摘要
小细胞肺癌(small cell lung cancer, SCLC)是所有肺癌中恶性程度最高的一种类型，在肺癌中约占为15%～20%。小细胞肺癌具有倍增时间短，高侵袭性、高复发性及放化疗敏感的生物学特点，大多数诊断时已为广泛期，局限期仅占1/3，而局限期和广泛期小细胞肺癌5年生存率仅为10%和2%。近20年来，小细胞肺癌的治疗并未取得明显进展，尽管患者初始治疗敏感，但大多数患者最终均因复发或进展而死亡。如何提高小细胞肺癌治疗效果已成为重要研究课题。分子靶向治疗已在多种肿瘤治疗中取得成功，但在小细胞肺癌中尚未取得突破，主要原因为发病机制不清。因此，从癌基因异常表达分析入手，明确小细胞肺癌的分子病理机制，以探索更加合适的靶点，将可能为小细胞肺癌的治疗带来突破性进展。
研究背景：
Fli-1（Friend leukemia virus integration1,)属于Ets （E26transformation-specific）家族的转录因子，最早由Ben-David等在Friend病毒诱导的小鼠红白血病细胞中发现。Fli-1即可以通过特异性序列结合多种基因的启动子或增强子调节基因的转录活性而发挥转录因子的功能，还可以通过蛋白质之间的相互作用来调节其它基因的活性。在正常生理条件下，Fli-1主要存在于造血系统和血管内皮细胞中，在造血和血管内皮生成等方面具有重要调节作用。本研究课题组前期工作中已经在红白血病中取得重要突破，发现在红白血病细胞系中Fli-1发挥促进细胞增殖，抑制细胞凋亡的功能。深入研究发现SHIP-1为Fli-1直接调控的靶基因，Fli-1通过抑制SHIP-1的转录活性，促使红细胞分化受阻，诱导红白血病的发生。这些研究显示Fli-1在肿瘤发生发展中发挥了癌基因的功能，提示其可能是一个良好的治疗靶点。在实体瘤方面，Fli-1在90%的尤文氏肉瘤中以EWS/Fli-1融合基因的形式存在，直接促进了细胞向恶性转化和尤文氏肉瘤的发生。近年来，我们及其他学者的研究显示Fli-1在恶性黑色素瘤、乳腺癌、卵巢癌及结直肠癌等实体肿瘤存在高表达。但Fli-1在实体瘤中的具体生物学功能，特别是该基因的分子作用机理尚不清楚。对Fli-1调控肿瘤发生发展机制的阐明有赖于对其参与调控的信号通路以及下游靶基因的识别，但目前对此所知甚少，迫切需要进一步深入探索。
研究目的：
1.筛查Fli-1在实体瘤中的表达谱。
2.分析Fli-1与小细胞肺癌临床特征的相关性。
3.全面评估Fli-1在小细胞肺癌发生发展中所发挥的生物学功能。
4.探讨Fli-1促癌的分子机制，揭示Fli-1参与调控的信号通路以及下游靶基因。
研究方法：
通过免疫组化的方法筛查Fli-1在实体瘤中的表达谱，特别是在小细胞肺癌中的表达情况，统计学方法分析Fli-1与小细胞肺癌临床特征的相关性。应用小RNA干扰的方法对Fli-1进行瞬时干涉，MTT、细胞计数法、流式细胞仪、Annexin V/PI染色、Transwell和软琼脂克隆形成实验评估Fli-1对细胞增殖、细胞周期、凋亡、迁移及克隆形成能力的影响。建立逆转录病毒稳定干涉Fli-1的NCI-H446细胞模型，在整体动物水平研究Fli-1对裸鼠成瘤能力的影响。应用CO-IP(Co-Immunoprecipitation)、荧光素酶活性测试、western blot、RT-PCR等方法对分子机制进行探讨。
研究结果：
1．Fli-1在多种肿瘤中高表达，并且以小细胞肺癌最显著，且Fli-1的表达水平与肿瘤的分期呈正相关。
2．Fli-1具有促进小细胞肺癌细胞增殖、细胞周期、克隆形成和抑制凋亡的功能，说明Fli-1对肿瘤的起始、形成及恶性表型的维持发挥了重要的作用。
3．在整体动物实验中，发现Fli-1具有促进裸鼠体内成瘤能力的作用，在体内水平验证Fli-1的促癌功能。
4．Fli-1能够与Smad3蛋白发生相互作用，并能抑制Smad3的活性。
5．Fli-1可以明显抑制Smad3的磷酸化水平，但对其蛋白的本底水平没有影响。
6. Fli-1能够抑制Smad3下游靶基因的转录水平，参与调控TGF-β/smad信号通路。
研究结论：
本项目通过于组织水平、细胞水平、动物水平和分子水平上完成了对Fli-1基因功能的研究，揭示了Fli-1在小细胞肺癌中发挥促进细胞增殖、细胞周期、克隆形成及抑制细胞凋亡的促癌基因功能。并进一步探讨了Fli-1参与调控的信号通路，阐明了Fli-1通过直接与Smad3相互作用抑制其活性，调控Smad3下游靶基因的转录，进而参与调控TGF-β/smad信号通路。本研究揭示了新的肿瘤发生发展的分子机制，并为TGF-β/smad信号通路的完整研究提供了有力的新线索。此外，本研究也为开发以Fli-1为靶点的小细胞肺癌分子靶向治疗的研究提供了理论依据和实验基础。
The molecular mechanism of Fli-1promotes development of smallcell lung cancer through regulating TGFβ/smad signaling pathway
Small cell lung cancer (SCLC) is regarded as the most malignanttumor in all types of lung cancers, accounts for approximately15%-20%.Small cell lung cancer shared common characteristics of short populationdoubling time, high invasion and recurring, and sensitivity toradiotherapy and chemotherapy. The five-year survival of small cell lungcancer is10%for extended stage and2%for limited stage because manypeople are diagnosed at extended stage, limited stage were found only inabout one-third of patients. In the last two decades, there is littleprogression on treatment of small cell lung cancer. Although patientswith small cell lung cancer are sensitive to initial treatment, most patientsare going to be recurrent or progressive and due to die. It has been amajor research subjects to improve the therapeutic effects of small celllung cancer. Molecular targeting therapy have been successful in manykinds of tumor treatment, but failed in small cell lung cancer because themechanism of disease is unclear. Therefore, the molecular pathogenesisof small cell lung cancer is need to confirm further which should proceedwith analyzing of abnormal expression of oncogene proteins andexploring more appropriate molecular targets, which may bring breakthroughs to treatment of small cell lung cancer.
Research background:
Fli-1(Friend leukemia virus integration1) is an Ets (E26transformation-specific) transcription factor family member, was firstidentified as a target of proviral integration in F-MuLV-induced mouseerythroleukemia by Ben-david. As a transcription factor, Fli-1couldcombined with many the promoter regions and enhancers of the gene, orregulated transcription through protein-protein interaction. Under thephysiological conditions, Fli-1is mainly expressed on blood stem cellsand endothelial cells and plays an important regulatory role in endothelialcells growth. In the preceding work of our project group, we found Fli-1could promoted cell growgh inhibited and cell death. The study alsorevealed that SHIP-1is a direct target of Fli-1. The overexpression ofFli-1inhibited SHIP-1activities and induced erythroleukemiaprogression. These study show Fli-1play an oncogene role in tumorgeneration and development, may be a good therapeutic target. On solidtumor side, Fli-1was presented in almost90%Ewing sarcoma asEWS/Fli-1fusion gene, which directly contribute to cell transforminginto malignancy, then led to Ewing sarcoma. In recent years, our andothers study results showed Fli-1was high expressed in melanomas,breast cancer, ovarian cancer and colorectal cancer. However, the specificbiological functions of Fli-1in solid tumor and molecular mechanism of Fli-1remains unknown. Studies towards elucidating the mechanisms ofFli-1regulating tumor generation and development depends onidentification of the signaling pathway and target genes, However, weknew very little about it, and pressing need further deeper exploration andresearch.
Research objectives:
1. Screen the expression pattern of Fli-1in solid tumor.
2. Analyze the correlation between the expression level of Fli-1andclinical data.
3. Complete assess the function of Fli-1in generation and development ofsmall cell lung cancer.
4. Discuss the mechanism of Fli-1promotes cancer, clarify the signalpathway and target genes regulated by Fli-1.
Research method:
Screen the expression pattern of Fli-1in solid tumor usedimmunohistochemical staining, especially in small cell lung cancer.Analyze the correlation between the expression level of Fli-1and clinicaldata by statistical method. Down-regulation of Fli-1by small interferingRNA (siRNA), MTT, cell count, flow cytometry, Annexin V/PI stain,Transwell and soft gar assay were used to assess the affection of Fli-1incell growth, cell cycle, apoptosis, migrate and ability of colony forming.Establish NCI-H446cell model of stable interference Fli-1with retrovirus to study the impact of Fli-1on tumor formation in nude mice atwhole animal level. CO-IP(Co-Immunoprecipitation), luciferase reporterassay, western blot and RT-PCR methods were used to discuss themechanism.
Research result:
1. Fli-1was expressed in multiple tumors, and small cell lung cancer isthe most significant. The expression level of Fli-1was correlated withtumor stage.
2. Fli-1played a role of promote cell growth, cell cycle, colony formingand inhibit cell apoptosis, illustrated Fli-1have important function intumor initiate, formation and malignant phenotype maintain.
3. On the whole animal level, Fli-1could significantly inhibited in vivotumorigenic ability.
4. Fli-1interacts with Smad3and could significantly inhibited activity ofSmad3.
5. Fli-1could inhibited phosphorylation of Smad3but do not affectbackground level of Smad3.
6. Fli-1regulated the transcriptional activity of Smad3target genes andthe TGF-β/smad signal pathway.
Research conclusions:
This study completed the research on gene function of Fli-1at thecellular level, molecular level and animal level and revealed Fli-1could promote cell proliferation and colony forming ability and inhibitapoptosis as a cancer-promoting gene in small cell lung cancer. Weexplored molecular mechanism and demonstrated that Fli-1interacts withSmad3and inhibited its activity and the transcriptional activity ofdownstream target genes, and then involved regulation of TGFβ cellularsignal transduction pathway. Therefore, this research contributed tofurther understanding the molecular mechanism involved in tumorgeneration and development and provide new powerful clue forthoroughly research of TGF-β/smad signal pathway. Addition, our studyalso provided theoretical and experimental base for molecular targettherapy which specifically target Fli-1in small cell lung cancer.
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