基于清热解毒功效的二甲双胍对前列腺癌细胞迁移的抑制作用及其机制研究

发布时间：2017-12-28 07:10

本文关键词：基于清热解毒功效的二甲双胍对前列腺癌细胞迁移的抑制作用及其机制研究　出处：《大连医科大学》2017年博士论文　论文类型：学位论文

【摘要】：一、背景据Global Burden of Disease Cancer Collaboration于2016年底在JAMA Oncology报道的数据显示,前列腺癌已经成为全球男性人群中最常见的恶性肿瘤,2015年约有160万例。我国全国肿瘤登记中心(NCCR)主任陈万青教授等发表在CA:A Cancer Journal for Clininicians期刊上的数据显示,2015年我国约有2.66万人死于前列腺癌,同时新增发病人数约达到6.03万;在我国虽然男性发病率最高的是肺癌和消化道癌(包括食道癌、胃癌和肝癌),但近10年来肺癌的发病率较为稳定,食道癌、胃癌和肝癌的发病率都有所下降,而前列腺癌的发病率和死亡率却都在持续上升。在原发性前列腺癌早期阶段,五年相对生存率接近100%,但是发生远端转移之后,相对生存率骤降至28%。《素问·宣明五气篇》中提到“肾主骨”,《素问·阴阳应象大论》也指出“肾生骨髓”,肾精化生为阴血以滋养骨髓和骨质,使骨质生长坚实;若肾有疽毒产生,则随血入髓伤骨。然肾主生殖,作为肾系统病变的前列腺癌,其远端转移最易发生在骨,约占91.1%。由于前列腺癌转移到骨或其他器官,到目前为止还是难以治愈的,所以死亡率很高。因此,找到有效的方法来抑制前列腺癌细胞迁移和侵袭,阻止其转移到其他器官,遏止痈疽之毒扩散,将是至关重要的。熏倒牛(Biebersteinia heterostemon Maxim.)和山羊豆(Galega officinalis L.)中的主要活性成分是山羊豆碱,但是由于山羊豆碱毒性较大,随后据其发挥降糖作用的分子结构,进一步合成一系列衍生物,通过药效和毒性筛选,最后获得了毒性小的双胍类衍生物,即二甲双胍。二甲双胍具有良好的降糖效果,这继承了山羊豆(在中世纪欧洲曾被用作缓解糖尿病病症)的优点,如今已经成为治疗2型糖尿病的一线药物。而从中医角度来说,消渴症阴虚为本、燥热为标,反映了二甲双胍具有清热而防阴津亏损之功效。熏倒牛是我国一种古老草药,它在《吾三卷香》和《晶珠本草》中都有记载,称其“花序和幼果”入药,性味苦寒,可治“痈疽疔疮”,有清热解毒及散肿止痛之效。二甲双胍继承了熏倒牛清热解毒之功效,显现出其消除痈疽和对热毒性肿瘤的潜在疗效,尤其是近年来多项流行病学研究和越来越多的证据表明二甲双胍能够降低包括前列腺癌在内的多种癌症风险,提高癌症患者的生存率。我们的前期研究表明二甲双胍能够通过诱导细胞周期阻滞和凋亡来抑制涎腺癌体外生长,通过体外和体内实验证明了二甲双胍能够靶向c-myc癌基因来抑制前列腺癌生长。细胞迁移是癌转移的关键步骤,推测二甲双胍有可能通过不依赖它对癌细胞增殖和凋亡的调控,而直接抑制前列腺癌的转移。已有报道二甲双胍可以通过抑制mtor和雄性激素受体等信号通路来抑制前列腺癌的生长。但是,对二甲双胍的抗癌特性还知之甚少,二甲双胍对前列腺癌细胞迁移的直接作用分子机制尚待研究。二、目的研究二甲双胍对前列腺癌细胞迁移的影响及其特异性分子机制,以便为精准抑制前列腺癌的转移找到特异性的靶点,进一步支持和加快推进二甲双胍作为清热解毒药物单独(单味药)或联合(配伍组方)用药治疗前列腺癌。三、方法1.利用细胞划痕实验检测二甲双胍对两种转移性前列腺癌细胞迁移的影响我们选取了两种不同的转移性前列腺癌细胞系(成骨性的c4-2b和溶骨性的pc-3),用二甲双胍处理24h(pc-3)或48h(c4-2b),同时利用细胞划痕实验检测二甲双胍处理对细胞迁移的情况。2.二甲双胍对前列腺癌细胞中suv39h1基因表达调控的研究2.1利用实时定量pcr检测二甲双胍处理后两种前列腺癌细胞(pc-3和c4-2b)中的suv39h1mrna水平;2.2利用放线菌素d实验,验证二甲双胍对suv39h1mrna水平的调控是通过抑制suv39h1的转录过程还是通过促进其mrna降解实现的;2.3利用放线菌酮实验,验证二甲双胍对suv39h1的调控是直接的还是间接的;2.4利用免疫印迹实验,检测二甲双胍处理24、48和72h后前列腺癌细胞中suv39h1的蛋白水平。3.建立稳定过表达细胞系,研究suv39h1基因过表达对前列腺癌细胞c4-2b迁移能力的影响3.1稳定过表达suv39h1或suv39h1Δset细胞系的建立;3.2利用免疫印迹实验和细胞免疫荧光实验检测二甲双胍处理对过表达细胞系中suv39h1表达的影响;3.3利用细胞划痕实验检测suv39h1和suv39h1Δset过表达对前列腺癌细胞迁移能力的影响;3.4利用结晶紫染色,检测suv39h1和suv39h1Δset过表达对前列腺癌细胞增殖的影响。4.采用前列腺癌组织微阵列(tissuemicroarray,tma)检测suv39h1和h3k9me2/3的蛋白水平利用免疫组织化学染色,检测人正常前列腺组织和不同分期前列腺癌组织中suv39h1和h3k9me2/3的蛋白水平。5.利用crispr-cas9技术敲除和稳定转染重新表达的方法,研究suv39h1对前列腺癌细胞迁移的调控作用5.1利用crispr-cas9技术敲除前列腺癌细胞pc-3中suv39h1基因,建立稳定表达的细胞系;5.2利用细胞划痕实验和transwell实验,检测suv39h1基因敲除对前列腺癌细胞pc-3细胞迁移能力的影响;5.3利用结晶紫染色,检测suv39h1基因敲除对前列腺癌细胞pc-3增殖的影响;5.4利用细胞划痕实验,检测二甲双胍对pc-3wt细胞和suv39h1敲除细胞迁移能力的抑制作用差异;5.5在suv39h1敲除细胞系的基础上,通过稳定转染分别携带人suv39h1和suv39h1Δset基因的逆转录病毒,实现suv39h1和suv39h1Δset基因的重新表达;5.6利用细胞划痕实验,检测suv39h1和suv39h1Δset基因的重新表达对敲除细胞迁移能力的影响。6.通过转录组测序,分析suv39h1基因敲除对前列腺癌细胞中基因表达和信号通路的影响6.1提取pc-3wt和suv39h1敲除细胞(ko1)总rna,进行转录组测序;6.2对转录组测序数据进行基因差异表达分析和生物学通路分析;6.3利用免疫印迹实验,验证suv39h1敲除对整合蛋白αv和β1表达水平的影响。7.fak(黏着斑激酶)及其磷酸化p-fak(y397)水平的检测利用免疫印迹实验,检测suv39h1敲除、重表达和二甲双胍处理的细胞中的fak及其磷酸化p-fak(y397)水平。8.抑制性转录因子的挖掘针对转录组测序的数据和encode项目的tfbschip-seq数据,利用r编程语言进行挖掘,结合功能分析,进而获得目的抑制性转录因子。四、结果1.二甲双胍对前列腺癌细胞迁移和suv39h1基因表达的影响1.1二甲双胍抑制前列腺癌细胞的迁移。利用细胞划痕实验,我们发现二甲双胍处理后,两种前列腺癌细胞系pc-3和c4-2b的细胞迁移能力都显著下降,分别降低了26%和59%。1.2二甲双胍抑制前列腺癌细胞中suv39h1的表达。利用实时定量pcr检测发现,二甲双胍处理48h,pc-3和c4-2b细胞中suv39h1的mrna和蛋白水平都明显降低。利用放线菌素d和放线菌酮实验显示,二甲双胍对suv39h1表达的调控是通过间接抑制suv39h1的转录实现的。2.suv39h1过表达对前列腺癌细胞迁移的影响以及临床相关性研究2.1suv39h1过表达促进了c4-2b前列腺癌细胞的迁移。通过稳定转染的方式,我们利用前列腺癌细胞系c4-2b,建立了稳定过表达suv39h1或suv39h1Δset的细胞系。利用细胞划痕实验,我们发现suv39h1过表达细胞的迁移能力显著提高,而suv39h1Δset过表达细胞的迁移能力没有显著变化;并且细胞增殖实验显示,suv39h1或suv39h1Δset过表达并未影响细胞的增殖能力。2.2suv39h1的表达水平与人前列腺癌的发展转移具有显著的临床相关性。对前列腺癌组织微阵列免疫组织化学染色后,我们发现suv39h1和h3k9me2/3水平与患者前列腺癌的发展和分期显著正相关;尤其在转移性前列腺癌组织中,suv39h1和h3k9me2/3都异常高表达。3.suv39h1基因敲除和重新表达对前列腺癌细胞迁移的影响3.1suv39h1敲除导致pc-3细胞迁移能力降低。利用crispr-cas9技术,我们将前列腺癌细胞系pc-3的suv39h1基因敲除,建立了两株独立的suv39h1敲除细胞系。对这两株敲除细胞系的细胞迁移检测发现,suv39h1的敲除显著降低了pc-3细胞的迁移能力。而这两株suv39h1敲除细胞系的增殖能力未发生显著变化。另外,利用细胞划痕实验对二甲双胍处理的pc-3wt和ko细胞迁移进行了检测,结果显示二甲双胍对suv39h1敲除细胞迁移的抑制作用变得不再显著。3.2suv39h1的重新表达使敲除细胞的迁移能力得到恢复。利用稳定转染的方式,我们在suv39h1敲除细胞系(ko1)中重新表达suv39h1或suv39h1Δset。根据transwell实验结果显示,suv39h1的重新表达使敲除细胞的迁移能力得到恢复,而suv39h1Δset的重新表达并未显著提高敲除细胞的迁移能力。4.suv39h1调控前列腺癌细胞迁移的分子机制研究4.1suv39h1调控整合素-fak信号通路。对转录组测序数据进行差异基因表达分析和生物学通路影响分析,我们发现与细胞运动和迁移有关的细胞粘附分子(celladhesionmolecules,cams)和ecm-受体交互作用(ecm-receptorinteraction)都在受suv39h1敲除影响最大的10个生物学通路之中,并发现其中的itgav和itgb1转录水平明显下调,经免疫印迹实验验证,其编码的整合蛋白αv和β1水平在敲除细胞和二甲双胍处理的细胞中也都显著降低。同时,整合素的直接下游分子黏着斑激酶(focaladhesionkinase,fak),在第397个酪氨酸(y397)的磷酸化水平在敲除细胞和二甲双胍处理细胞中都显著降低,并且在重表达细胞系中也与suv39h1的表达水平变化相一致。4.2抑制性转录因子介导了suv39h1对itgav和itgb1的表达调控。通过生物学分析结合功能分析挖掘,最后得到了6个介导suv39h1对itgav和itgb1调控的抑制性转录因子。五、结论本研究首次揭示了SUV39H1调控前列腺癌细胞运动迁移的分子机制;证明了SUV39H1通过整合素-FAK信号通路介导了清热解毒药二甲双胍对前列腺癌细胞迁移的抑制作用。该研究表明SUV39H1是抑制前列腺癌细胞迁移和侵袭的新靶标。
[Abstract]:Background: according to the data reported by JAMA Oncology at the end of 2016, according to the data of Global Burden of Disease Cancer Collaboration, prostate cancer has become the most common malignancy in the global male population, and there are about 1 million 600 thousand cases in 2015. China's National Cancer Registration Center (NCCR) director Professor Chen Wanqing published in the CA:A Cancer Journal for Clininicians journal data show that about 26 thousand and 600 people died of prostate cancer in China in 2015, at the same time the additional number of patients reached 60 thousand and 300; in China, although the male incidence was highest in lung cancer and cancer of the digestive tract (including esophageal cancer, gastric cancer and liver cancer), but the incidence rate of lung cancer in recent 10 years is relatively stable, esophageal cancer, gastric cancer and liver cancer incidence rates have declined, the sustained rise in morbidity and mortality of prostate cancer are. In the early stage of primary prostate cancer, the relative survival rate of five years was close to 100%, but the relative survival rate dropped to 28% after the distal metastasis. "Q - five" vision mentioned in the "kidney main bone", "basic questions, be as big of yin and Yang" also pointed out that "kidney procreates marrow", the kidney is Yin and blood to nourish the bone marrow and bone growth, solid; if kidney carbuncle poison, with blood into the injured bone marrow. However, as the renal disease of the kidney, the distal metastasis of the kidney is most likely to occur in the bone, accounting for about 91.1%. Because prostate cancer is transferred to bone or other organs, it is still difficult to cure so far, so the death rate is very high. Therefore, to find effective method to inhibit cancer cell migration and invasion of prostate, prevent the metastasis to other organs, prevent ulcer toxin diffusion, will be crucial. Smoked down cattle (Biebersteinia heterostemon Maxim.) and galegas (Galega officinalis L.) is the main active ingredient of galegine, but due to galegine toxicity, then according to its molecular structure of hypoglycemic effect, further synthesized a series of derivatives through screening, pharmacodynamics and toxicity, finally obtained double guanidine derivatives low toxicity, namely, metformin. Metformin has a good hypoglycemic effect. It inherited the advantage of goats (which was used as a mediating disease in medieval Europe), and now has become the first-line treatment for type 2 diabetes. From the point of view of traditional Chinese medicine, the yin deficiency of thirst is the standard of the yin deficiency, which reflects the effect of the metformin in clearing away heat and preventing the loss of yin and Tianjin. Smoked down cattle is a kind of ancient Chinese medicine, it is recorded in the three volume of "my sweet" and "jingzhubencao", called the "inflorescence and fruit" medicine, taste of bitter cold, cure "carbuncle furuncle, detoxification and loose swelling and pain effect. Metformin inherited smoked down cattle detoxification effect, showing its elimination and ulcer of noxious heat tumor therapeutic potential, especially in recent years, a number of epidemiological studies and increasing evidence suggests that metformin can reduce a variety of cancers including prostate cancer risk, improve the survival rate of cancer patients. Our previous studies indicate that metformin can inhibit the growth of salivary gland cancer in vitro by inducing cell cycle arrest and apoptosis. In vitro and in vivo experiments have proved that metformin can target c-myc oncogene to inhibit prostate cancer growth. Cell migration is a key step in cancer metastasis. It is speculated that metformin may directly inhibit the metastasis of prostate cancer without regulating its proliferation and apoptosis. It has been reported that metformin can inhibit the growth of prostate cancer by inhibiting the signaling pathways such as mTOR and androgen receptor. However, little is known about the anticancer properties of metformin, and the molecular mechanism of metformin's direct effect on the migration of prostate cancer cells remains to be studied. Objective to study the effect of metformin, two prostate cancer cell migration and its specific molecular mechanism, so as to inhibit the metastasis of prostate cancer to find the precise target specificity, further support and accelerate the metformin as Qingrejiedu drugs alone (single drug) or combined (prescription) drug for the treatment of prostate cancer. Three, 1. methods by cell scratch assay of metformin in two metastatic prostate cancer cell migration effect we selected two different metastatic prostate cancer cell lines (c4-2b osteoblastic and osteolytic PC-3), 24h treatment with metformin (PC-3) or 48h (c4-2b), at the same time by cell scratch test detection of metformin treatment on cell migration. The study of the regulation of 2.1 using real-time quantitative PCR detection of metformin after treatment of two prostate cancer cell 2. metformin on the expression of suv39h1 gene in prostate cancer cell lines (PC-3 and c4-2b) in the suv39h1mrna level; 2.2 by actinomycin D experiment, verify the regulation on the level of suv39h1mrna by metformin inhibits suv39h1 transcription or by promoting the implementation of the degradation of mRNA; 2.3 by cycloheximide experiments verify the regulation of metformin on suv39h1 is directly or indirectly; 2.4 by Western blotting, the protein level of metformin treatment for 24, 48 and 72h after the detection of suv39h1 in prostate cancer cells. 3. establish the stable expression cell line of suv39h1 gene over expression effects on prostate cancer cell migration ability of c4-2b 3.1 suv39h1 or suv39h1 set stable cell lines expressing; 3.2 using Western blot and immunofluorescence assay of metformin treatment on the effects of over expression of suv39h1 expression in cell lines by cell scratch; 3.3 the detection of suv39h1 and suv39h1. The effect of overexpression of set on prostate cancer cell migration; 3.4 by using crystal violet staining, the detection of suv39h1 and suv39h1. The effect of overexpression of set on proliferation of prostate cancer cells. 4., prostate cancer tissue microarray (tissuemicroarray, TMA) was used to detect the protein level of suv39h1 and h3k9me2/3. Immunohistochemical staining was used to detect normal prostate tissues and different tissues.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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