant protein and prepared the anti-tumstatin polyclonal antibody.
Methods:
(1)The sequence encoding tumstatin was amplified by RT-PCR and then inserted into the prokaryotic expression vector pTriEx-4 by TA clone.
(2) The positive plasmid constructs named pTriEx-tumstatin were finally transformed into E.coli JM109 (DE3) for expression which was induced by IPTG The expressed fusion protein was identified by SDS-PAGE and western blot with anti His.tag monoclonal antibody.
(3) The recombinant protein was purified by nickel chelate affinity column, refolded by dialyzing and concentrated by PEG8000. The concentrated solution was analyzed through SDS-PAGE and western blot.
(4) Tumstatin/Adjuvant mixture was used to immunize Balb/c mice and collected murine antiserum. The antibodies in the serum were titrated by ELISA and tested by western blot.
Results:
(1) The cDNA encoding human tumstatin was amplified.
(2) The prokaryotic expression plasmid pTriEx-tumstatin was constructed sucessfully and confirmed by restriction endonuclease digestion and DNA sequencing.
(3) Tumstatin was expressed significantly in E.coli, and then purified, refolded and concentrated effectively. SDS-PAGE and western blot confirmed the identity of the fusion protein.
(4) The anti-tumstatin polyclonal antibody was prepared successfully. ELISA and western blot showed that the antibody could immunobind to recombinant human tumstatin protein.
Conclusions:
The anti-tumstatin polyclonal antibody was prepared successfully using the recombinant tumstatin protein as the immunogen. It was the necessary agent for subsequent research.
PART TWO THE RELATIONSHIP BETWEEN TUMSTATIN GENE EXPRESSION AND RENAL CARCINOMA
Objective:
To detect the expression of tumstatin in renal carcinoma and construct a human tumstatin eukaryotic expression plasmid to examine its effect on the proliferation of vascular endothelial cells and renal cancer cells.
Methods:
(1) Semiquantitative RT-PCR and western blot analysis were performed to detect the expression level of tumstatin in renal cancer tissues at the mRNA and protein level.
(2) Semiquantitative RT-PCR and western blot analysis were performed to detect the expression levels of tumstatin mRNA and protein in renal cancer cells.
(3) Constructed eukaryotic expression plasmid pcDNA-tumstatin and identified by restriction enzyme digestion and DNA sequencing.
(4) Transfected plasmid pcDNA-tumstatin into human umbilical vein endothelial cell line ECV304 and human renal carcinoma cell line ACHN. The expression of tumstatin in the two cell lines by RT-PCR and western blot.
(5) CCK-8 assay was used to analyze the effect of pcDNA-tumstatin on the growth of ECV304 and ACHN cells.
(6) Cell cycle distribution of ECV304 was detected by flow cytometiy analysis. The effect of tumstatin expression on cell cycle regulated protein cyclin D1 was measured by western blot analysis
Results:
(1) The tumstatin mRNA levels in renal carcinoma tissues (0.57±0.75) were significantly lower than in normal tissues (1.08±1.08) (P<0.05). Western blot also showed that tumstatin protein is expressed at low levels in most of the tumor tissues (1.41±0.34) when compared with the corresponding normal tissues (1.82±0.48) (P<0.05).
(2) RT-PCR and western blot showed that tumstatin mRNA and protein levels in ACHN cells were significantly lower than in HEK293 cells.
(3) Restriction endonuclease digestion result showed that the direction and length of inserted tumstatin fragment in pcDNA-tumstatin was right, further confirmed by DNA sequencing.
(4) The results of RT-PCR and western blot showed that tumstatin was highly expressed in both ECV304 and ACHN cells transfected with pcDNA-tumstatin.
(5) The proliferation trend was significantly decreased in ECV304 cells transfected with pcDNA-tumstatin, compared with cells transfected with empty vector and the untransfected cells. However, there is no remarkable effect on ACHN cells.
(6) Tumstatin expression induced ECV304 cells to arrest at G1 phase and decreased the expression of cyclin D1.
Conclusions:
The expression of tumstatin is down-regulated in renal carcinoma, indicating that changes in tumstatin gene expression are related to the development of renal cancer. pcDNA3.1 (+)-mediated overexpression of tumstatin inhibits endothelial cell proliferation specifically by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1. Administration of tumstatin using a gene therapy approach might represent a promising treatment option for renal cancer treatment.
PART THREE ORNITHINE DECARBOXYLASE GENE EXPRESSION REGULATES THE EXPRESSION OF TUMSTATIN
Objective:
To investigate the relationship between ODC gene expression and tumstatin expression.
Methods:
(1) Semiquantitative RT-PCR and western blot analysis were used to detect the expression levels of ODC and tumstatin in renal cancer tissues.
(2) The expression levels of ODC and tumstatin in cancer cells (human renal cancer cell ACHN and human lung cancer cell A549) were detected by semiquantitative RT-PCR and western blot analysis.
(3) Constructed ODC-overexpressing plasmid pcDNA-ODC and identified by restriction enzyme digestion and DNA sequencing.
(4) The antisense ODC expressing vector pcDNA-ODCr was transformed into E.coli to amplify, confirmed by restriction enzyme digestion and DNA sequencing.
(5) Transfected plasmid pcDNA3.1 and pcDNA-ODC into HEK293 cells with LipofectamineTM 2000, used G418 to select positive cells.3-4 weeks later, picked out the clone, digested with trypsinase and continued culturing to establish the mock transfectants and ODC transfectants.
(6) The expression levels of ODC and tumstatin protein were examined by RT-PCR and western blot analysis using total cell RNA and cell lysates from HEK293 cells treated by PBS、pcDNA3.1、pcDNA-ODC、pcDNA-ODC and pcDNA-ODCr, putrescine.
(7) Semiquantitative RT-PCR was used to detect the expression levels of ODC and tumstatin mRNA in HEK293 cells treated like above.
(8) Two luciferase reporter plasmids of tumstatin gene promotor pGL-tumstatin2.2kb and pGL-tumstatin0.5kb were constructed. Cotransfected pGL-tumstatin2.2kb and pRL-TK into HEK293 cells treated like above to observe the effect of ODC overexpression on tumstatin promoter activity by Dual Luciferas Reporter gene assay.
(9) CCK-8 assay was used to analyze the effect of conditioned media from HEK293 cells treated like above on the growth of ECV304.
Results:
(1) RT-PCR showed that thirty-two of thirty-eight cancer tissues overexpressed ODC mRNA compared with the normal renal tissues. And twenty-four of thirty-two ODC-overexpressing human renal cancers showed suppressed tumstatin mRNA expression. The result indicated that there was a correlation between ODC gene expression and tumstatin expression.
(2) RT-PCR showed that the expression of tumstatin was remarkably suppressed in the ODC-overexpressing cell lines ACHN and A549, compared with the corresponding normal cells (HEK293 and HELF), as determined by western blot.
(3) The ODC-overexpressing plasmid pcDNA-ODC was constructed sucessfully and confirmed by restriction endonuclease digestion and DNA sequencing.
(4) Restriction endonuclease digestion showed that the length of inserted ODC antisense RNA fragment is right. DNA sequencing further confirmed that the pcDNA-ODCr plasmid delivered a 120bp fragment complementary to the initiation codon of ODC gene.
(5) The results of RT-PCR and western bolt showed that the positive ODC-overexpressing cells selected by G418 highly expressed ODC mRNA and protein.
(6) ODC mRNA and protein were overexpressed in ODC transfectants, and the elevated ODC expression level returns to that of mock transfectants, while there were no changes in putrescine treated group, as comared with PBS treated group and mock transfectants. The expression of tumstatin in ODC transfectants and putrescine treated group at both the mRNA and protein levels was significantly suppressed. The suppression of tumstatin expression was rescued after pcDNA-ODCr transfected. VEGF mRNA expression levels in all HEK293 treated remained unchanged.
(7) The tumstatin promoter luciferase reporter plasmids pGL-tumstatin2.2kb and pGL-tumstatin0.5kb were constructed successfully. Both of them have promoter activity and pGL-tumstatin2.2kb presented stronger promoter activity than pGL-tumstatin0.5kb. Moreover, the activity of pGL-tumstatin2.2kb decreased about 45.8%、45.2% by ODC overexpression and putrescine respectively.
(8) Conditioned media from ODC transfectants and putrescine treated group induced 1.41±0.07 and 1.46±0.07 fold ECV304 cell proliferation respectively, compared with media from the mock transfectants (P<0.05).
Conclusions:
ODC overexpression and administration of putrescine could inhibite tumstatin expression which maybe the underlying mechanism that the elevated ODC expression levels facilitate endothelial cell proliferation (angiogenesis).
肿瘤抑素(tumstatin)是继血管抑素(angiostatin)和内皮抑素(endostatin)之后新发现的基底膜来源的肿瘤血管生成抑制因子；来源于Ⅳ型胶原α3链的C末端,由244个氨基酸组成,分子量为28kD。肿瘤抑素不但能够抑制血管内皮细胞增殖,诱导内皮细胞凋亡,进而抑制血管生成,从而抑制肿瘤生长；而且能够直接抑制肿瘤细胞增生,促进肿瘤细胞凋亡。在几种内源性血管生成抑制剂(angiostatin,endostatin,canstatin,restin,tumstatin)中,肿瘤抑素具有最强的抗血管生成效应,呈现良好的应用前景。
肾癌是最常见的一种肾实质肿瘤,其发病率在泌尿系肿瘤中占第二位,病因尚不清楚。目前肾癌的根治首选手术治疗,术后五年存活率为40%：而放疗和化疗只能作为辅助治疗方法,不能使肿瘤彻底控制。这些传统的治疗方法在治疗肿瘤的同时也损害机体正常的组织细胞,而肿瘤抗血管生成疗法只抑制病理性的肿瘤血管生成,不影响机体正常的血管生成,有望成为治疗肿瘤的有力武器之一。肿瘤抑素主要分布于肾小球基底膜,肺泡血管基底膜等,其与肾癌的关系尚未见报道。本文从组织和细胞水平研究了肿瘤抑素基因表达与肾癌的关系,并构建了肿瘤抑素真核表达载体以探讨其用于基因治疗的可行性。
鸟氨酸脱羧酶(ornithine decarboxylase, ODC)能催化鸟氨酸脱羧生成腐胺,是多胺合成途径中的第一个限速酶。现已发现在多种人类癌细胞和组织中ODC含量及活性明显升高,其与细胞转化、肿瘤侵袭和血管生成有关。为验证ODC过表达是否通过影响肿瘤抑素的表达来调控血管生成,本文首先检测了ODC和肿瘤抑素在肾组织和细胞中表达的相关性,然后筛选出含ODC过表达载体的HEK293细胞株,从mRNA、蛋白质、细胞增殖、启动子水平检测了ODC对肿瘤抑素表达的调控作用。研究结果表明ODC过表达能够抑制肿瘤抑素的表达,提示肿瘤组织中ODC过表达抑制肿瘤抑素的表达,是其促进血管生成的机制之
本研究过程中需要肿瘤抑素抗体,但课题开始时肿瘤抑素抗体尚未商品化,故本文首先构建了肿瘤抑素原核表达载体,在大肠杆菌中进行了高效表达,表达的包涵体蛋白经变性、纯化、复性后免疫小鼠,制备鼠抗人肿瘤抑素多克隆抗体,经ELISA和western blot验证其能够与肿瘤抑素特异性结合。这为本文的完成提供了实验材料,为后续研究奠定了基础。
第一部分人肿瘤抑素原核表达载体的构建、表达、纯化及其多克隆抗体的制备
[目的]
构建人肿瘤抑素基因原核表达载体,诱导表达后纯化获得重组人肿瘤抑素融合蛋白,制备鼠抗人肿瘤抑素抗体,为后续研究奠定基础。
[方法]
1.RT-PCR扩增肿瘤抑素的编码序列,TA克隆后NcoI、XhoI双酶切回收目的片段,插入到原核表达载体pTriEx-4中,构建重组表达质粒pTriEx-tumstatin。
2.将pTriEx-tumstatin表达质粒转化大肠杆菌E.coli JM109(DE3), IPTG诱导肿瘤抑素融合蛋白表达,SDS-PAGE电泳鉴定表达产物。
3.根据亲和层析原理,采用Ni SepharoseTM 6 Fast Flow纯化出重组人肿瘤抑素蛋白,透析复性,PEG8000浓缩,SDS-PAGE及western blot鉴定纯化产物。
4.纯化的肿瘤抑素融合蛋白与免疫佐剂混合,作为免疫原,免疫小鼠,取血清制备鼠抗人肿瘤抑素多抗；ELISA及western blot测定抗血清的效价及特异性。
[结果]
1.RT-PCR扩增出肿瘤抑素编码序列(735bp)。
2.重组表达质粒pTriEx-tumstatin构建成功,酶切鉴定及DNA序列分析证明该质粒含有肿瘤抑素全部编码序列,开放阅读框架正确。
3. pTriEx-tumstatin转化入JM109(DE3)诱导表达,表达产物经纯化、复性、浓缩,SDS-PAGE及western blot鉴定,分子量约为30kD,与预期结果相符。
4.制备出鼠抗人肿瘤抑素多克隆抗体,并测定了其效价及特异性。
[结论]
成功构建了含有人肿瘤抑素基因编码序列的原核表达载体pTriEx-tumstatin,并在E.coli JM109(DE3)中高效表达,经Ni SepharoseTM 6 Fast Flow纯化得到肿瘤抑素融合蛋白；以其为免疫原制备鼠抗人肿瘤抑素多抗,为后续的研究工作奠定了基础。
第二部分肿瘤抑素基因表达与肾癌关系的研究
[目的]
检测肿瘤抑素在肾癌组织和细胞中的表达水平；构建人肿瘤抑素基因真核表达载体,观察其对血管内皮细胞及肾癌细胞增殖的作用。
[方法]
1.半定量RT-PCR和western blot分别检测肾癌标本中肿瘤抑素mRNA和蛋白的表达情况。
2.半定量RT-PCR和western blot检测肾癌细胞(ACHN)和正常肾细胞(HEK293)中肿瘤抑素mRNA和蛋白的表达差异。
3.构建含肿瘤抑素基因的真核表达质粒(pcDNA-tumstatin),酶切鉴定并测序分析。
4.以脂质体LipofectamineTM 2000介导重组质粒pcDNA-tumstatin转染血管内皮细胞ECV304和人肾癌细胞ACHN,RT-PCR及western blot鉴定其在这两种细胞中的表达情况。
5.用CCK-8细胞活性检测法检测pcDNA-tumstatin转染后对ECV304和ACHN细胞增殖的影响。
6.流式细胞术检测pcDNA-tumstatin转染ECV304细胞后对细胞周期分布的影响,western blot检测肿瘤抑素基因表达对细胞周期调节蛋白cyclin D1表达的影响。
[结果]
1.肾组织标本中肿瘤抑素mRNA和蛋白的表达丰度以均数±标准差表示,结果显示：肾癌组织中肿瘤抑素mRNA和蛋白的表达量明显低于正常肾组织中的表达量(P<0.05)。
2.RT-PCR和western blot结果显示：肾癌细胞ACHN中肿瘤抑素mRNA和蛋白的表达量明显低于胚肾HEK293细胞中的表达量(P<0.05)。
3.经限制性内切酶酶切鉴定,pcDNA-tumstatin中肿瘤抑素基因片段大小符合；DNA测序显示序列及插入方向正确。
4.RT-PCR及western blot结果显示pcDNA-tumstatin能够在血管内皮细胞ECV304和肾癌细胞ACHN中表达。
5. pcDNA-tumstatin转染后可明显抑制血管内皮细胞ECV304的增殖,而对肾癌细胞ACHN的增殖无影响。
6.流式细胞术结果显示pcDNA-tumstatin转染的ECV304细胞周期阻滞在G1期；western blot结果显示肿瘤抑素基因表达抑制G1期主要的细胞周期蛋白cyclin D1的表达。
[结论]
肾癌组织和细胞中肿瘤抑素mRNA和蛋白表达下调,表明肿瘤抑素的表达变化可能与肾癌的进展有关。真核表达载体pcDNA3.1(+)介导的肿瘤抑素过表达可特异性抑制血管内皮细胞增殖,诱导其细胞周期阻滞于G1期,并且该阻滞作用与细胞周期蛋白cyclin D1表达下调有关,初步证实肿瘤抑素可作为肾癌治疗的治疗剂,并通过基因治疗手段来持续给药。
第三部分鸟氨酸脱羧酶基因表达对肿瘤抑素表达调控作用的研究
[目的]
构建鸟氨酸脱羧酶过表达载体pcDNA-ODC;建立ODC过表达的HEK293细胞株；研究ODC基因表达对肿瘤抑素表达的调控作用。
[方法]
1.半定量RT-PCR检测肾癌组织中ODC和肿瘤抑素的表达情况。
2.半定量RT-PCR和western blot检测癌细胞(肾癌细胞ACHN和肺癌细胞A549)中ODC和肿瘤抑素的表达情况。
3.构建含ODC基因的过表达质粒pcDNA-ODC,酶切鉴定并测序分析。
4.重新转化与ODC基因翻译起始位点互补的ODC反义真核表达质粒pcDNA-ODCr,酶切鉴定并测序分析。
5.以脂质体LipofectamineTM 2000介导pcDNA3.1质粒和pcDNA-ODC重组质粒分别转染人胚肾细胞HEK293,利用G418进行筛选,3-4周后,挑取单克隆、消化接种后继续培养建立稳转细胞株,并以western blot鉴定。
6.实验分为5组,分别为：PBS组、pcDNA3.1空载体稳转组、pcDNA-ODC稳转组、pcDNA-ODC与pcDNA-ODCr共转染组和腐胺处理组。分别提取各组HEK293细胞总蛋白,western blot检测ODC和肿瘤抑素蛋白的表达情况。
7.分别提取上述五组细胞总RNA,半定量RT-PCR检测ODC和肿瘤抑素mRNA的表达情况。
8.构建肿瘤抑素启动子的荧光素酶报告基因质粒(pGL-tumstatin2.2kb和pGL-tumstatin0.5kb),利用脂质体将pGL-tumstatin2.2kb质粒与内参照质粒pRL-TK共转染上述五组细胞,荧光素酶报告基因分析检测启动子活性。
9.获取上述五组细胞的条件培养基,作用于血管内皮细胞ECV304,CCK-8检测其对血管内皮细胞增殖的影响。
[结果]
1.半定量RT-PCR结果显示：38例肾癌标本中,有32例ODC mRNA是过表达的；在这32例ODC过表达的标本中有24例肿瘤抑素mRNA的表达量低于正常组织中的表达量。统计学分析ODC过表达与肿瘤抑素表达下调有关。
2.半定量RT-PCR和western blot结果显示：肾癌细胞ACHN、肺癌细胞A549中ODC mRNA和蛋白的表达量明显高于相应的正常细胞(人胚肾细胞HEK293和人胚肺细胞HELF),而肿瘤抑素mRNA和蛋白的表达量明显低于相应的正常细胞。统计学分析二者存在相关性。
3.经限制性内切酶和DNA测序鉴定,pcDNA-ODC质粒中,ODC基因片段大小符合,插入方向和序列正确。
4.经酶切鉴定和测序分析,所转化的pcDNA-ODCr质粒,目的片段基因序列及插入方向正确,说明所转化的pcDNA-ODCr质粒是携带与ODC翻译起始位点互补的基因序列的ODC反义真核表达质粒。
5.RT-PCR和western blot结果显示：筛选到的含pcDNA-ODC质粒的细胞能够高水平表达ODC。
6.RT-PCR和western blot结果显示：相对于空白对照组和空载体组,pcDNA-ODC稳转组中ODC mRNA和蛋白的表达量明显升高,在转染pcDNA-ODCr的pcDNA-ODC稳转细胞组中ODC mRNA和蛋白的表达量接近空白对照组,腐胺处理组中ODC表达无明显变化；而肿瘤抑素mRNA和蛋白的表达量在pcDNA-ODC稳转组和腐胺处理组中明显下降,转染pcDNA-ODCr的pcDNA-ODC稳转细胞组中肿瘤抑素mRNA和蛋白的表达量恢复至正常水平。RT-PCR结果显示：各处理组中血管内皮生长因子VEGF mRNA的表达量保持不变。
7.构建了肿瘤抑素基本启动子(2149bp)和已报道的共用启动子(530bp)的荧光素酶报告质粒(pGL-tumstatin2.2kb和pGL-tumstatin0.5kb),荧光素酶报告基因分析结果显示两者都具有启动子活性,其中pGL-tumstatin2.2kb活性明显,故选择该报告质粒进行下一步实验。结果显示ODC过表达和外源性腐胺的给予可抑制肿瘤抑素启动子活性,抑制率分别为45.8%、45.2%。
8.培养的血管内皮细胞ECV304中加入从各处理组获得的条件培养基继续培养18h后用CCK-8检测,结果显示：与PBS处理组相比,来自pcDNA-ODC稳转组和腐胺处理组的条件培养基能够诱导1.41±0.07、146±0.07倍的血管内皮细胞增殖；而来自空载体稳转组和pcDNA-ODCr转染的pcDNA-ODC稳转细胞组的条件培养基对血管内皮细胞增殖无明显影响。
[结论]
成功构建了ODC过表达的真核表达质粒pcDNA-ODC,并筛选出能够高表达ODC的人胚肾HEK293细胞株；ODC基因过表达和外源性腐胺的增加能够抑制肿瘤抑素基因的表达,从而使血管内皮细胞增殖加快,提示ODC过表达可能通过抑制肿瘤抑素表达而促进血管生成。
全文摘要总结
本课题成功构建了肿瘤抑素的原核表达载体pTriEx-tumstatin,并在大肠杆菌E.coli JM109(DE3)中高效表达了带有His.tag的融合蛋白,其纯化后作为免疫原,制备出鼠抗人肿瘤抑素多抗；利用该抗体研究发现肾癌组织和细胞中肿瘤抑素的表达量明显低于其在正常肾组织和细胞中的表达量,提示肿瘤抑素基因表达下调与肾癌的进展有关。利用表达肿瘤抑素的真核表达载体pcDNA-tumstatin,体外证明其能够特异性抑制血管内皮细胞增殖,从而抑制肿瘤生长。进一步分子机理研究证明,肿瘤抑素过表达可能是通过抑制细胞周期蛋白cyclin D1基因表达而使血管内皮细胞增殖停滞于G1期,为肾癌基因治疗研究提供了理论依据。
鸟氨酸脱羧酶(ODC)是多胺合成途径中的第一个限速酶。ODC过表达能够促进血管生成。肾癌组织和细胞中ODC的表达升高,而肿瘤抑素的表达下降,者有相关性。利用含ODC过表达载体的稳转细胞,进一步研究证明ODC基因过表达和腐胺水平增加能够抑制肿瘤抑素表达,从而使血管内皮细胞增殖加快,提示ODC过表达可能通过抑制肿瘤抑素表达而调控血管生成。
Tumor is one of the most common and frequently encountered diseases. In particular, malignant tumor is the leading cause of influencing healthy. Nowadays, with the population aging, the morbidity and mortality of tumor continue to show an upward tendency in China. Despite having some progress, searching for highly sensitive, specific and efficient treatment for tumor remains difficult, because of the diversity of etiological factor and complexity of tumor cells. In 1971, Folkman proposed that tumor growth and progression relied on neovasculature. And then anti-agiogenic therapy became a hot research subject.
Tumstatin is a novel endogenous inhibitor of angiogenesis, the same as angiostatin and endostatin, liberated from basement membrane. It is the bioactive NC I domain (28KD) of ColⅣα3, composed of 244 amino acids. Tumstatin can specifically suppress proliferation of endothelial cells (EC), induce apoptosis of EC and inhibit pathological angiogenesis and tumor growth. Among the type of endogenous angiogenesis inhibitors discovered recently such as arresten, endostatin, castatin; tumstatin is the most powerful inhibitor. Therefore, it is regarded as a promising therapeutic candidate in the control of tumor angiogenesis and growth.
Renal carcinoma is one of the most common malignant tumors originating from renal parenchyma with high and growing incidence. The definite etiopathogenesis of renal cancer is unkown. At present, early detection, diagnosis and treatment with surgery (radical or partial nephrectomy) have yielded positive results. However, overall five year survival rate for this disease is around 40%. Radiotherapy and chemotherapy, adjunctive therapies for cancer treatment, can't cure tumor completely. And these traditionary therapies may also damage the normal tissues and cells. Nevertheless, anti-agiogenic therapy inhibits pathological angiogenesis specifically, while angiogenesis associated with development and tissue repair are unaffected. So, it becomes the potent treatment for cancer with good prospect. The distribution of tumstatin is limited to glomerular basement membrane, alveolar capillary basement membrane and the vascular basement membrane of several organs. So far, there is no information on the relationship between tumstatin and renal carcinoma. Our article revealed the tumstatin gene expression in renal cancer with mRNA and protein level to observe the relatioship between tumstatin gene expression and the pathogenesis of renal cancinoma. Moreover, we constructed a eukaryotic expression plasmid containing tumstatin gene to explore the feasibility for the utilization of gene therapy.
Ornithine decarboxylase (ODC) is the first rate-limiting enzyme of polyamine biosynthesis, catalyzing the decarboxylation of ornithine to produce putrescine. ODC, overexpressed in various cancers, is associated with cell transformation, tumor invasion and angiogenesis. In order to study whether ODC overexpression facilitate angiogenesis by inhibiting tumstatin expression, we first analyzed the relationship between ODC expression levels and tumstatin production in renal tissues and cells, and then established the ODC-overexpression HEK293 cells to examine the effect of ODC overexpression on tumstatin expression and vascular endothelial cell proliferation. Our results showed that ODC overexpression can supress the expression of tumstatin, which indicated that ODC promoting tumor angiogenesis by suppressing tumstatin expression in many cancers.
In this study, we required anti-tumstatin antibody for sequential experiment, but there is no commercial antibody of tumstatin then. Therefore, we constructed the human tumstatin expression plasmid, purified and refolded the expressed fusion protein which was used to immunize the Balb/c mouse as the immunogen, and then prepared an anti-tumstatin polyclonal antibody. ELISA and western blot showed that the prepared antibody could combine the tumstatin protein specifically. The preparation of the polyclonal antibody facilitated the following study.
PART ONE PROKARYOTIC EXPRESSION OF HUMAN TUMSTATIN GENE AND PREPARATION OF POLYCLONAL ANTI-TUMSTATIN ANTIBODY
Objective:
To construct the human tumstatin prokaryotic expression plasmid, purified the recombinant protein and prepared the anti-tumstatin polyclonal antibody.
Methods:
(1)The sequence encoding tumstatin was amplified by RT-PCR and then inserted into the prokaryotic expression vector pTriEx-4 by TA clone.
(2) The positive plasmid constructs named pTriEx-tumstatin were finally transformed into E.coli JM109 (DE3) for expression which was induced by IPTG The expressed fusion protein was identified by SDS-PAGE and western blot with anti His.tag monoclonal antibody.
(3) The recombinant protein was purified by nickel chelate affinity column, refolded by dialyzing and concentrated by PEG8000. The concentrated solution was analyzed through SDS-PAGE and western blot.
(4) Tumstatin/Adjuvant mixture was used to immunize Balb/c mice and collected murine antiserum. The antibodies in the serum were titrated by ELISA and tested by western blot.
Results:
(1) The cDNA encoding human tumstatin was amplified.
(2) The prokaryotic expression plasmid pTriEx-tumstatin was constructed sucessfully and confirmed by restriction endonuclease digestion and DNA sequencing.
(3) Tumstatin was expressed significantly in E.coli, and then purified, refolded and concentrated effectively. SDS-PAGE and western blot confirmed the identity of the fusion protein.
(4) The anti-tumstatin polyclonal antibody was prepared successfully. ELISA and western blot showed that the antibody could immunobind to recombinant human tumstatin protein.
Conclusions:
The anti-tumstatin polyclonal antibody was prepared successfully using the recombinant tumstatin protein as the immunogen. It was the necessary agent for subsequent research.
PART TWO THE RELATIONSHIP BETWEEN TUMSTATIN GENE EXPRESSION AND RENAL CARCINOMA
Objective:
To detect the expression of tumstatin in renal carcinoma and construct a human tumstatin eukaryotic expression plasmid to examine its effect on the proliferation of vascular endothelial cells and renal cancer cells.
Methods:
(1) Semiquantitative RT-PCR and western blot analysis were performed to detect the expression level of tumstatin in renal cancer tissues at the mRNA and protein level.
(2) Semiquantitative RT-PCR and western blot analysis were performed to detect the expression levels of tumstatin mRNA and protein in renal cancer cells.
(3) Constructed eukaryotic expression plasmid pcDNA-tumstatin and identified by restriction enzyme digestion and DNA sequencing.
(4) Transfected plasmid pcDNA-tumstatin into human umbilical vein endothelial cell line ECV304 and human renal carcinoma cell line ACHN. The expression of tumstatin in the two cell lines by RT-PCR and western blot.
(5) CCK-8 assay was used to analyze the effect of pcDNA-tumstatin on the growth of ECV304 and ACHN cells.
(6) Cell cycle distribution of ECV304 was detected by flow cytometiy analysis. The effect of tumstatin expression on cell cycle regulated protein cyclin D1 was measured by western blot analysis
Results:
(1) The tumstatin mRNA levels in renal carcinoma tissues (0.57±0.75) were significantly lower than in normal tissues (1.08±1.08) (P<0.05). Western blot also showed that tumstatin protein is expressed at low levels in most of the tumor tissues (1.41±0.34) when compared with the corresponding normal tissues (1.82±0.48) (P<0.05).
(2) RT-PCR and western blot showed that tumstatin mRNA and protein levels in ACHN cells were significantly lower than in HEK293 cells.
(3) Restriction endonuclease digestion result showed that the direction and length of inserted tumstatin fragment in pcDNA-tumstatin was right, further confirmed by DNA sequencing.
(4) The results of RT-PCR and western blot showed that tumstatin was highly expressed in both ECV304 and ACHN cells transfected with pcDNA-tumstatin.
(5) The proliferation trend was significantly decreased in ECV304 cells transfected with pcDNA-tumstatin, compared with cells transfected with empty vector and the untransfected cells. However, there is no remarkable effect on ACHN cells.
(6) Tumstatin expression induced ECV304 cells to arrest at G1 phase and decreased the expression of cyclin D1.
Conclusions:
The expression of tumstatin is down-regulated in renal carcinoma, indicating that changes in tumstatin gene expression are related to the development of renal cancer. pcDNA3.1 (+)-mediated overexpression of tumstatin inhibits endothelial cell proliferation specifically by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1. Administration of tumstatin using a gene therapy approach might represent a promising treatment option for renal cancer treatment.
PART THREE ORNITHINE DECARBOXYLASE GENE EXPRESSION REGULATES THE EXPRESSION OF TUMSTATIN
Objective:
To investigate the relationship between ODC gene expression and tumstatin expression.
Methods:
(1) Semiquantitative RT-PCR and western blot analysis were used to detect the expression levels of ODC and tumstatin in renal cancer tissues.
(2) The expression levels of ODC and tumstatin in cancer cells (human renal cancer cell ACHN and human lung cancer cell A549) were detected by semiquantitative RT-PCR and western blot analysis.
(3) Constructed ODC-overexpressing plasmid pcDNA-ODC and identified by restriction enzyme digestion and DNA sequencing.
(4) The antisense ODC expressing vector pcDNA-ODCr was transformed into E.coli to amplify, confirmed by restriction enzyme digestion and DNA sequencing.
(5) Transfected plasmid pcDNA3.1 and pcDNA-ODC into HEK293 cells with LipofectamineTM 2000, used G418 to select positive cells.3-4 weeks later, picked out the clone, digested with trypsinase and continued culturing to establish the mock transfectants and ODC transfectants.
(6) The expression levels of ODC and tumstatin protein were examined by RT-PCR and western blot analysis using total cell RNA and cell lysates from HEK293 cells treated by PBS、pcDNA3.1、pcDNA-ODC、pcDNA-ODC and pcDNA-ODCr, putrescine.
(7) Semiquantitative RT-PCR was used to detect the expression levels of ODC and tumstatin mRNA in HEK293 cells treated like above.
(8) Two luciferase reporter plasmids of tumstatin gene promotor pGL-tumstatin2.2kb and pGL-tumstatin0.5kb were constructed. Cotransfected pGL-tumstatin2.2kb and pRL-TK into HEK293 cells treated like above to observe the effect of ODC overexpression on tumstatin promoter activity by Dual Luciferas Reporter gene assay.
(9) CCK-8 assay was used to analyze the effect of conditioned media from HEK293 cells treated like above on the growth of ECV304.
Results:
(1) RT-PCR showed that thirty-two of thirty-eight cancer tissues overexpressed ODC mRNA compared with the normal renal tissues. And twenty-four of thirty-two ODC-overexpressing human renal cancers showed suppressed tumstatin mRNA expression. The result indicated that there was a correlation between ODC gene expression and tumstatin expression.
(2) RT-PCR showed that the expression of tumstatin was remarkably suppressed in the ODC-overexpressing cell lines ACHN and A549, compared with the corresponding normal cells (HEK293 and HELF), as determined by western blot.
(3) The ODC-overexpressing plasmid pcDNA-ODC was constructed sucessfully and confirmed by restriction endonuclease digestion and DNA sequencing.
(4) Restriction endonuclease digestion showed that the length of inserted ODC antisense RNA fragment is right. DNA sequencing further confirmed that the pcDNA-ODCr plasmid delivered a 120bp fragment complementary to the initiation codon of ODC gene.
(5) The results of RT-PCR and western bolt showed that the positive ODC-overexpressing cells selected by G418 highly expressed ODC mRNA and protein.
(6) ODC mRNA and protein were overexpressed in ODC transfectants, and the elevated ODC expression level returns to that of mock transfectants, while there were no changes in putrescine treated group, as comared with PBS treated group and mock transfectants. The expression of tumstatin in ODC transfectants and putrescine treated group at both the mRNA and protein levels was significantly suppressed. The suppression of tumstatin expression was rescued after pcDNA-ODCr transfected. VEGF mRNA expression levels in all HEK293 treated remained unchanged.
(7) The tumstatin promoter luciferase reporter plasmids pGL-tumstatin2.2kb and pGL-tumstatin0.5kb were constructed successfully. Both of them have promoter activity and pGL-tumstatin2.2kb presented stronger promoter activity than pGL-tumstatin0.5kb. Moreover, the activity of pGL-tumstatin2.2kb decreased about 45.8%、45.2% by ODC overexpression and putrescine respectively.
(8) Conditioned media from ODC transfectants and putrescine treated group induced 1.41±0.07 and 1.46±0.07 fold ECV304 cell proliferation respectively, compared with media from the mock transfectants (P<0.05).
Conclusions:
ODC overexpression and administration of putrescine could inhibite tumstatin expression which maybe the underlying mechanism that the elevated ODC expression levels facilitate endothelial cell proliferation (angiogenesis).
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