【作者】 熊震；

【导师】 杨仕明；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 硕士

【摘要】 研究背景及目的恶性肿瘤是当前严重影响人类健康的最重要的疾病之一。而肿瘤的侵袭和转移是导致肿瘤患者死亡的主要原因。肿瘤的早期诊断和早期治疗固然是肿瘤患者获得长期生存的主要途径,但许多就诊的患者多已发生转移,而常规的手术和放、化疗疗效有限。如何有效延长这类患者的生存期显得尤为重要。肝素酶(Heparanase,Hpa)是目前发现的唯一能降解细胞外基质(Extracellular matrix,ECM)及基底膜(Base memebrance,BM)中乙酰肝素蛋白多糖(heparan sulfateproteoglycans,HSPG)的内源性糖苷内切酶。在正常组织中除淋巴细胞、骨髓等呈低表达外,在正常成熟的非免疫组织中(如心、肺、肝脏、骨骼肌及胰腺等)均不表达,但是在几乎所有的转移性恶性肿瘤细胞中普遍存在。目前的研究表明,肝素酶在诱导肿瘤血管生成、促进肿瘤侵袭和转移方面具有重要作用,而抑制肝素酶的表达可明显抑制肿瘤的增殖及转移。因此肝素酶作为一种肿瘤基因治疗的靶点已经受到越来越多的关注。RNA干扰(RNA interference,RNAi)是指小分子双链RNA(siRNA)转录后基因沉默。siRNA主要通过特异地与序列互补的mRNA结合,并促使其降解。RNAi技术近年来发展非常迅速,尤其是在2001年《Science》杂志首次报道siRNA可以诱导哺乳动物细胞特异性基因沉默后,RNAi逐渐成为生物医学研究中的一项重要的实验工具。目前RNAi技术已基本替代传统的反义核酸技术诱导特异性基因沉默而广泛应用到基因功能鉴定、基因表达转录后调控等热门领域,并为多种疾病的基因治疗提供了新的思路。基于以上分析,本实验拟通过RNAi技术诱导Hpa在HepG2肝癌细胞中沉默,并研究沉默Hpa后的HepG2细胞在肿瘤的生长、侵袭等各方面的变化情况,为Hpa作为肿瘤基因治疗的靶点提供理论依据。研究方法1.通过Genebank找到人肝素酶mRNA序列,应用网络在线工具设计3条干扰序列,经BLAST设计一组无关序列为阴性对照序列;设计包含干扰序列及无关序列的dsDNA并进行生物合成;双酶切pGenesil-1质粒:将合成的dsDNA克隆至酶切质粒;测序鉴定后进行质粒抽提及纯化。2.培养HepG2细胞,采用脂质体法将各重组干扰质粒稳定转染至HepG2细胞:G418抗性筛选3周;从各组细胞中挑取单克隆扩大培养。3.采用Real Time RT-PCR和Western blot分别检测干扰及未干扰的HepG2细胞肝素酶的表达,筛选有效的干扰序列。4.采用不同的实验技术检测肝素酶RNA干扰后HepG2细胞增殖能力、细胞周期、克隆形成能力、侵袭能力及体内成瘤能力的变化。实验结果1.通过www.genscript.com网站的Online tools找到三条肝素酶干扰序列,分别为:No.1(1214-1232):5′-GGCTATCTCTTCTGTTCAA-3′,No.2(167-185):5′-TCCTGTCCGTCACCATrGA-3′,No.3(611-629):5′.CTCAGTTGC TCC TGG ACTA-3′,及阴性对照序列5′.ACTACCGTTGTATAGGTGT-3′:并成功将包含各干扰序列及阴性对照序列的dsDNA克隆至质粒pGenesil-1,经测序鉴定后,证实所连入的序列与设计序列一致,分别命名为pGenesil-1/Hpa/siRNA-1、pGenesil-1/Hpa/siRNA-2、pGenesil-1/Hpa/siRNA-3、pGenesil-1/Hpa/siRNA-N。2.采用脂质体法将上述各组干扰质粒成功转染至HepG2肝癌细胞当中,经G418抗性筛选后,得到阳性克隆:扩大培养后各细胞株分别命名为HepG2/RNAi/1-1、HepG2/RNAi/2-2、HepG2/RNAi/3-1、HepG2/RNAi/3-3、HepG2/RNAi/N。3.Real Time RT-PCR结果表明,干扰组HepG2/RNAi/1-1、HepG2/RNAi/2-2及HepG2/RNAi/3-3中Hpa在mRNA表达水平明显下调,而HepG2/RNAi/3-1中的Hpa表达与阴性对照组和HepG2组无差别;Westem blot检测结果表明HepG2/RNAi/1-1与HepG2/RNAi/3-3两组中Hpa蛋白表达水平较HepG2组和HepG2/RNAi/N组显著下调,抑制效率分别达到57%、71%。4.采用Western blot检测不同代数干扰细胞肝素酶的表达,结果表明,在10代以前,Hpa/siRNA-1和Hpa/siRNA-3可以有效抑制HepG2肝癌细胞肝素酶蛋白的表达。5.MTT实验表明HepG2/RNAi/1-1与HepG2/RNAi/3-3组细胞增殖能力明显低于HepG2组和HepG2/RNAi/N组;平皿克隆形成实验表明HepG2/RNAi/1-1与HepG2/RNAi/3-3组单个细胞形成克隆的能力与HepG2组和HepG2/RNAi/N组相比显著降低(34±4,26±5 vs 138±7,123±22,p＜0.05);流式细胞生长周期实验表明,与HepG2和HepG2/RNAi/N细胞相比,HepG2/RNAi/1-1与HepG2/RNAi/3-3 Go/G1期细胞比例增加,而S期细胞比例下降;Transwell体外侵袭实验表明,HepG2/RNAi/1-1与HepG2/RNAi/3-3细胞穿膜数较HepG2和HepG2/RNAi/N细胞穿膜数明显减少(85.1±9.1±78.1±10.4 VS182.2±9.7,183.5±9.3,p＜0.05);裸鼠成瘤实验显示,HepG2、HepG2/RNAi/N细胞成瘤率为100%,虽然HepG2/RNAi/1-1细胞成瘤亦为100%,但裸鼠皮下肿瘤体积较HepG2、HepG2/RNAi/N细胞明显缩小(0.099±0.030 vs 0.585±0.135,0.690±0.099,p＜0.01),而HepG2/RNAi/3-3细胞裸鼠皮下未见肿瘤形成。结论1.通过生物信息学技术,选取3条人肝素酶RNA干扰序列及1条阴性对照序列;通过基因重组技术,成功构建3种肝素酶RNAi载体及阴性对照载体:通过脂质体转染及G418抗性筛选,成功筛选出不同载体的抗性克隆;通过Realtime Time PCR和Westernblot技术,成功筛选出肝素酶有效干扰序列:即No.1(1214.1232)5′-GGCTATCTCTTCTGTTCAA-3′,No.3(61l-629)5′-CTCAGTTGCTCCTGGACTA-3′;并确定在10代以前,上述干扰序列可以有效地抑制HepG2肝癌细胞肝素酶的表达。2.体外及体内实验证实,经过有效干扰后的HepG2肝癌细胞在细胞生长速度、单个细胞克隆形成能力、体外侵袭能力以及裸鼠皮下成瘤能力等方面均较HepG2细胞和阴性对照组显著下降。以上研究表明,我们所筛选的人肝素酶干扰序列能有效抑制肝癌的增殖、侵袭以及成瘤能力,为肝素酶RNA干扰在中晚期肝癌及其他肿瘤中的治疗作用提供了理论依据。更多还原

【Abstract】 Background and ObjectiveMalignant tumor is one of the most severe deseases that are threatening human health. Tumor invasion and metastasis are main causes leading to patients’ death.Although early diagnosis and treatment would be the best way for patients obtaining long term survival, many patients are found to be metastasized and with little hope on surgery,chemotherapy and radiotherapy.Hence how to last the survival time of these patients is becoming an ever important goal.Heparanase（Hpa）is the only endogenous endo-glycosidase found by now that is able to degradate heparan sulfate proteoglycans（HSPG） contained in extracellular matrix（ECM） and base memebrance（BM）.In normal cells,except lymphocyte and bone marrow cells, heparanase is not expressed in most mature nonimmune tissue（such as heart,lung,liver, skeletal muscle and pancreas）.However,it exists in almost all metastatic malignant tumor cells.Studies have shown that down-regulating expression of heparanase could obviously inhibit proliferation and metastasis of tumor cells.Heparanase is gaining more and more attention as a new target for tumor gene therapy.RNA interference（RNAi） is gene silencing after the transcription of micromolecule double strands RNA（siRNA）.siRNA selectively combines with it’s complementary mRNA and induces it’s degradation.The technology of RNAi has developed very fast recently.Specially after the report,on 2001 Science,of siRNA successfully inducing selective mammalian cell gene silencing,RNAi gradually becomes an important tool for biomedical research.Now RNAi technology has been vastly use in many hot field such as gene function test,post-transcriptional control of gene expression,replacing the conventional antisense nucleic acids method inducing specific gene silencing.It provides a new possibility of gene therapy for many kinds of desease. Based on above analysis,this study uses RNAi technology to induce Hpa silencing in liver cancer cell line HepG2,investigating the Hpa silenced HepG2 status on tumor growth, invasion and so on,providing theoretical basis for target choosing of tumor gene therapy.Method1.Heparanase mRNA sequence is available in Genebank.We selected 3 interference sequences through on line design tool in web site,and a group of independence sequence by BLAST as negative control group.We further designed dsDNA including interference sequences and independence sequence for synthesis.Then double restricted enzyme digested pGenesil-1 was ligated with synthesized dsDNA.After being verified by sequencing we had it amplified and extracted.2.HepG2 liver cancer cells were stablely transfected with each recombinant interference plasmid by DOTAP lipofection method.After 4 weeks of G418 selection,drug resistant clones were picked from each group for amplified culturing and were named as: HepG2/RNAi/N,HepG2/RNAi/1-1,HepG2/RNAi/2-2,HepG2/RNAi/3-1 and HepG2/RNAi/3-3,respectively.3.Real Time RT-PCR and Western blot were carried out to screen effective interference sequences.4.Changes of biological behaviour of HepG2 after heparanase RNA interference were investigated respectively on proliferation,cell cycle distribution,clone formation,invasion and tumorigenesis in nude mice.Result1.Three interference sequences were determined by online tools of www. genscript.com.They are:No.1（1214-1232）:5’ -GGCTATCTCTTCTGTTCAA-3’,No.2 （167-185）:5’ -TCCTGTCCGTCACCATTGA-3’,No.3（611-629）:5’ -CTCAGTTGCT CCTGGACTA-3’,and negative control sequence 5’ -ACTACCGTTGTATAGGTGT-3’; dsDNA that including interference and control sequences were successfully cloned into pGenesil-1 plasmid and confirmed correct by sequencing.These plasmids were named as pGenesil-1/Hpa/siRNA-1,pGenesil-1 /Hpa/siRNA-2,pGenesil-1/Hpa/siRNA-3 and pGenesil-1/Hpa/siRNA-N,respectively.2.Each group of recombinant interference plasmids was transfected into HepG2 cells by DOTAP lipofection method.After 3 weeks of G418 selection,drug resistant clones were picked and named as HepG2/RNAi/1-1,HepG2/RNAi/2-2,HepG2/RNAi/3-1, HepG2/RNAi/3-3 and HepG2/RNAi/N cells respectively.3.Real Time RT-PCR result showed that compared with HepG2 and HepG2/RNAi/N cells,mRNA level of Hpa was significantly down-regulated in the group of HepG2/RNAi/1-1,HepG2/RNAi/2-2 and HepG2/RNAi/3-3 cells except HepG2/RNAi/3-1 cells.Western blot result showed that Hpa protein level in HepG2/RNAi/1-1 and HepG2/RNAi/3-3 was significantly decreased compared with HepG2 and HepG2/RNAi/N cells.The inhibiting rate of heparanase protein was 57%and 71%respectively.This results confirm that sequence 5’- GGCTATCTCTTCTGTTCAA-3’（1214-1232） and 5’-CTCAG TTGCTCCTGGACTA-3’（611-629） are the effective interference sequences of heparanase.4.Western blot testing various generations of interfered cells for heparanase protein level showed that before the 10th generation,No.1（1214-1232） and No.3（611-629） interference sequences could effectively inhibit expression of heparanase protein.5.MTT result showed that cell proliferation ability of HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells was significantly lower than that of HepG2 and HepG2/RNAi/N cells.Plate clone formation experiment demonstrated that single cell clone formation capacity of HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells was obviously lower than that of HepG2 and HepG2/RNAi/N cells（34±4,26±5 vs 138±7,123±22,p＜0.05）.Flow cytometry demonstrated that No.1（1214-1232） and No.3（611-629） interference sequence could increase HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells arresting in G₀/G₁ and decrease proliferative index（PI） of HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells.Transwell in vitro invasion experiment showed that penetrated cell number in HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells was much lower than in HepG2 and HepG2/RNAi/N cells（85.1±9.1, 78.1±10.4 vs 182.2±9.7,183.5±9.3,p＜0.05）.Tumorigenesis in nude mice experiment demonstrated that HepG2 and HepG2/RNAi/N cells had a tumor formation ratio of 100%. Although HepG2/RNAi/1-1 cells also had a tumor formation ratio of 100%,but its subcutaneous tumor volume is much smaller than of HepG2 and HepG2/RNAi/N cells （0.099±0.030 vs 0.585±0.135,0.690±0.099,p＜0.01）,while no subcutaneous tumor formed in group of HepG2/RNAi/3-3 cells.Conclusion1.Through bioinformatics technology,we choose three heparanase RNA interference sequences and a negative control sequence.We successfully construct three heparanase RNAi plasmids which are then stablely transfected into HepG2 liver cancer cells.Drug resistant clones are picked out after G418 selection for 4 weeks.By realtime time RT-PCR and Western blot,we successfully screened that 5’-GGCTATCT CTTCTGTTCAA-3’ （1214-1232） and 5’-CTCAGTTGCTCCTGGACTA-3’（611-629） are the effective interference sequences of heparanase.Western blot further confirms that before the 10th generation,above interference sequences could effectively inhibit expression of heparanase protein in HepG2 cells.2.A serial of in vivo and in vitro study indicated that after effectively interfered by the above sequences,the cell growth velocity,single cell clone formation capacity,in vitro invasion capacity and tumorigenesis in nude mice are significantly decreased in HepG2/RNAi/1-1 and HepG2/RNAi/3-3 cells compared with HepG2 and HepG2/RNAi/N cells.The above results suggest that the heparanase RNAi sequences we screened can effectively inhibit HepG2 liver cancer cells in proliferation,invasion,and tumorigenesis in nude mice.The above heparanase RNAi sequences may therefore represent interesting candidates for the treatment of patients with advanced liver cancer and possibly also with other malignanices.更多还原