【作者】 衣翠华；

【导师】 侯明；

【作者基本信息】 山东大学 ， 内科血液病， 2007， 博士

【摘要】 HER2(Her2／neu)原癌基因是人类表皮生长因子受体2(Human Epidermal Growth Factor Receptor-type 2)的编码基因，所编码的蛋白为185kDa，并具有酪氨酸激酶活性。HER2在人类乳腺癌、卵巢癌、NSCLC、胃癌、结直肠癌等多种癌症中存在扩增及过量表达，并与肿瘤的侵袭性表型及生存期短密切相关。HER2癌基因的致癌机制包括：抑制凋亡，促进存活；促进肿瘤新生血管生成；增加肿瘤细胞的侵袭力等。HER2在20-25％的浸润性乳腺癌中存在过表达，并具有以下特点：(1)HER2水平与乳腺癌的发病及预后具有很强的相关性；(2)HER2在人类癌细胞中的基因扩增水平远远高于正常组织；(3)HER2在肿瘤细胞中以很高的比例存在，高表达HER2的肿瘤通常显示均一的、强烈的免疫组化染色；(4)HER2过表达不仅见于肿瘤原发灶，在转移灶中也有过表达。上述特点提示，抗HER2治疗会以大多数的肿瘤细胞为靶点，而对正常组织的毒性较低，并可能对所有部位的肿瘤都有效。因而HER2在乳腺癌中已成为重要的治疗靶点。目前抗HER2治疗的策略有：(1)以HER2蛋白为靶点的治疗，包括外源性阻断抗体、干扰受体异二聚体化抗体、抗HER2疫苗、酪氨酸激酶抑制剂、细胞内单链抗体等，其中曲妥珠单抗(Trastuzumab，Herceptin)是已进入临床应用的人源化单克隆抗体，与化疗联合能增加有效率、延长疾病进展时间及生存期。(2)以HER2基因为靶点的治疗，包括HER2基因启动子转录抑制剂、反义寡核苷酸(antisense oligonucleotides，ASOs)、核酶技术、反义RNA技术等。每种策略各有其优缺点。RNA干扰(RNA interference，RNAi)现象是由与靶基因序列同源的双链RNA(double-stranded RNA，dsRNA)引发的广泛存在于生物体中的序列特异性基因转录后的沉默过程。利用RNAi技术可以使mRNA发生特异性的降解，导致其相应的基因沉默，这种转录后基因沉默机制(post-transcriptional gene silencing，PTGS)最终抑制蛋白表达。dsRNA在细胞内发挥作用的是21-23核苷酸长的小分子干扰RNA片段(small interfering RNAs，siRNAs)。由于RNAi对染色体DNA序列的复制和转录过程不产生任何影响，具有高度的序列特异性和抑制基因表达的高效性，而且RNAi的效应可以在不同细胞间长距离传递和维持，或在某些生物中具有遗传性，所以迅速成为一种功能强大的研究哺乳动物中选择性抑制特异性基因表达和研究基因功能的工具。本研究目的：探讨RNAi技术沉默HER2基因的效果，以及HER2基因沉默对肿瘤细胞Taxol敏感性、Survivin、VEGF基因表达的影响。研究内容分两部分：1．siRNA表达质粒沉默HER2基因及其效果：GenBank查找HER2基因的mRNA序列(GenBank ID：NC-000017)，根据siRNA的设计原则，设计出3个针对HER2进行干扰作用的靶点，进行Blast基因同源性分析以保证基因沉默的特异性，RNA Draw软件分析mRNA二级空间结构。靶序列1(基因序列23194-23214)：5′-TCTCTGCGGTGGTTGGCATTC-3′，命名为H1；靶序列2(基因序列6945-6966)：5′-GGGAAACCTGGAACTCACCTAC-3′，命名为H2；靶序列3(基因序列27229-27251)：5′-AAGGGGCTGGCTCCGATGTATTT-3′，命名为H3；无义对照目标基因序列：5′-GACTTCATAAGGCGCATGC-3′，命名为CON。人工合成各单链DNA片段，退火形成发夹状siRNA DNA双链，插入到pGenesil-1 vector U6的BamHⅠ和EcoRⅠ之间。重组体转化大肠杆菌DH5α，筛选kana抗性克隆，提取质粒，分别对构建的表达载体pGenesil-1质粒(H1、H2、H3和CON)进行测序，经测序鉴定，确认得到的序列为所需序列。采用METAFECTENE转染试剂转染HER2高表达乳腺癌细胞株SK-BR-3及卵巢癌细胞株SK-OV-3，转染后72小时，实验细胞加入G418终浓度为250μg／ml的完全RPMI-1640培养液。筛选3周后大部分细胞死亡，存活的细胞为转染质粒稳定表达的细胞。细胞筛选出来后，采用RT-PCR和Western Blot法观察HER2基因的沉默效果；CCK-8比色分析检测细胞体外增殖活力及对Taxol的敏感性；流式细胞仪检测细胞周期。HER2特异性siRNA稳定转染SK-BR-3细胞后，SK-BR-3细胞形态学上表现为皱缩、变圆、脱落；筛选出的阳性克隆生长非常慢，难以形成单层贴壁状态，表明3个特异性HER2 siRNA对SK-BR-3细胞均有显著的生长抑制作用。但因细胞生长缓慢，未能扩大培养进行进一步检测。HER2特异性siRNA稳定转染SK-OV-3细胞后，细胞形态与亲本细胞相似，但生长速度较缓慢。RT-PCR和Western Blot检测结果显示：转染了特异性HER2 siRNA的细胞HER2 mRNA及p185蛋白水平均明显低于亲本细胞及转染无义对照序列的细胞；CCK-8比色分析显示HER2基因沉默的细胞存活分数明显低于HER2基因高表达的细胞(P=0.000)；流式细胞仪细胞周期分析也显示，HER2基因沉默的细胞处于凋亡状态及非增殖期的比例明显增加(P均＜0.01)，而处于合成期的比例却显著减少(P≤0.001)，细胞周期主要阻滞于G0／G1期。HER2基因沉默的SK-OV-3细胞Taxol剂量反应曲线右移，IC50值也明显升高，对Taxol的敏感性下降。2．HER2基因沉默对Survivin、VEGF基因表达的影响：采用RT-PCR和Western Blot法检测转染了特异性HER2 siRNA的SK-OV-3细胞Survivin与VEGF mRNA及相应蛋白的表达水平。结果显示HER2基因沉默的SK-OV-3细胞Survivin与VEGF mRNA及相应蛋白的表达均明显受抑制。结论：1．HER2特异性siRNA表达质粒对SK-BR-3细胞具有显著的生长抑制作用。2．HER2特异性siRNA表达质粒能够从mRNA及蛋白水平稳定、持久地沉默SK-OV-3细胞的HER2基因。3．HER2基因沉默可使SK-OV-3细胞增殖减慢，凋亡率增加，细胞周期阻滞于G0／G1期。4．HER2基因沉默可使SK-OV-3细胞对Taxol的敏感性下降。以HER2基因为靶点的RNAi治疗与Taxol化疗联合治疗卵巢癌需慎重。5．HER2基因沉默可在转录及翻译水平上抑制SK-OV-3卵巢癌细胞株Survivin与VEGF基因的表达。HER2的抑制凋亡、促进肿瘤新生血管生成作用至少部分是通过上调Survivin与VEGF基因来实现的。更多还原

【Abstract】 The HER2 gene is a member of the epidermal growth factor (EGF)-receptor (EGFR) family and encodes a 185 kDa protein with tyrosine kinase activity. The amplification or enhanced transcription of the HER2 gene is associated with the oncogenesis or increased severity of several types of cancers. The HER2 protein can dimerize with other members of the EGFR family, and it is thought that overexpression of the HER2 gene product provides mitogenic signals to tumor cells, resulting in increased growth potential and rendering them resistant to apoptotic factors. Overexpression of HER2 occurs primarily through amplification of the wild-type HER2 gene and is associated with poor disease-free survival and may be associated with resistance to certain types of chemotherapy.HER2 has become an important therapeutic target in breast cancer for several reasons. (1) HER2 levels correlate strongly with the pathogenesis and prognosis of breast cancer. (2) The level of HER2 in human cancer cells with gene amplification is much higher than that in normal adult tissues, potentially reducing the toxicity of HER2 -targeting drugs. (3) HER2 is present in a very high proportion of tumor cells, and tumors with high expression often show uniform, intense immunohistochemical staining, suggesting that anti- HER2 therapy would target most cancer cells in a given patient. (4) HER2 overexpression is found in both the primary tumor and metastatic sites, indicating that anti- HER2 therapy may be effective in all disease sites.HER2-targeting strategies include:(1) Strategies target HER2 protein, include exogenous blocking antibodies, heterdimerization disrupting antibodies, endogenous blocking antibodies induced by HER2 specific vaccines, tyrosine kinase inhibitors, single chain antibodies, etc. Trastuzumab, a humanized monoclonal antibody against the extracellular domain of HER2, is currently used for clinical therapy. Trastuzumab combined with chemotherapy increases response rates, time to progression, and survival. However, the majority of cancers that initially respond to trastuzumab begin to progress again within 1 year. (2) Strategies target HER2 gene, such as transcription repressors, antisense oligonucleotides, ribozymes, antisense RNA, etc. Each strategy has its advantages and disadvantages.RNA interference (RNAi) is a form of post-transcriptional gene silencing(PTGS) in which double-stranded RNA(ds RNA) induces degradation of the homologous endogenous transcripts, mimicking the effect of the reduction, or loss, of gene activity. Small interfering RNAs (siRNAs) of approximately 21-23bp mediate this sequence-specific mRNA degradation. RNAi blocks the expression of endogenous gene in a sequence-specific and high-effective manner. Its effects can be delivered between cells, even are hereditary. These characteristics make it a high-selective and powerful strategy for gene therapy and gene functional study.In our study, we constructed 3 siRNAs against separate regions of the HER2 mRNA, whose sequences are 5’-TCTCTGCGGTGGTTGGCATTC -3’ (named H1), 5’-GGGAAACCTGGAACTCACCTAC -3’ (named H2), 5’-AAGGGGCTGGCTCCGATGTATTT -3’ (named H3), separately; and one nonsense control sequence 5’-GACTTCATAAGGCGCATGC -3’ (named CON). They were directed into plasmids- pGenesil-1 vector. DNA sequencing of the plasmids verified the successful construction of 3 HER2-specific and one control siRNA vectors. Then, they were transfected into HER2- positive tumor cells, SK-BR-3 and SK-OV-3, with METAFECTENE. Positive clones of infected cells were selected for 21 days using G418 (250μg /ml). The ability of siRNAs to downregulate HER2 expression was detected by RT-PCR and Western blot. CCK- 8(Cell counting kit- 8) colorimetry was used for growth assays and taxol (paclitaxel) resistance detection. Cell cycle analysis was done with flow cytometry.After stable transfection with HER2-specific siRNAs, SK-BR-3 cells became wrinkled, rounded, some sheded from the growing surface under microscopy. The positive clones grew too slow to form a layer on the growing surface, which indicated significant growth suppression of HER2-specific siRNAs to SK-BR-3 cells. Further detections to these clones were not done because of the difficulty to acquire enough cells.Stable transfection with HER2-specific siRNAs in SK-OV-3 cells resulted in slower growth while similar morphology to parental cells. Detections with RT-PCR and Western Blot showed sequence-specific decrease in HER2 mRNA and protein levels. Survival fractions of HER2 gene silenced cells were statistically decreased compared with HER2 gene highly expressed cells (P =0.000). Cell cycle analysis showed increased numbers of HER2 gene silenced cells in non-proliferative and apoptotic stages (P all lower than 0.01), while decreased number in synthetic stage compared with those of HER2 gene highly expressed cells (P ≤0.001). The cell cycles of HER2 gene silenced cells mainly arrested at G0/G1. siRNAs treatment also resulted in markedly increased cellular taxol resistance in SK-OV-3 cells, consistent with a powerful RNA silencing effect.Knockdown of HER2 expression by siRNAs was also associated with decreased expression of apoptosis inhibitor survivin and the pro-angiogenic vascular endothelial growth factor(VEGF), suggesting that HER2 stimulates tumor growth at least in part by regulating apoptosis and angiogenesis. siRNA-mediated gene silencing of HER2 and decreasing the expressions of survivin and VEGF may be a useful therapeutic strategy for HER2-overexpressing breast or ovarian cancer.Conclusions: 1. Transfection with HER2-specific siRNA plasmids can suppress the growth of SK-BR-3 cells.2. Transfection with HER2-specific siRNA plasmids resulted in sequence-specific decrease in HER2 mRNA and protein levels stably and permanently in SK-OV-3 cells.3. HER2 gene silencing by HER2-specific siRNAs transfection resulted in antiproliferative and apoptotic responses in SK-OV-3 cells, caused cell cycle arrest at G0/G1.4. HER2 gene silencing also resulted in markedly increased cellular taxol resistance in SK-OV-3 cells.5. Knockdown of HER2 expression by siRNAs was also associated with decreased expression of apoptosis inhibitor survivin and the pro-angiogenic vascular endothelial growth factor(VEGF), suggesting that HER2 stimulates tumor growth at least in part by regulating apoptosis and angiogenesis.更多还原