siRNA靶向诱导肝素酶启动子甲基化对胃癌侵袭转移调控作用的研究
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摘要
肝素酶(Heparanase，HPA)是肿瘤发生、浸润和转移过程中的关键酶之一，是裂解硫酸乙酰肝素蛋白多糖的惟一酶类，通过降解硫酸肝素，破坏细胞外基质和基底膜完整性而发挥作用，并参与肿瘤血管生成，与肿瘤的侵袭转移密切相关。启动子区域CpG岛甲基化状态的改变与癌症发生发展过程中相关基因的转录状态在表观遗传学上面密切相关。甲基化作为表观遗传学改变的分子标志可以为癌症的早期检测、预防和治疗起到重大作用。本实验研究目的是以肿瘤侵袭、转移相关性基因肝素酶为靶标，明确胃癌组织和胃癌细胞株中肝素酶启动子区域的甲基化状态，筛选出诱导肝素酶启动子甲基化的siRNA序列，并建立肿瘤特异性siRNA转导体系；在细胞和整体水平，明确靶向siRNA诱导肝素酶启动子甲基化及转录沉默对胃癌细胞侵袭、转移的调控作用，为运用表观遗传学方法调控胃癌的侵袭、转移提供全新的视野。本研究拟提取胃癌组织、细胞株的基因组DNA，经亚硫酸氢钠修饰，设计甲基化引物，运用PCR、克隆测序等分子生物学方法检测肝素酶启动子CpG岛甲基化状态，确定甲基化干扰位点。靶向诱导肝素酶启动子甲基化及转录沉默的siRNA筛选。设计特异性siRNA，转染肿瘤细胞，采用巢式甲基化特异性PCR、亚硫酸氢盐修饰DNA序列分析检测siRNA对肝素酶启动子甲基化的诱导作用，采用Western Blot、实时定量PCR检测肝素酶在siRNA干扰后转录水平基因沉默前后的变化情况。实验结果发现在胃癌组织和细胞系中肝素酶启动子区域呈现低甲基化状态，且随肿瘤细胞分化程度的降低呈现不同程度的去甲基化，而相应的肝素酶表达则随分化程度的降低而增多，两者之间有明显的相关性；在siRNA干扰后不同分化程度胃癌细胞系后，肝素酶启动子区域甲基化状态发生明显变化，在特定干扰位点发生甲基化，而相应肝素酶表达则明显降低，但与分化程度无明显相关性。本实验通过检测胃癌中肝素酶基因启动子区域甲基化状态，发现其与肿瘤发生发展密切相关，且与肿瘤分化程度具有一致性，而且肝素酶在肿瘤中的表达情况也随甲基化状态发生改变，这与肝素酶在不同分化程度的胃癌组织中的表达情况相一致，说明肝素酶基因启动子区域甲基化状态与肝素酶表达密切相关，而肝素酶的表达情况与胃癌组织的侵袭性和转移恶能也密切相关，通过siRNA靶向干扰肿瘤侵袭、转移相关性基因肝素酶的启动子区域促使其甲基化可以明显降低肝素酶的表达，从而可以有效的从表观遗传学角度降低肿瘤的侵袭和转移。这为肿瘤的早期发现和有效治疗及预防转移提供了新的理论依据和实践方案。
第一部分
肝素酶在胃癌中表达情况及临床意义的研究
目的探讨肝素酶(heparanase)在胃癌组织和不同分化程度的胃癌细胞系中的表达情况，分析其表达情况的差异与胃癌临床病理特征的关系。
方法收集72例胃癌手术后组织标本，采用免疫组织化学链霉亲和素-生物素-过氧化物酶复合物法(streptavidinbiotin peroxidase complex，SABC)，免疫荧光方法(immunofluorescence，IF)，逆转录-聚合酶链反应(RT-PCR)和免疫蛋白印记法(Western Blot)检测胃癌组织和细胞系中肝素酶mRNA和蛋白的表达，并结合临床病理资料进行回顾分析。
结果72例胃癌组织中肝素酶的表达率为免疫组化77．8％(56／72)、RT-PCR 93．1％(67／72)和Western Blot 83．3％(60／72)。肝素酶蛋白表达与胃癌的肿瘤浸润深度、淋巴结转移、TNM分期相关(P＜0．05)。而与患者年龄、性别、肿瘤部位及大小关系之间的差异无统计学意义(P＞0．05)。RT-PCR与Western blot结果显示，肝素酶mRNA和蛋白在3株胃癌细胞株中均有表达，但表达水平有差异，其中以TMK-1中表达最强，SGC-7901次之，MKN-28表达最弱。
结论胃癌中肝素酶mRNA和蛋白表达增多，其与胃癌淋巴结转移、TNM分期、分化程度等临床病理资料密切相关；肝素酶表达对对胃癌的预测和预后的判断有一定的参考价值，为其诊疗提供了相当明确的理论依据。
第二部分
肝素酶启动子区域在胃癌中甲基化情况的解析
目的以肿瘤侵袭、转移相关性基因肝素酶为靶标，探讨并明确胃癌组织和胃癌细胞株中肝素酶启动子区域的甲基化状态。
方法提取不同不同分化程度和病理类型胃癌组织及细胞株基因组DNA，经亚硫酸氢钠修饰，专业软件设计甲基化引物，运用甲基化PCR(MSP)、甲基化克隆测序(BSP)等分子生物学方法检测肝素酶基因启动子区域CpG岛甲基化状态，并初步确定甲基化预干扰位点。
结果甲基化PCR检测肝素酶启动子区域发现在胃癌组织和细胞系中肝素酶启动子区域呈现低甲基化状态，且随肿瘤细胞分化程度的降低呈现不同程度的去甲基化，其中以TMK-1中表现非甲基化频率最强，SGC-7901次之，MKN-28最弱。亚硫酸氢盐修饰DNA序列(BSP)分析检测启动子区域14个CG位点的甲基化状态，不是所有的有非甲基化的组织中所出现甲基化的频率一致，且出现非甲基化的位点也并不固定。
结论肝素酶基因启动子区域在肿瘤组织中较正常组织呈现低甲基化状态，在包含转录因子结合元件EGR1附近的14个CG位点的甲基化频率随肿瘤细胞分化程度的降低而呈现不同程度的降低，但其具体去甲基化位点分布不一致，且并不固定。不同分化程度的肿瘤细胞株中肝素酶基因启动子区域的甲基化检测结果与组织标本中的甲基化频率相一致，为进一步体外探讨肝素酶基因启动子区域甲基化对胃癌肿瘤生物学行为的作用奠定了研究基础。
第三部分
siRNA靶向干扰肝素酶启动子对胃癌中甲基化影响情况的研究
目的研究siRNA干扰肝素酶启动子区域对胃癌中甲基化状态的影响情况，并筛选出诱导肝素酶启动子甲基化的siRNA序列，建立肿瘤特异性siRNA转导体系。
方法构建靶向诱导肝素酶基因启动子区域甲基化及转录沉默的短发卡小干扰RNA(shRNA)表达质粒pGenesil-1，转染表达肝素酶的不同分化程度的胃癌细胞株MKN-28(高分化)、SGC-7901(中分化)、TMK-1(低分化)，以实现对肝素酶表达的沉默，并对所设计不同位点序列siRNA进行筛选，为设计特异性siRNA转导体系和进一步探讨肝素酶在胃癌侵袭与转移中的作用打下坚实的基础。
结果重组质粒测序结果与Genebank中的肝素酶基因cDNA序列相符，转染MKN-28、SGC-7901、TMK-1细胞后，荧光显微镜下可观察到绿色荧光蛋白的表达；转染后甲基化PCR检测检测肝素酶启动子区域甲基化状态显示在site1、site2转染序列组细胞中均出现甲基化趋势，而在对照组中检测甲基化未见明显变化。TMK-1和SGC-7901细胞在site1、site2转染序列组中均出现甲基化趋势，而MKN-28细胞则只在site2转染序列组中有甲基化趋势。而在其他转染序列组中均未见明显甲基化状态的改变。
结论靶向肝素酶基因启动子区域诱导其甲基化shRNA表达载体构建无误，并针对不同甲基化位点筛选有效诱导肝素酶基因启动子区域甲基化的siRNA，随肿瘤细胞分化程度的降低，siRNA诱导其甲基化的频率升高，这为靶向特定性基因启动子区域CpG岛，而不是其编码区的siRNA能在人类细胞中诱导该基因DNA甲基化，并导致该基因在转录水平产生基因沉默提供了理论研究依据，为进一步阐明其作用机制建立了研究模型，并为将来高效精确靶向表观遗传调控基因表达奠定了一定的研究基础。
第四部分
靶向诱导肝素酶启动子甲基化对胃癌中肝素酶表达情况影响的研究
目的探讨靶向siRNA诱导肝素酶启动子甲基化对肝素酶表达的影响及对胃癌细胞侵袭、转移的调控作用，并探讨及其作用机制。
方法绘制转染前后各细胞组生长曲线，MTT法检测细胞增殖活力，通过Western Blot、实时定量PCR检测肝素酶在siRNA干扰前后转录水平基因沉默前后的变化情况，Transwell侵袭小室侵袭实验和黏附实验观察转染后各组细胞的侵袭能力的改变，评价siRNA诱导肝素酶启动子区域甲基化对肝素酶表达的表观遗传学调控作用及对胃癌侵袭转移的表观遗传调控作用。
结果转染siRNA干扰各组细胞后RT-PCR和Western Blot检测肝素酶表达结果显示，site1、site2转染序列组干扰效果最强，但其相应的肝素酶表达并不随细胞分化程度的降低而降低，其与肝素酶启动子区域甲基化状态各组之间的变化情况呈同向的趋势，两者之间有明显的相关性，有统计学意义(P＜0．01)。siRNA干扰后细胞生长周期检测细胞增殖情况结果显示各转染序列组中site1、site2转染序列组对细胞的增殖抑制作用最为明显，site3、site4组次之，而site5组作用最弱。细胞侵袭实验中site1、site2转染序列组穿膜细胞数显著低于空白对照组(48±4)(P＜0．05)，site3、site4组次之，而site5组作用最弱。细胞黏附实验中site1、site2转染序列组黏附细胞数显著低于空白对照组(48±4)(P＜0．05)，site3、site4组次之，而site5组作用最弱。
结论肝素酶基因启动子区域siRNA干扰不同分化程度胃癌细胞系后，肝素酶表达发生明显的变化，但并不随分化程度降低而降低，与分化程度无明显相关性。肝素酶表达的变化趋势与其在不同分化程度的胃癌组织中的表达情况不相一致的结果显示通过诱导肝素酶基因启动子区域甲基化虽然能够降低肝素酶的表达，但是其与肝素酶基因启动子区域甲基化频率的相关性并不具有统计学意义。其具体原因可能与所诱导的甲基化位点本身的功能以及诱导的甲基化的频率变化幅度有关，这涉及到siRNA诱导基因DNA甲基化的具体作用机制，有待进一步研究来证实。通过siRNA靶向干扰肿瘤侵袭、转移相关性基因肝素酶的启动子区域促使其甲基化可以明显降低肝素酶的表达，从而可以有效的从表观遗传学角度降低肿瘤的侵袭和转移，这为肿瘤的早期发现和有效治疗及预防转移提供了新的理论依据和实践方案。
Heparanase plays a key role in promoting tumor angiogenesis,invasiveness andmetastasis.This predominant enzyme is primarily responsible for cleaving heparin sulphate,the main polysaccharide constituent of extracellular matrix and basement membrane,thushaving become a novel target of tumor therapy.The change of status of methylation in thepromoter CpG island epigenetic relates to the transcription condition of coherent gene inthe process of tumor angiogenesis and development.Methylation could play an importantrole in detecting the tumor at early stage,and preventing and treating the tumor as anepigenetic molecules marker.
Heparanase is overexpressed in gastric carcinomas and significantly associated withthe tumor invasion and matsatasis.This study is to examine the expression of heparanase inthe gastric carcinomas specimens and diverse differential gastric celllines,and to explorethe status of methylation in the promoter CpG island,screen the siRNA sequence that couldinduce methylation of the promoter of heparanase,set up the tumor specific transduction system of siRNA mediated by tumor-specific promoter,identify the regulation effect totumor invasiveness and metastasis of siRNA inducing methylation targeting the promoterof heparanase in cell and integrity level,provide a new visual field to regulate tumorinvasiveness and metastasis by using epigenetic methods.
The alteration of status of methylation in the promoter CpG island was detected bynMSP and BSP assays.The change of heparanase expression of gastric cancer cell linesafter siRNA interfere was investigated by Western Blot,real time PCR.The invasiveability was detected by Transwell assay,the adhesiveness ability was evaluated byadherence test.
The expressions of heparanase mRNA and protein were significantly descend aftersiRNA interfering each group by pGenesil-1_site1,_site2,the other sites were notsignificantly descend meanwhile.The change tendency was not identical with thedifferentiation of the tumor cells,but was identical with the change of frequency ofmethylaiton in each group.The status of methylation in the promoter CpG island ofheparanase was hypomethylation,the frequence of methylation in the region of Thetranscription factor,early growth response 1 (EGR1)is associated with the inducibletranscription of the heparanase gene of diverse differentiation degree,however,the specificsite and the frequence of methylation was not identical.The frequence of methylation ingastric cancer cells lines were identical with the status of gastric tissues,so it was toestablish rationale to investigate the effects of methylation of promoter CpG island ofheparanase to gastric oncobiology.
The status of methylation in heparanase gene promoter region was significantlycorrelated with expression of heparanase.It reveal that the the status of methylation play animportant role in tumor invasiveness and metastasis.By using siRNA,the invasive abilitywas weaken in each cell groups,and the adherence ability was also suppressed.Thisresearch demonstrate the methylation in heparanase gene promoter could depress theexpression of heparanase,thus,it could effectively inhibit the tumor invasiveness and metastasis and provide a novel rationale and strategy for tumor therapy and preventingmetastasis and detecting tumor in the early stage.
PartⅠ
Retrospective Study on Expression of Heparanase in Gastric Carcinomaand Clinical Significance
Objective:To investigate the expressions of heparanase in the gastric carcinomasspecimens and diverse differential gastric cell lines,and to explore theircorrelation to clinical pathological features of gastric carcinoma.
Methods:The expressions of heparanase proteins in 72 cases of gastric carcinoma anddiverse differential gastric cancer cell lines were detected by SABCimmunohistochemistry and immunofluorescence.Analyses the relationshipbetween the heparanase and gastric clinicpathological features,and evaluatethe effects of heparanase in tumor invasiveness and metastasis by concordanceat gene and protein level.
Results:56 of the 72 colorectal carcinoma specimens (77.8%)were positive inheparanase by immunohistochemistry and 93.1% (67/72)by RT-PCR and83.3% (60/72)by Western Blot,the expression of heparanase was correlatedwith invasion depth and lymph node metastasis and TNM stage of gastriccarcinoma(P＜0.05).Heparanase expression was detected significantly in all ofgastric cancer cell lines,the order of heparanase expression is TMK-1＞SGC-7901＞MKN-28(P＜0.05).
Conclusion:The expression of the heparanase in gastric carcinoma tissues and diversedifferential gastric cancer cell lines may be associated with its progression andmetastasis and differentiation degree and TNM stage but not with otherclinicpathological features.Molecular targets of heparanase may also beimportant factors in the metastasis of gastric cardinoma and it could provideidentifying evidence to gastric diagnose and treat.
PartⅡ
Analysis of the Status of Methylation in the Region of Heparanase Promoter inGastric Carcinoma
Objective:To explore the status of methylation in the promoter CpG islands of heparanase,and to screen the CG sites for methylation.
Methods:Extract the DNA of genome of gastric carcinoma and diverse differentialgastric cancer cell lines,hydrosulfite modify the nucleic acid,detect the statusof methylation in the promoter CpG island by MSP and BSP methods.
Results:It was hypomethylation in the promoter CpG islands of heparanase by usingMSP detecting the region of the promoter,and significant relevance was foundbetween the status change and the differentiation degree.The order offrequency of methylaiton was TMK-1＞SGC-7901＞MKN-28(P＜0.05).Theresults of DNA sequencing were identical with this tendency.The frequency ofmethylaiton was not at equal pace in the diverse differentiation tissues and thesite of methylaiton was neither fixed in one place.
Conclusion:The status of methylation in the promoter CpG island of heparanase washypomethylation,the frequence of methylation in the region of Thetranscription factor,early growth response 1 (EGR1)is associated with theinducible transcription of the heparanase gene of diverse differentiation degree,however,the specific site and the frequence of methylation was not identical.The frequence of methylation in gastric cancer cells lines were identical withthe status of gastric tissues,so it was to establish rationale to investigate theeffects of methylation of promoter CpG island of heparanase to gastriconcobiology.
PartⅢ
The study of the effects of siRNA targeting heparanase gene promoter to methylationin gastric cancer cell lines
Objective:To screen the siRNA sequence that could induce methylation of the promoter ofheparanase,set up the tumor specific transduction system of siRNA mediatedby tumor-specifi-promoter.
Methods:The shRNA oligonucleotides targeting for heparanase gene promoter weresynthesized and cloned into pGenesil-1 to generate shRNA eukaryoticexpression vectors.The recombinant named as pGenesil-1_site1,_site2,_site3,_site4,and _site5.shRNA expression plasmids were identificated bysequencing.The eukaryotic expression vectors were transfected into diversedifferentiation cells:MKN-28(weI1)、SGC-7901(moderately)、TMK-1(poor).The green fluorescent protein (GFP)was detected by fluorescence microscope,and the silencing effects of the recombinant vectors were determined byRT-PCR.Screen the siRNA sequence sites for constructing the specifictransduction system and investigate the effects of heparanase in gastriccarcinoma subsequently.
Results:The recombinant sequence identified by sequencing was the same as thetargeting one.In the MKN-28、SGC-7901、TMK-1 cells transfected with therecombinant vectors,the expression of green fluorescent protein (GFP)wasdetected,the silencing effect of pGenesil-1_site1,_site2 was more evident thanother recombinant vectors.The methylaiton status of promoter was changedsignificant in pGenesil-1_site1,_site2 at SGC-7901、TMK-1 cells groups,andMKN-28 cells group was only significant in pGenesil-1_site2,the other groupsand control group were not significant in the changes of methylaiton status ofpromoter.
Conclusion:shRNA recombinant vector was established successfully by RNAi techniqueand effectively transfected into 3 diverse differentiation degree gastric cancercells lines.Screen the effectively siRNA sequence targeting differentmethylation sites to induce methylation of promoter.It provided the rationalefor siRNA effects of inducing methylation of DNA in gene promoter regionwhile not effects of the silencing gene expressing by targeting the encodingregion of DNA.
PartⅣ
The study of the effects of siRNA targeting heparanase gene promoter to theexpression of heparanase in gastric cancer cell lines
Objective:To investigate the role of siRNA targeting heparanase gene promoter inregulating the expression of heparanase and the tumor invasion and matsatasis.
Methods:The alteration of status of methylation in the promoter CpG islands wasdetected by nMSP and BSP assays.The change of heparanase expression ofgastric cancer cell lines after siRNA interfere was investigated by Western Blot,real time PCR.The invasive ability was detected by Transwell assay,theadhesiveness ability was evaluated by adherence test.
Results:The cell growth curve show that the growth was inhibited by siRNA inpGenesil-1_site1,_site2 sequence in each cells groups,and the order of the cellgrowth was_site1,_site2＞_site3,_site4＞_site5,the control group.In cellsinvasive test,cells of _site1,_site2 groups migrated into the gap more thanother groups and control group at same time-points after inducing the lesion.The counts of parental cells penetrating through membrane were 113±7,significantly more than 48±4 in control group (P＜0.05).The adherence cells of_site1,_site2 groups was 0.83±0.02,significantly higher than 0.38±0.01 incontrol group (P＜0.05).
Conclusion:siRNA suppress the expression of heparanase in the differentiation gastriccancer cells lines,and the change was not identical with the differentiation ofthe tumor cells,the research results show that inducing methylation of DNA inheparanase gene promoter region could suppress the expression of heparanase,but it was not significantly correlated with the frequency of methylation.Theactual reason need subsequently research to define.
第一部分
肝素酶在胃癌中表达情况及临床意义的研究
目的探讨肝素酶(heparanase)在胃癌组织和不同分化程度的胃癌细胞系中的表达情况，分析其表达情况的差异与胃癌临床病理特征的关系。
方法收集72例胃癌手术后组织标本，采用免疫组织化学链霉亲和素-生物素-过氧化物酶复合物法(streptavidinbiotin peroxidase complex，SABC)，免疫荧光方法(immunofluorescence，IF)，逆转录-聚合酶链反应(RT-PCR)和免疫蛋白印记法(Western Blot)检测胃癌组织和细胞系中肝素酶mRNA和蛋白的表达，并结合临床病理资料进行回顾分析。
结果72例胃癌组织中肝素酶的表达率为免疫组化77．8％(56／72)、RT-PCR 93．1％(67／72)和Western Blot 83．3％(60／72)。肝素酶蛋白表达与胃癌的肿瘤浸润深度、淋巴结转移、TNM分期相关(P＜0．05)。而与患者年龄、性别、肿瘤部位及大小关系之间的差异无统计学意义(P＞0．05)。RT-PCR与Western blot结果显示，肝素酶mRNA和蛋白在3株胃癌细胞株中均有表达，但表达水平有差异，其中以TMK-1中表达最强，SGC-7901次之，MKN-28表达最弱。
结论胃癌中肝素酶mRNA和蛋白表达增多，其与胃癌淋巴结转移、TNM分期、分化程度等临床病理资料密切相关；肝素酶表达对对胃癌的预测和预后的判断有一定的参考价值，为其诊疗提供了相当明确的理论依据。
第二部分
肝素酶启动子区域在胃癌中甲基化情况的解析
目的以肿瘤侵袭、转移相关性基因肝素酶为靶标，探讨并明确胃癌组织和胃癌细胞株中肝素酶启动子区域的甲基化状态。
方法提取不同不同分化程度和病理类型胃癌组织及细胞株基因组DNA，经亚硫酸氢钠修饰，专业软件设计甲基化引物，运用甲基化PCR(MSP)、甲基化克隆测序(BSP)等分子生物学方法检测肝素酶基因启动子区域CpG岛甲基化状态，并初步确定甲基化预干扰位点。
结果甲基化PCR检测肝素酶启动子区域发现在胃癌组织和细胞系中肝素酶启动子区域呈现低甲基化状态，且随肿瘤细胞分化程度的降低呈现不同程度的去甲基化，其中以TMK-1中表现非甲基化频率最强，SGC-7901次之，MKN-28最弱。亚硫酸氢盐修饰DNA序列(BSP)分析检测启动子区域14个CG位点的甲基化状态，不是所有的有非甲基化的组织中所出现甲基化的频率一致，且出现非甲基化的位点也并不固定。
结论肝素酶基因启动子区域在肿瘤组织中较正常组织呈现低甲基化状态，在包含转录因子结合元件EGR1附近的14个CG位点的甲基化频率随肿瘤细胞分化程度的降低而呈现不同程度的降低，但其具体去甲基化位点分布不一致，且并不固定。不同分化程度的肿瘤细胞株中肝素酶基因启动子区域的甲基化检测结果与组织标本中的甲基化频率相一致，为进一步体外探讨肝素酶基因启动子区域甲基化对胃癌肿瘤生物学行为的作用奠定了研究基础。
第三部分
siRNA靶向干扰肝素酶启动子对胃癌中甲基化影响情况的研究
目的研究siRNA干扰肝素酶启动子区域对胃癌中甲基化状态的影响情况，并筛选出诱导肝素酶启动子甲基化的siRNA序列，建立肿瘤特异性siRNA转导体系。
方法构建靶向诱导肝素酶基因启动子区域甲基化及转录沉默的短发卡小干扰RNA(shRNA)表达质粒pGenesil-1，转染表达肝素酶的不同分化程度的胃癌细胞株MKN-28(高分化)、SGC-7901(中分化)、TMK-1(低分化)，以实现对肝素酶表达的沉默，并对所设计不同位点序列siRNA进行筛选，为设计特异性siRNA转导体系和进一步探讨肝素酶在胃癌侵袭与转移中的作用打下坚实的基础。
结果重组质粒测序结果与Genebank中的肝素酶基因cDNA序列相符，转染MKN-28、SGC-7901、TMK-1细胞后，荧光显微镜下可观察到绿色荧光蛋白的表达；转染后甲基化PCR检测检测肝素酶启动子区域甲基化状态显示在site1、site2转染序列组细胞中均出现甲基化趋势，而在对照组中检测甲基化未见明显变化。TMK-1和SGC-7901细胞在site1、site2转染序列组中均出现甲基化趋势，而MKN-28细胞则只在site2转染序列组中有甲基化趋势。而在其他转染序列组中均未见明显甲基化状态的改变。
结论靶向肝素酶基因启动子区域诱导其甲基化shRNA表达载体构建无误，并针对不同甲基化位点筛选有效诱导肝素酶基因启动子区域甲基化的siRNA，随肿瘤细胞分化程度的降低，siRNA诱导其甲基化的频率升高，这为靶向特定性基因启动子区域CpG岛，而不是其编码区的siRNA能在人类细胞中诱导该基因DNA甲基化，并导致该基因在转录水平产生基因沉默提供了理论研究依据，为进一步阐明其作用机制建立了研究模型，并为将来高效精确靶向表观遗传调控基因表达奠定了一定的研究基础。
第四部分
靶向诱导肝素酶启动子甲基化对胃癌中肝素酶表达情况影响的研究
目的探讨靶向siRNA诱导肝素酶启动子甲基化对肝素酶表达的影响及对胃癌细胞侵袭、转移的调控作用，并探讨及其作用机制。
方法绘制转染前后各细胞组生长曲线，MTT法检测细胞增殖活力，通过Western Blot、实时定量PCR检测肝素酶在siRNA干扰前后转录水平基因沉默前后的变化情况，Transwell侵袭小室侵袭实验和黏附实验观察转染后各组细胞的侵袭能力的改变，评价siRNA诱导肝素酶启动子区域甲基化对肝素酶表达的表观遗传学调控作用及对胃癌侵袭转移的表观遗传调控作用。
结果转染siRNA干扰各组细胞后RT-PCR和Western Blot检测肝素酶表达结果显示，site1、site2转染序列组干扰效果最强，但其相应的肝素酶表达并不随细胞分化程度的降低而降低，其与肝素酶启动子区域甲基化状态各组之间的变化情况呈同向的趋势，两者之间有明显的相关性，有统计学意义(P＜0．01)。siRNA干扰后细胞生长周期检测细胞增殖情况结果显示各转染序列组中site1、site2转染序列组对细胞的增殖抑制作用最为明显，site3、site4组次之，而site5组作用最弱。细胞侵袭实验中site1、site2转染序列组穿膜细胞数显著低于空白对照组(48±4)(P＜0．05)，site3、site4组次之，而site5组作用最弱。细胞黏附实验中site1、site2转染序列组黏附细胞数显著低于空白对照组(48±4)(P＜0．05)，site3、site4组次之，而site5组作用最弱。
结论肝素酶基因启动子区域siRNA干扰不同分化程度胃癌细胞系后，肝素酶表达发生明显的变化，但并不随分化程度降低而降低，与分化程度无明显相关性。肝素酶表达的变化趋势与其在不同分化程度的胃癌组织中的表达情况不相一致的结果显示通过诱导肝素酶基因启动子区域甲基化虽然能够降低肝素酶的表达，但是其与肝素酶基因启动子区域甲基化频率的相关性并不具有统计学意义。其具体原因可能与所诱导的甲基化位点本身的功能以及诱导的甲基化的频率变化幅度有关，这涉及到siRNA诱导基因DNA甲基化的具体作用机制，有待进一步研究来证实。通过siRNA靶向干扰肿瘤侵袭、转移相关性基因肝素酶的启动子区域促使其甲基化可以明显降低肝素酶的表达，从而可以有效的从表观遗传学角度降低肿瘤的侵袭和转移，这为肿瘤的早期发现和有效治疗及预防转移提供了新的理论依据和实践方案。
Heparanase plays a key role in promoting tumor angiogenesis,invasiveness andmetastasis.This predominant enzyme is primarily responsible for cleaving heparin sulphate,the main polysaccharide constituent of extracellular matrix and basement membrane,thushaving become a novel target of tumor therapy.The change of status of methylation in thepromoter CpG island epigenetic relates to the transcription condition of coherent gene inthe process of tumor angiogenesis and development.Methylation could play an importantrole in detecting the tumor at early stage,and preventing and treating the tumor as anepigenetic molecules marker.
Heparanase is overexpressed in gastric carcinomas and significantly associated withthe tumor invasion and matsatasis.This study is to examine the expression of heparanase inthe gastric carcinomas specimens and diverse differential gastric celllines,and to explorethe status of methylation in the promoter CpG island,screen the siRNA sequence that couldinduce methylation of the promoter of heparanase,set up the tumor specific transduction system of siRNA mediated by tumor-specific promoter,identify the regulation effect totumor invasiveness and metastasis of siRNA inducing methylation targeting the promoterof heparanase in cell and integrity level,provide a new visual field to regulate tumorinvasiveness and metastasis by using epigenetic methods.
The alteration of status of methylation in the promoter CpG island was detected bynMSP and BSP assays.The change of heparanase expression of gastric cancer cell linesafter siRNA interfere was investigated by Western Blot,real time PCR.The invasiveability was detected by Transwell assay,the adhesiveness ability was evaluated byadherence test.
The expressions of heparanase mRNA and protein were significantly descend aftersiRNA interfering each group by pGenesil-1_site1,_site2,the other sites were notsignificantly descend meanwhile.The change tendency was not identical with thedifferentiation of the tumor cells,but was identical with the change of frequency ofmethylaiton in each group.The status of methylation in the promoter CpG island ofheparanase was hypomethylation,the frequence of methylation in the region of Thetranscription factor,early growth response 1 (EGR1)is associated with the inducibletranscription of the heparanase gene of diverse differentiation degree,however,the specificsite and the frequence of methylation was not identical.The frequence of methylation ingastric cancer cells lines were identical with the status of gastric tissues,so it was toestablish rationale to investigate the effects of methylation of promoter CpG island ofheparanase to gastric oncobiology.
The status of methylation in heparanase gene promoter region was significantlycorrelated with expression of heparanase.It reveal that the the status of methylation play animportant role in tumor invasiveness and metastasis.By using siRNA,the invasive abilitywas weaken in each cell groups,and the adherence ability was also suppressed.Thisresearch demonstrate the methylation in heparanase gene promoter could depress theexpression of heparanase,thus,it could effectively inhibit the tumor invasiveness and metastasis and provide a novel rationale and strategy for tumor therapy and preventingmetastasis and detecting tumor in the early stage.
PartⅠ
Retrospective Study on Expression of Heparanase in Gastric Carcinomaand Clinical Significance
Objective:To investigate the expressions of heparanase in the gastric carcinomasspecimens and diverse differential gastric cell lines,and to explore theircorrelation to clinical pathological features of gastric carcinoma.
Methods:The expressions of heparanase proteins in 72 cases of gastric carcinoma anddiverse differential gastric cancer cell lines were detected by SABCimmunohistochemistry and immunofluorescence.Analyses the relationshipbetween the heparanase and gastric clinicpathological features,and evaluatethe effects of heparanase in tumor invasiveness and metastasis by concordanceat gene and protein level.
Results:56 of the 72 colorectal carcinoma specimens (77.8%)were positive inheparanase by immunohistochemistry and 93.1% (67/72)by RT-PCR and83.3% (60/72)by Western Blot,the expression of heparanase was correlatedwith invasion depth and lymph node metastasis and TNM stage of gastriccarcinoma(P＜0.05).Heparanase expression was detected significantly in all ofgastric cancer cell lines,the order of heparanase expression is TMK-1＞SGC-7901＞MKN-28(P＜0.05).
Conclusion:The expression of the heparanase in gastric carcinoma tissues and diversedifferential gastric cancer cell lines may be associated with its progression andmetastasis and differentiation degree and TNM stage but not with otherclinicpathological features.Molecular targets of heparanase may also beimportant factors in the metastasis of gastric cardinoma and it could provideidentifying evidence to gastric diagnose and treat.
PartⅡ
Analysis of the Status of Methylation in the Region of Heparanase Promoter inGastric Carcinoma
Objective:To explore the status of methylation in the promoter CpG islands of heparanase,and to screen the CG sites for methylation.
Methods:Extract the DNA of genome of gastric carcinoma and diverse differentialgastric cancer cell lines,hydrosulfite modify the nucleic acid,detect the statusof methylation in the promoter CpG island by MSP and BSP methods.
Results:It was hypomethylation in the promoter CpG islands of heparanase by usingMSP detecting the region of the promoter,and significant relevance was foundbetween the status change and the differentiation degree.The order offrequency of methylaiton was TMK-1＞SGC-7901＞MKN-28(P＜0.05).Theresults of DNA sequencing were identical with this tendency.The frequency ofmethylaiton was not at equal pace in the diverse differentiation tissues and thesite of methylaiton was neither fixed in one place.
Conclusion:The status of methylation in the promoter CpG island of heparanase washypomethylation,the frequence of methylation in the region of Thetranscription factor,early growth response 1 (EGR1)is associated with theinducible transcription of the heparanase gene of diverse differentiation degree,however,the specific site and the frequence of methylation was not identical.The frequence of methylation in gastric cancer cells lines were identical withthe status of gastric tissues,so it was to establish rationale to investigate theeffects of methylation of promoter CpG island of heparanase to gastriconcobiology.
PartⅢ
The study of the effects of siRNA targeting heparanase gene promoter to methylationin gastric cancer cell lines
Objective:To screen the siRNA sequence that could induce methylation of the promoter ofheparanase,set up the tumor specific transduction system of siRNA mediatedby tumor-specifi-promoter.
Methods:The shRNA oligonucleotides targeting for heparanase gene promoter weresynthesized and cloned into pGenesil-1 to generate shRNA eukaryoticexpression vectors.The recombinant named as pGenesil-1_site1,_site2,_site3,_site4,and _site5.shRNA expression plasmids were identificated bysequencing.The eukaryotic expression vectors were transfected into diversedifferentiation cells:MKN-28(weI1)、SGC-7901(moderately)、TMK-1(poor).The green fluorescent protein (GFP)was detected by fluorescence microscope,and the silencing effects of the recombinant vectors were determined byRT-PCR.Screen the siRNA sequence sites for constructing the specifictransduction system and investigate the effects of heparanase in gastriccarcinoma subsequently.
Results:The recombinant sequence identified by sequencing was the same as thetargeting one.In the MKN-28、SGC-7901、TMK-1 cells transfected with therecombinant vectors,the expression of green fluorescent protein (GFP)wasdetected,the silencing effect of pGenesil-1_site1,_site2 was more evident thanother recombinant vectors.The methylaiton status of promoter was changedsignificant in pGenesil-1_site1,_site2 at SGC-7901、TMK-1 cells groups,andMKN-28 cells group was only significant in pGenesil-1_site2,the other groupsand control group were not significant in the changes of methylaiton status ofpromoter.
Conclusion:shRNA recombinant vector was established successfully by RNAi techniqueand effectively transfected into 3 diverse differentiation degree gastric cancercells lines.Screen the effectively siRNA sequence targeting differentmethylation sites to induce methylation of promoter.It provided the rationalefor siRNA effects of inducing methylation of DNA in gene promoter regionwhile not effects of the silencing gene expressing by targeting the encodingregion of DNA.
PartⅣ
The study of the effects of siRNA targeting heparanase gene promoter to theexpression of heparanase in gastric cancer cell lines
Objective:To investigate the role of siRNA targeting heparanase gene promoter inregulating the expression of heparanase and the tumor invasion and matsatasis.
Methods:The alteration of status of methylation in the promoter CpG islands wasdetected by nMSP and BSP assays.The change of heparanase expression ofgastric cancer cell lines after siRNA interfere was investigated by Western Blot,real time PCR.The invasive ability was detected by Transwell assay,theadhesiveness ability was evaluated by adherence test.
Results:The cell growth curve show that the growth was inhibited by siRNA inpGenesil-1_site1,_site2 sequence in each cells groups,and the order of the cellgrowth was_site1,_site2＞_site3,_site4＞_site5,the control group.In cellsinvasive test,cells of _site1,_site2 groups migrated into the gap more thanother groups and control group at same time-points after inducing the lesion.The counts of parental cells penetrating through membrane were 113±7,significantly more than 48±4 in control group (P＜0.05).The adherence cells of_site1,_site2 groups was 0.83±0.02,significantly higher than 0.38±0.01 incontrol group (P＜0.05).
Conclusion:siRNA suppress the expression of heparanase in the differentiation gastriccancer cells lines,and the change was not identical with the differentiation ofthe tumor cells,the research results show that inducing methylation of DNA inheparanase gene promoter region could suppress the expression of heparanase,but it was not significantly correlated with the frequency of methylation.Theactual reason need subsequently research to define.
引文
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