重组腺病毒介导的RNA干扰对肝肿瘤细胞的干预实验研究
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摘要
背景与目的：人肝细胞癌是世界范围最高发的恶性肿瘤之一，亚洲和非洲尤其高发，估计每年有五十万新发病人和一百万死亡病人。肝癌的病因已经研究得比较清楚，在中国主要是乙型病毒感染，近年来酒精性肝病有上升趋势，另外黄曲霉素、糖尿病、代谢性肝病、体重过大等都是危险因素。肝癌是多因素多步骤发病，涉及多个基因变异最终导致肝细胞恶变。由于肝癌致病原因多样发病机制复杂，其确切分子机制至今尚不十分清楚。各种致病因子导致肝细胞反复受损、反复修复，可能导致基因变异，例如细胞癌基因激活、抑癌基因失活、DNA配对错误、染色体损伤、生长因子和血管生长因子过度表达等。肝癌与肝硬化关系密切，70-90％的肝癌病人有肝硬化。肝癌的治疗可分为四类：外科治疗(肿瘤切除和肝移植)，经皮介入治疗(乙醇注射、射频热剥蚀)，经血管介入治疗(栓塞、化学药物灌注、化学药物栓塞)和药物治疗(包括基因和免疫治疗)。有望治愈肿瘤的方法包括肿瘤切除、肝移植和经皮介入治疗。经过选择的病人接受经动脉介入治疗可提高部分病人的生存时间。药物治疗和常规放射治疗没有明显疗效。外科治疗方面，不合并肝硬化的肝癌病人接受手术切除是危险性相对比较低，治疗效果比较肯定的选择，遗憾的是大多数病人合并肝硬化，切除范围受到限制，切除范围过大容易导致手术后肝衰竭。肝功能正常、肿瘤单发、无临床症状的肝癌病人手术效果比较好，五年生存率可以达到70％。但是肿瘤成功切除五年后，有70％的病人硬化的肝脏可能再发生癌症。肝移植应该是治疗肝癌比较有效的方法，能一次性切除肿瘤和硬化的肝脏，还能防止肿瘤复发，但是如果肿瘤体积大或者多发则治疗效果不佳，另外供肝来源不足也极大地限制了该方法的使用，该治疗方法的推广应用还面临着伦理、社会和法律等诸多问题。经皮介入治疗仅仅适用于瘤体比较小而且不能手术切除的肝癌病人，可通过乙醇注射、射频或微波热疗激光热疗、及冷冻等方法进行，使用范围有限。经动脉栓塞或化学栓塞治疗适用于既不能手术切除又不能通过经皮途径治疗的肝癌病人。有15-50％的病人对该治疗方法部分有效，与保守治疗比较能延长病人生存时间，肝功能差的病人无益处。总之传统治疗方法治疗效果不十分理想，病人预后差。
随着分子生物学的进展人们观察到一些基因改变与肝癌有关，例如：p53变异、c-myc和cyclin D1过度表达，杂合性缺失(loss of heterozygosity LOH)伴有肿瘤抑制基因失活，肝癌细胞中1p，4q，6q，8p，13q，16q and 17p位点经常发生LOH，另外几个生长因子也与肝癌的发生有关，包括转化生长因子α和β等。但是肝癌发生的分子机制尚不十分明了。
近来有越来越多的证据表明表皮生长因子(EGFR)是有希望的肿瘤治疗靶点，许多肿瘤显示EGFR异常增强或结构性EGFR表达。有报道人肝细胞癌EGFR表达，与肿瘤的侵袭性生长特性有关。尤其是低分化肝细胞癌EGFR过表达，是预后不良的指标，它与肿瘤早期复发和肝外转移正相关。因此EGFR是治疗肝细胞癌新方法的有希望的靶点。
近几年研究发现EGF/EGFR系统对肝细胞生长有强刺激作用。表皮生长因子受体(EGFR)是原癌基因erbB-1的表达产物，属Ⅰ型跨膜酪氨酸激酶糖蛋白生长因子受体，分子量约为170KD。配体与EGFR结合后，启动其信号转导，引起下游一系列的级联反应，主要通过两条途径将信号传递至细胞核，一条是Ras→Raf→MAPK(有丝分裂原活化蛋白激酶)途径，通过c-jun、c-fos将信号传导至核内激活AP-1；另一条是PI3K(磷脂酰肌醇3激酶)→PKC→IKK途径，使IκBα磷酸化后导致NF-κB移位至核内，最终将胞外信号传导至核内，促使细胞增殖、血管形成及抑制调亡，当EGFR过度表达或表达失调时会导致肿瘤形成。研究显示肝细胞癌常有EGFR过度表达或异常表达，抑制EGFR系统能抑制有EGFR表达的人肝癌细胞生长。目前，以EGFR为目标的治疗方略集中在细胞外配体结合部、细胞内酪氨酸激酶和配体。
临床前研究和临床研究显示多种单克隆抗体阻断细胞外EGFR与其配体的结合，具有一定抗肿瘤作用。在第一代抗EGFR抗体Cetuximab刚刚经过欧洲和美国使用核准几星期后，第二代抗EGFR抗体已经在澳大利亚进入最初的临床试验阶段。两篇发表于生物化学杂志的文章，提到这种新一代的EGFR抗体806，可以在正常细胞和癌细胞上辨别EGFR分子。目前市面上已经有一个抗EGFR抗体，更多的抗体正在进行临床试验。尽管这些抗EGFR抗体在患者体内表现出抗肿瘤的活性，但是它们离理想还有一段距离，因为它们多数无法区别癌细胞和正常细胞的EGFR。
这些第一代抗体会造成副作用，因为它们也把正常的组织当作攻击目标，虽然这些副作用是温和的，但却经常发生。更重要的是，第一代抗体的临床应用和能力均受到限制。806抗体的动物研究显示，它可以针对过度表达EGFR的癌细胞产生抑制作用。
Erlotinib是一种可口服的EGFR-TK活性抑制物，作用具有可逆性。Erlotinib的作用机制是竞争性抑制ATP与受体TK部位的结合，从而抑制EGFR的自动磷酸化。FDA已经批准该药为处方药。有研究显示Erlotinib导致的EGFR-TK抑制能通过诱导凋亡和使细胞周期停止在G1／S转变期而抑制人肝癌细胞生长。上述研究都是在EGFR形成后，再对其本身或配体与其结合后的下游过程进行干预。其主要不足是不能减少EGFR的生成，抑制作用不完全、不持久。
RNAi是指由内源性或外源性的双链RNA(double strain ribose nucleic acid dsRNA)介导，并引起细胞内同源靶基因表达沉默或抑制的效应，这一现象属于转录后的基因沉默机制。在RNAi反应过程中，双链RNA被核糖核酶-Ⅲ(Dicer酶)切割为21～23个核苷酸长度的小干扰核糖核酸(small interference ribose nucleic acid siRNA)，siRNA再与含有核酸内切酶的核酶复合物结合形成RNA诱导沉默复合体RISC(ribose nucleic acid induced silence complex RISC)，进而识别并和与siRNA具有完全互补序列的同源mRNA结合，将之裂解，引起基因沉默，从而特异高效率地抑制目的基因蛋白的表达。有研究表明，带有shRNA(小发夹RNA)表达框架的载体可以取得较高的RNA干扰效应，然而其作为抗肝细胞癌药物的潜能仍需实验验证。本实验的目的：1．用EGFR shRNA质粒载体对HepG2细胞(人肝癌细胞)进行体外RNA干扰实验，验证EGFR基因沉默效果。2．构建表达EGFRshRNA重组腺病毒，评价它对HepG2细胞生长抑制的效果。
方法：1．用重组pSIREN-hERa质粒转染HepG2细胞，通过RT-PCR、WesternBlot、MTT及流式细胞仪检测其RNA干扰后细胞的EGFR蛋白、mRNA表达改变及细胞周期和细胞增殖性能的变化，验证EGFR基因沉默效果。
2．将质粒载体中的U6 shRNA表达框架切下，插入到腺病毒DNA载体中，构建重组腺病毒载体(recombinant pAdeno-hER~(RNAi) DNA vector)，经人胚肾293细胞包装后获得用于RNA干扰的重组腺病毒(Adeno-hER~(RNAi) virus)。通过XhoⅠ酶切分析及PCR分析，鉴定重组腺病毒是否含有插入的目的序列。用100 pfu／cell的重组腺病毒感染HepG2细胞，分析处理后细胞的生物学特征变化，评价重组腺病毒的RNA干扰效率。
结果：1．DNA序列测定结果证实重组质粒载体pSIREN-hERs构建正确，插入序列位置正确。PCR及酶切鉴定结果，重组腺病毒含有目的插入序列。2．重组质粒pSIREN-hERa转染HepG2细胞后，RT-PCR，Western-blot，MTT及流式细胞仪检测结果显示：处理后细胞的EGFR mRNA、EGFR蛋白表达量下降，细胞增殖活性下降，细胞周期停滞及凋亡增加。3．重组腺病毒pAdeno-hER~(RNAi) virus感染HepG2细胞后Real Time RT-PCR、Western-blot及流式细胞仪检测结果显示：pAdeno-hER~(RNAi) virus处理后细胞的EGFR mRNA、EGFR蛋白表达量明显下降，凋亡增加，其中重组腺病毒处理后细胞的EGFR mRNA表达量比对照组下降了77.85％。
结论：重组腺病毒pAdeno-hER~(RNAi) virus介导的EGFR基因的RNA干扰可以下调EGFR基因的mRNA表达，通过降低细胞增殖活性，使细胞周期停滞及增加凋亡抑制肿瘤细胞的生长，该技术有望成为肝细胞癌及其它EGFR高表达肿瘤的有效辅助治疗方法。
Background and Objective:Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world and is estimated to cause half a million deaths annually. The incidence of HCC is dramatically increasing in the USA, Europe and Asia, due to high prevalence of chronic hepatitis B and hepatitis C virus infections, alcohol disease, diabetes and obesity. Unfortunately, the majority of patients suffer from advanced disease at presentation. Therefore curative local ablation, surgical resection of HCC, or liver transplantation can be achieved only in a minority of patients. Local tumor destruction, chemoembolization or systemic chemotherapy are the treatment options of advanced HCC. However, overall survival is poor. Apart from chemoembolization, which improves survival in well-selected patients with unresectable HCC, palliative treatment options do not appear to greatly improve overall survival. Therefore, innovative treatment approaches are urgently needed. Recently, evidence has been accumulated that the epidermal growth factor receptor (EGFR) is a promising target for cancer therapy. A great variety of tumors show abnormal, enhanced and/or constitutive expression of EGFR. Several reports indicate that EGFRs are expressed frequently in human HCC, most likely contributing to the aggressive growth characteristics of these tumors. Especially in poorly differentiated HCCs, EGFR overexpression has been demonstrated to be a negative prognostic factor, since it positively correlated with early tumor recurrence and the occurrence of extrahepatic metastasis. Hence, the EGFR is a promising target for innovative treatment strategies in HCC. Epidermal growth factor receptor (EGFR or erbB1) is a glycoprotein with a molecular weight of 170 000 with an intrinsic tyrosinespecific protein kinase, which is stimulated upon EGF binding. Activation of EGF receptor tyrosine kinase results in the generation of a number of intracellular signals that culminate in cell proliferation. The known downstream effectors of EGF receptor include PI3-K, RAS-RAF-MAPK signal pathways, and protein kinase C signaling pathways. EGF receptor signaling involves in cell growth, angiogenesis and DNA repair. Deregulated and excessive expression of EGFR, the transmembrane receptor tyrosine kinase , is a feature and/or cause of a wide range of human cancers, especially in epithelium cell originated carcinomas. Blockade of EGF receptor signaling pathway represents a new perspective on the development of novel and selective anticancer therapies. Although considerable progress has been made in the application of EGF receptor—targeted antibodies and small molecule tyrosinekinase inhibitors, none of these agents is curative.Current therapies of its expression are still need to be improved. In RNA interference (RNAi), duplexes of 21-23 nt RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. It is approved that vectors containing shRNA expressing cassette could induce good result of RNAi. RNAi has shown enormous promise in gene silencing, the potential of which in developing new methods for cancer therapy is still need tested. In this study we want firstly to filtrate an effective RNAi sequence by reconstructing shRNA expressing plasmid wector ,using which to investigate the biological features of A431 cells(human epithelium tumor cell) testing the efficiency of RNAi in vitro. Then to establish a shRNA expressing adenovirus from the U6 expressing cassette to further confirm the effect of RNAi in HepG2 cells (cancer cell line of human heaptocarcinoma) in vitro . METHODS: 1. RT-PCR、 Western Blot、 FCM(flow cytometry ) and MTT were performed on the HepG2 cell line transfected by recombinant pSIREN-hERa to test the biology alterations of phenotype in HepG2 cells in vitro. pSIREN-hERs consisted of three 69 base-pair oligos coding small hairpin RNA containing 19bp of sense sequence and of antisense sequence which specifically targeting EGFR gene which were inserted into the downstream of U6 promoter in RNAi-Ready pSIREN-Shuttle Vector to reconstruct RNAi expressing recombinant
plasmid vector for gene of EGFR ( recombinant pSIREN-hERs). 2. Transferred U6 shRNA expressing cassette from pSIREN-hERa to BD Adeno-X~(TM) Viral DNA to construct recombinant pAdeno-hER~(RNAi)DNA vector . Recombinant pAdeno-hER~(RNAi)DNA vector was packaged into HEK293 (human embryonic kidney) cells. Recombinant adenovirus ( Adeno-hER~(RNAi)virus) was harvested by lysing transfected cells. The presence of inserter expression cassette in recombinant adenovirus were verified by restriction analysis with Xho I digestion and PCR Analysis. HepG2 cells were infected by 100 MOI of recombinant virus for 72 hrs. Real-Time RT-PCR, Western-blot were applied to evaluate the efficiency of RNAi induced by recombinant virus.
Results: 1. Sequencing analysis results demonstrated that 69 bp oligos were inserted into expected site and the insertion sequence was exactly correct in recombinant pSIREN-hER vectors. recombinant pAdeno-hER~(RNAi) DNA vector and recombinant Adeno-hER~(RNAi)virus were verified by restriction and PCR Analysis that they have aim gene. 2. The mRNA expressing level of erbB-1 was depressed with recombinant pSIREN-hERa by RT-PCR analysis compared with control group in HepG2 cells treated. Western-blot and FCNM of HepG2 cell treated with recombinant pSIREN-hERa indicated that the recombinant pSIREN-hERa decreased the level of EGFR protein obviously with cell proliferation reduced due to induction of apoptosis and cell cycle arrest. 3. The results of by using recombinant virus of 100 MOI to infect HepG2 cells showed that mRNA expressing level of erbB-1 was depressed by 77.85% compared with control group ; a similar result was observed by western-blot.
CONCLUSION: siRNA-mediated inhibition of EGFR gene induced by shRNA expressing Adeno-hER~(RNAi)virus is capable of suppressing EGF receptor expression with significantly inhibiting cellular proliferation and tumor growth due to cell cycle arrest and apoptosis induced may constitute a useful approach in the treatment of human liver cancer and other EGFR over expressing carcinomas.
随着分子生物学的进展人们观察到一些基因改变与肝癌有关，例如：p53变异、c-myc和cyclin D1过度表达，杂合性缺失(loss of heterozygosity LOH)伴有肿瘤抑制基因失活，肝癌细胞中1p，4q，6q，8p，13q，16q and 17p位点经常发生LOH，另外几个生长因子也与肝癌的发生有关，包括转化生长因子α和β等。但是肝癌发生的分子机制尚不十分明了。
近来有越来越多的证据表明表皮生长因子(EGFR)是有希望的肿瘤治疗靶点，许多肿瘤显示EGFR异常增强或结构性EGFR表达。有报道人肝细胞癌EGFR表达，与肿瘤的侵袭性生长特性有关。尤其是低分化肝细胞癌EGFR过表达，是预后不良的指标，它与肿瘤早期复发和肝外转移正相关。因此EGFR是治疗肝细胞癌新方法的有希望的靶点。
近几年研究发现EGF/EGFR系统对肝细胞生长有强刺激作用。表皮生长因子受体(EGFR)是原癌基因erbB-1的表达产物，属Ⅰ型跨膜酪氨酸激酶糖蛋白生长因子受体，分子量约为170KD。配体与EGFR结合后，启动其信号转导，引起下游一系列的级联反应，主要通过两条途径将信号传递至细胞核，一条是Ras→Raf→MAPK(有丝分裂原活化蛋白激酶)途径，通过c-jun、c-fos将信号传导至核内激活AP-1；另一条是PI3K(磷脂酰肌醇3激酶)→PKC→IKK途径，使IκBα磷酸化后导致NF-κB移位至核内，最终将胞外信号传导至核内，促使细胞增殖、血管形成及抑制调亡，当EGFR过度表达或表达失调时会导致肿瘤形成。研究显示肝细胞癌常有EGFR过度表达或异常表达，抑制EGFR系统能抑制有EGFR表达的人肝癌细胞生长。目前，以EGFR为目标的治疗方略集中在细胞外配体结合部、细胞内酪氨酸激酶和配体。
临床前研究和临床研究显示多种单克隆抗体阻断细胞外EGFR与其配体的结合，具有一定抗肿瘤作用。在第一代抗EGFR抗体Cetuximab刚刚经过欧洲和美国使用核准几星期后，第二代抗EGFR抗体已经在澳大利亚进入最初的临床试验阶段。两篇发表于生物化学杂志的文章，提到这种新一代的EGFR抗体806，可以在正常细胞和癌细胞上辨别EGFR分子。目前市面上已经有一个抗EGFR抗体，更多的抗体正在进行临床试验。尽管这些抗EGFR抗体在患者体内表现出抗肿瘤的活性，但是它们离理想还有一段距离，因为它们多数无法区别癌细胞和正常细胞的EGFR。
这些第一代抗体会造成副作用，因为它们也把正常的组织当作攻击目标，虽然这些副作用是温和的，但却经常发生。更重要的是，第一代抗体的临床应用和能力均受到限制。806抗体的动物研究显示，它可以针对过度表达EGFR的癌细胞产生抑制作用。
Erlotinib是一种可口服的EGFR-TK活性抑制物，作用具有可逆性。Erlotinib的作用机制是竞争性抑制ATP与受体TK部位的结合，从而抑制EGFR的自动磷酸化。FDA已经批准该药为处方药。有研究显示Erlotinib导致的EGFR-TK抑制能通过诱导凋亡和使细胞周期停止在G1／S转变期而抑制人肝癌细胞生长。上述研究都是在EGFR形成后，再对其本身或配体与其结合后的下游过程进行干预。其主要不足是不能减少EGFR的生成，抑制作用不完全、不持久。
RNAi是指由内源性或外源性的双链RNA(double strain ribose nucleic acid dsRNA)介导，并引起细胞内同源靶基因表达沉默或抑制的效应，这一现象属于转录后的基因沉默机制。在RNAi反应过程中，双链RNA被核糖核酶-Ⅲ(Dicer酶)切割为21～23个核苷酸长度的小干扰核糖核酸(small interference ribose nucleic acid siRNA)，siRNA再与含有核酸内切酶的核酶复合物结合形成RNA诱导沉默复合体RISC(ribose nucleic acid induced silence complex RISC)，进而识别并和与siRNA具有完全互补序列的同源mRNA结合，将之裂解，引起基因沉默，从而特异高效率地抑制目的基因蛋白的表达。有研究表明，带有shRNA(小发夹RNA)表达框架的载体可以取得较高的RNA干扰效应，然而其作为抗肝细胞癌药物的潜能仍需实验验证。本实验的目的：1．用EGFR shRNA质粒载体对HepG2细胞(人肝癌细胞)进行体外RNA干扰实验，验证EGFR基因沉默效果。2．构建表达EGFRshRNA重组腺病毒，评价它对HepG2细胞生长抑制的效果。
方法：1．用重组pSIREN-hERa质粒转染HepG2细胞，通过RT-PCR、WesternBlot、MTT及流式细胞仪检测其RNA干扰后细胞的EGFR蛋白、mRNA表达改变及细胞周期和细胞增殖性能的变化，验证EGFR基因沉默效果。
2．将质粒载体中的U6 shRNA表达框架切下，插入到腺病毒DNA载体中，构建重组腺病毒载体(recombinant pAdeno-hER~(RNAi) DNA vector)，经人胚肾293细胞包装后获得用于RNA干扰的重组腺病毒(Adeno-hER~(RNAi) virus)。通过XhoⅠ酶切分析及PCR分析，鉴定重组腺病毒是否含有插入的目的序列。用100 pfu／cell的重组腺病毒感染HepG2细胞，分析处理后细胞的生物学特征变化，评价重组腺病毒的RNA干扰效率。
结果：1．DNA序列测定结果证实重组质粒载体pSIREN-hERs构建正确，插入序列位置正确。PCR及酶切鉴定结果，重组腺病毒含有目的插入序列。2．重组质粒pSIREN-hERa转染HepG2细胞后，RT-PCR，Western-blot，MTT及流式细胞仪检测结果显示：处理后细胞的EGFR mRNA、EGFR蛋白表达量下降，细胞增殖活性下降，细胞周期停滞及凋亡增加。3．重组腺病毒pAdeno-hER~(RNAi) virus感染HepG2细胞后Real Time RT-PCR、Western-blot及流式细胞仪检测结果显示：pAdeno-hER~(RNAi) virus处理后细胞的EGFR mRNA、EGFR蛋白表达量明显下降，凋亡增加，其中重组腺病毒处理后细胞的EGFR mRNA表达量比对照组下降了77.85％。
结论：重组腺病毒pAdeno-hER~(RNAi) virus介导的EGFR基因的RNA干扰可以下调EGFR基因的mRNA表达，通过降低细胞增殖活性，使细胞周期停滞及增加凋亡抑制肿瘤细胞的生长，该技术有望成为肝细胞癌及其它EGFR高表达肿瘤的有效辅助治疗方法。
Background and Objective:Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world and is estimated to cause half a million deaths annually. The incidence of HCC is dramatically increasing in the USA, Europe and Asia, due to high prevalence of chronic hepatitis B and hepatitis C virus infections, alcohol disease, diabetes and obesity. Unfortunately, the majority of patients suffer from advanced disease at presentation. Therefore curative local ablation, surgical resection of HCC, or liver transplantation can be achieved only in a minority of patients. Local tumor destruction, chemoembolization or systemic chemotherapy are the treatment options of advanced HCC. However, overall survival is poor. Apart from chemoembolization, which improves survival in well-selected patients with unresectable HCC, palliative treatment options do not appear to greatly improve overall survival. Therefore, innovative treatment approaches are urgently needed. Recently, evidence has been accumulated that the epidermal growth factor receptor (EGFR) is a promising target for cancer therapy. A great variety of tumors show abnormal, enhanced and/or constitutive expression of EGFR. Several reports indicate that EGFRs are expressed frequently in human HCC, most likely contributing to the aggressive growth characteristics of these tumors. Especially in poorly differentiated HCCs, EGFR overexpression has been demonstrated to be a negative prognostic factor, since it positively correlated with early tumor recurrence and the occurrence of extrahepatic metastasis. Hence, the EGFR is a promising target for innovative treatment strategies in HCC. Epidermal growth factor receptor (EGFR or erbB1) is a glycoprotein with a molecular weight of 170 000 with an intrinsic tyrosinespecific protein kinase, which is stimulated upon EGF binding. Activation of EGF receptor tyrosine kinase results in the generation of a number of intracellular signals that culminate in cell proliferation. The known downstream effectors of EGF receptor include PI3-K, RAS-RAF-MAPK signal pathways, and protein kinase C signaling pathways. EGF receptor signaling involves in cell growth, angiogenesis and DNA repair. Deregulated and excessive expression of EGFR, the transmembrane receptor tyrosine kinase , is a feature and/or cause of a wide range of human cancers, especially in epithelium cell originated carcinomas. Blockade of EGF receptor signaling pathway represents a new perspective on the development of novel and selective anticancer therapies. Although considerable progress has been made in the application of EGF receptor—targeted antibodies and small molecule tyrosinekinase inhibitors, none of these agents is curative.Current therapies of its expression are still need to be improved. In RNA interference (RNAi), duplexes of 21-23 nt RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. It is approved that vectors containing shRNA expressing cassette could induce good result of RNAi. RNAi has shown enormous promise in gene silencing, the potential of which in developing new methods for cancer therapy is still need tested. In this study we want firstly to filtrate an effective RNAi sequence by reconstructing shRNA expressing plasmid wector ,using which to investigate the biological features of A431 cells(human epithelium tumor cell) testing the efficiency of RNAi in vitro. Then to establish a shRNA expressing adenovirus from the U6 expressing cassette to further confirm the effect of RNAi in HepG2 cells (cancer cell line of human heaptocarcinoma) in vitro . METHODS: 1. RT-PCR、 Western Blot、 FCM(flow cytometry ) and MTT were performed on the HepG2 cell line transfected by recombinant pSIREN-hERa to test the biology alterations of phenotype in HepG2 cells in vitro. pSIREN-hERs consisted of three 69 base-pair oligos coding small hairpin RNA containing 19bp of sense sequence and of antisense sequence which specifically targeting EGFR gene which were inserted into the downstream of U6 promoter in RNAi-Ready pSIREN-Shuttle Vector to reconstruct RNAi expressing recombinant
plasmid vector for gene of EGFR ( recombinant pSIREN-hERs). 2. Transferred U6 shRNA expressing cassette from pSIREN-hERa to BD Adeno-X~(TM) Viral DNA to construct recombinant pAdeno-hER~(RNAi)DNA vector . Recombinant pAdeno-hER~(RNAi)DNA vector was packaged into HEK293 (human embryonic kidney) cells. Recombinant adenovirus ( Adeno-hER~(RNAi)virus) was harvested by lysing transfected cells. The presence of inserter expression cassette in recombinant adenovirus were verified by restriction analysis with Xho I digestion and PCR Analysis. HepG2 cells were infected by 100 MOI of recombinant virus for 72 hrs. Real-Time RT-PCR, Western-blot were applied to evaluate the efficiency of RNAi induced by recombinant virus.
Results: 1. Sequencing analysis results demonstrated that 69 bp oligos were inserted into expected site and the insertion sequence was exactly correct in recombinant pSIREN-hER vectors. recombinant pAdeno-hER~(RNAi) DNA vector and recombinant Adeno-hER~(RNAi)virus were verified by restriction and PCR Analysis that they have aim gene. 2. The mRNA expressing level of erbB-1 was depressed with recombinant pSIREN-hERa by RT-PCR analysis compared with control group in HepG2 cells treated. Western-blot and FCNM of HepG2 cell treated with recombinant pSIREN-hERa indicated that the recombinant pSIREN-hERa decreased the level of EGFR protein obviously with cell proliferation reduced due to induction of apoptosis and cell cycle arrest. 3. The results of by using recombinant virus of 100 MOI to infect HepG2 cells showed that mRNA expressing level of erbB-1 was depressed by 77.85% compared with control group ; a similar result was observed by western-blot.
CONCLUSION: siRNA-mediated inhibition of EGFR gene induced by shRNA expressing Adeno-hER~(RNAi)virus is capable of suppressing EGF receptor expression with significantly inhibiting cellular proliferation and tumor growth due to cell cycle arrest and apoptosis induced may constitute a useful approach in the treatment of human liver cancer and other EGFR over expressing carcinomas.
引文
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