EphA7在原发性肝细胞癌中的表达及其体外抑制研究
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摘要
原发性肝细胞癌是世界范围内发病率最高的恶性肿瘤之一,位居恶性肿瘤相关死亡原因的第三位,其中中国原发性肝细胞癌患者年死亡人数>20万,约占世界的53%。近年来,随着人们对肝癌的日益关注和重视及相关研究的逐渐深入,肝癌的发病原因及高危因素已基本明确,其发生发展过程中所涉及的病理变化也逐渐清楚,对高危患者的筛查方法更是得到了不断完善,但肝癌患者的术后生存时间并未得到明显提高。造成这种结果的主要原因是肝癌患者的病情比较隐匿,确诊时多为晚期,导致只有少数人可以接受根治性手术切除,多数肝癌患者只能接受姑息性手术,或依赖于化疗、放疗和其他如肝动脉栓塞化疗、无水酒精注射、射频消融、微波凝固等疗法,但这些方法治疗后易发生肝癌复发且远期疗效不理想。因此,深入研究肝癌的转移、复发的机制并探索有效的治疗措施仍是当前肿瘤研究的重点。
目前,关于肝癌新的治疗理念是以外科治疗为主同时联合应用多种方法的综合治疗。研究证实,综合治疗较单一治疗在治疗效果上优势明显。在这方面,基因治疗可被视为一个潜在的辅助治疗措施,迄今为止,已经完成的临床实验均证实,基因治疗的副作用在大多数情况下处于可以接受的范围内。由于基因治疗的作用机制与传统的治疗不同,因此将基因治疗方法和传统治疗方案的合理组合可以达到协同治疗效应。此外,改良的传统的治疗方法如TACE和PEI可以将基因治疗的载体送入肝癌内部,从而增加有效剂量并尽可能减少非靶细胞的副作用。RNA干扰技术是研究功能基因组学最有前途的工具,目前被广泛的应用在基因研究和基因治疗中。如今,人们利用这种技术研究已知或可疑的因素在肿瘤形成中发挥的作用,来探索肿瘤形成过程中新的调节因子,从而为疾病的治疗提供新的选择。
Eph(erythropoietin producing hepatoma cell line)家族是酪氨酸蛋白激酶受体中的最大亚族,参与了胚胎发育、细胞生长、组织塑形、神经系统信号传递等重要的生理过程。EphA7作为该家族的重要一员,定位于染色体6q16.1靠近断裂点的位置,其基因同源性较高且在人体内分布广泛。EphA7受体及其配体Ephrin结合后可形成双向信号传导,参与脊椎动物胚胎的早期发育和轴突导向等许多重要的生理过程,其在中枢神经系统发育、大脑连接处的形成、神经嵴细胞导向以及运动神经轴突形成过程中发挥着关键作用。以往对EphA7的研究多集中在生理方面,近几年来,其在肿瘤中所起的作用也被人们日益关注如在肺癌、结肠癌、胃癌、前列腺癌、多形性恶性胶质瘤等多种人类恶性肿瘤中均发现EphA7的异常表达。此外,有证据显示EphA7在肿瘤血管的形成、肿瘤细胞的增殖、侵袭和转移过程中发挥着重要作用。但总的来说,对EphA7在肿瘤中的研究尚处于资料和认识的探索、积累阶段。目前,国内外有关EphA7在肝癌中的研究报道很少,仅做了初步筛查,未能形成系统性研究,且基于较少数量的临床标本,有可能会影响结果的可靠性,仍需进一步的研究加以证实。因此,对EphA7在人类原发性肝癌中表达的情况进行检测,并从基因水平探索其对肝癌细胞生物学行为的影响,对于了解EphA7在肝癌发生发展中的作用机制、指导临床工作具有重要的意义。
本研究对EphA7在人类原发性肝癌、癌旁肝组织及正常肝组织中的表达情况进行检测,并分析其与原发性肝细胞癌临床病理学因素之间的关系,对两者的相关性及临床意义作初步探讨。在此基础上,构建针对EphA7基因的siRNA真核表达载体并转染肝癌细胞SMMC-7721,观察RNA干扰抑制EphA7的表达对肝癌细胞SMMC-7721生长、增殖和侵袭等生物学行为的影响。然后将转染后的肝癌细胞在裸鼠中建立移植瘤模型,验证EphA7的下调对肝癌细胞在活体动物体内生长等的影响,奠定利用该基因治疗肝癌的实验基础,为了解EphA7在肝癌发生发展中的作用机制、靶向EphA7基因治疗肝癌提供理论依据。本研究分为以下四个部分：
第一部分EphA7基因和蛋白在肝癌中的表达及临床意义
目的
观察EphA7基因和蛋白在原发性肝细胞癌中的表达,分析EphA7的表达与肝癌临床病理特征的关系并探讨其临床意义。
方法
收集40例肝癌组织及其相应的癌旁肝组织和10例正常肝脏组织标本。40例肝癌患者中男23例,女17例。年龄41-58岁,平均年龄48.3岁。Ⅰ-Ⅱ级16例,Ⅲ-Ⅳ级的24例。Ⅰ-Ⅱ期26例,Ⅲ期14例。分别采用RT-PCR、实时荧光定量PCR、免疫组织化学和Western blot方法检测上述标本中EphA7基因和蛋白的表达情况,并分析其于原发性肝癌临床病理因素的关系。
结果
1.40例肝癌组织及相应的癌旁肝组织和10例正常肝脏组织中均有EphA7基因的表达,其表达量分别为20.0711±32.0232、4.5184±9.4738和4.1764±4.7193(F=5.399,P<0.05)。统计分析显示肝癌组织中EphA7基因的表达量显著高于癌旁肝组织(P=0.015)和正常肝组织(P=0.013),而在旁癌肝组织和正常肝组织中EphA7基因的表达差异无统计学意义(P=0.998)。
2. EphA7 mRNA的表达与肝癌患者的年龄、肿瘤大小、临床分期和甲胎蛋白水平等各项临床病理指标均无关(P>0.05),而与肿瘤的分化程度、门静脉癌栓和淋巴结转移有关(P<0.05)。分化程度低的肝癌患者EphA7 mRNA的表达明显高于分化程度高者(P=0.030),有门静脉癌栓的患者EphA7 mRNA表达明显高于无门静脉癌栓者(P=0.017),有淋巴结转移者EphA7 mRNA表达明显高于无淋巴结转移者(P=0.013)。
3. EphA7蛋白的阳性表达主要定位于肝细胞和肝癌细胞的胞浆,以及纤维间隔内血管壁的内皮细胞。其中肝癌细胞中EphA7蛋白的阳性表达较强,染色较深。癌旁肝组织及正常肝脏组织虽也可见到EphA7蛋白的阳性表达,但颜色普遍较浅。Western blot分析发现EphA7蛋白在肝癌组织、癌旁肝组织和正常组织中的表达量分别为0.5752±0.2590、0.4019±0.2234和0.3169±0.1594(P=7.826,P<0.05)。肝癌组织组织中EphA7蛋白的表达量显著高于癌旁肝组织(P=0.001)和正常肝组织(P=0.003),而在癌旁肝组织和正常肝组织中的其表达差异无统计学意义(P=0.309)。
4.肝癌组织中EphA7蛋白的表达与肝癌患者的年龄、肿瘤大小、临床分期等临床病理指标均无关(P>0.05),而与肿瘤的分化程度、有门静脉癌栓、淋巴结转移和AFP水平有关(P<0.05)。分化程度低的肝癌患者EphA7蛋白的表达明显高于分化程度高者(P=0.001),有门静脉癌栓的患者EphA7蛋白的表达明显高于无门静脉癌栓者(P=0.008),有淋巴结转移者EphA7蛋白的表达明显高于无淋巴结转移者(P=0.033),AFP水平高者EphA7蛋白的表达显著高于AFP水平低者(P=0.013)
结论
EphA7不仅参与了正常肝脏细胞的生长发育过程,还与肝脏细胞的恶性转化密切相关。其可能在肝癌的恶性转化、侵袭和转移等生物学行为中发挥重要作用。
第二部分pRNA-siEphA7干扰表达载体的构建与鉴定
目的
成功构建针对酪氨酸蛋白激酶受体EphA7基因的RNA干扰真核表达载体pRNA-siEphA7。
方法
根据Genbank上人类EphA7基因的核苷酸序列及siRNA的设计原则,选择3段序列：638-656nt、713-731nt和1456-1474nt作为干扰片段,所有寡核苷酸末端均加上与质粒载体pRNA-U6.1/Neo对应的酶切识别位点及转录终止子,人工合成针对EphA7的发夹样结构siRNA互补双链寡核苷酸。经过退火、质粒线性化以及体外重组等过程,构建抑制EphA7基因表达的干扰载体pRNA-siEphA7-1、2和3。将干扰载体转染入感受态大肠杆菌DH5α后进行克隆和筛选,PCR鉴定及基因测序验证干扰载体的准确性。
结果
1.新合成的正义及反义寡核苷酸,经退火处理形成双链互补结构。经DNA片段纯化及回收后,在1.5%的琼脂糖凝胶上进行电泳,在UVP凝胶成像分析仪上对照Marker进行验证,可见100bp附近的清晰条带,与设计的寡核苷酸大小118bp相吻合。
2.以空载体pRNA-U6.1/Neo为对照,使用插入鉴定引物P1和P2进行PCR扩增筛选鉴定,得到310bp左右的扩增片段的阳性克隆,证明已经插入pRNA-U6.1/Neo载体。
3.对3个重组质粒进行测序,结果证实插入的寡核苷酸序列正确,进一步证实重组质粒成功构建。
结论
构建了针对EphA7基因表达的干扰载体pRNA-siEphA7-1、2和3,经克隆、筛选及鉴定,证实其插入的寡核苷酸序列正确,进一步验证了其准确性并证实干扰载体pRNA-siEphA7构建成功。
第三部分RNA干扰抑制EphA7基因表达对肝癌细胞株SMMC-7721生物学特性的影响
目的
观察转染干扰载体pRNA-siEphA7对肝癌细胞SMMC-7721的增殖活性、细胞周期以及体外侵袭能力等生物学行为的影响,筛选出抑制效果最强的干扰表达载体。
方法
以转染空载体和未转染的肝癌细胞SMMC-7721作为对照,将构建好的3组干扰载体pRNA-siEphA7-1、2和3转染至肝癌细胞SMMC-7721,通过G418筛选的方法建立稳定转染的肝癌细胞SMMC-7721/siEphA7。利用Real-time PCR和Western blot技术检测转染干扰载体前后肝癌细胞SMMC-7721中EphA7基因和蛋白的变化,MTT法测定转染干扰载体前后肝癌细胞SMMC-7721的增殖活性的改变,流式细胞技术分析转染干扰载体前后肝癌细胞SMMC-7721细胞周期的变化,Transwell小室侵袭实验检测转染干扰载体前后对肝癌细胞SMMC-7721体外侵袭能力的影响。
结果
1.通过荧光倒置显微镜,可发现转染干扰载体pRNA-siEphA7-1、2和3、转染空载体的肝癌细胞SMMC-7721中均可见清晰的绿色荧光,证明其转染成功。而未转染的肝癌细胞SMMC-7721则不见荧光。
2.实时荧光定量PCR结果显示,转染干扰载体pRNA-siEphA7-1、2和3、转染空载体和未转染的的肝癌细胞SMMC-7721中EphA7基因的相对表达量分别为：0.1911±0.0153、0.1488±0.0199、0.0874±0.0123、0.4293±0.0533、0.4311±0.0410。经统计分析显示三组转染干扰载体的肝癌细胞中EphA7基因的表达明显低于空载体转染组和未转染组,差异有统计学意义(P<0.05),在三组转染干扰载体的肝癌细胞中,转染pRNA-siEphA7-3的肝癌细胞中EphA7基因的表达最低,与其他两组相比差异有统计学意义(P<0.05)。转染pRNA-siEphA7-1和2的两组肝癌细胞中EphA7基因的表达差异无统计学意义(P>0.05)。转染空载体和未转染载体的肝癌细胞中EphA7基因的表达差异无统计学意义(P>0.05)
3. Western blot结果显示转染干扰载体pRNA-siEphA7-1、2和3、转染空载体和未转染的的肝癌细胞SMMC-7721中EphA7蛋白的相对表达量分别为：0.2786±0.0392、0.3011±0.0372、0.2142±0.0147、0.5822±0.0291和0.5832±0.0154。三组转染干扰载体的肝癌细胞中EphA7蛋白的表达低于空载体转染组和未转染组,差异有统计学意义(P<0.05)。其中转染pRNA-siEphA7-3后对肝癌细胞中EphA7蛋白表达抑制最为明显,显著低于另外两个干扰载体转染组,差异有统计学意义(P<0.05)。转染pRNA-siEphA7-1和2的两组肝癌细胞之间,以及转染空载体和未转染的肝癌细胞之间EphA7蛋白的表达差异无统计学意义(P>0.05)
4.经MTT法检测转染干扰载体pRNA-siEphA7-1、2和3、转染空载体及未转染的肝癌细胞SMMC-7721的体外增殖能力并绘制生长曲线,发现第1天各组之间细胞增殖速度无明显差异(P>0.05)。自第2天开始,转染干扰载体的三组肝癌细胞增殖速度明显低于空载体转染组和未转染组(P<0.05),但转染干扰载体的三组肝癌细胞之间增殖速度无明显差异(P>0.05)。自第3天开始,转染pRNA-siEphA7-3的肝癌细胞增殖速度开始低于转染干扰载体pRNA-siEphA7-1和2的肝癌细胞,并持续至第7天(P<0.05)。自第5天开始,转染干扰载体pRNA-siEphA7-1的肝癌细胞增殖速度开始低于转染pRNA-siEphA7-2的肝癌细胞,并持续至第7天(P<0.05)。而空载体转染组和未转染组的肝癌细胞之间增殖速度始终没有统计学差异(P>0.05)。
5.流式细胞检测结果发现转染干扰载体pRNA-siEphA7-1、2和3、转染空载体和未转染载体的肝癌细胞SMMC-7721在G1期、G2期和S期的细胞分布比例基本相近,差异无统计学意义(P>0.05)。
6.Transwell小室侵袭实验结果发现转染干扰载体pRNA-siEphA7-1、2和3的肝癌细胞SMMC-7721穿过人工基底膜的细胞分别是23.6±3.65、27±5.57和13.4±3.43.pRNA-siEphA7-3转染组的肝癌细胞穿膜细胞数明显低于pRNA-siEphA7-1和2转染组的穿膜细胞数,差异有统计学意义(P<0.05)。而pRNA-siEphA7-1和2转染组的肝癌细胞穿膜细胞计数无统计学差异(P>0.05)。转染干扰载体的三组肝癌细胞穿膜细胞计数明显低于空载体转染组(68.6±6.50)和未转染组(72±9.30),差异有统计学意义(P<0.05)。空载体转染组和未转染组相比,其穿膜细胞计数差异无统计学意义(P>0.05)。
结论
转染干扰载体pRNA-siEphA7-1.2和3后,肝癌细胞SMMC-7721中EphA7基因及蛋白的表达明显受到抑制。EphA7的下调使肝癌细胞SMMC-7721的增殖活性明显降低、体外侵袭能力减弱,但细胞周期的变化不大。3组干扰载体中以pRNA-siEphA7-3(1456-1474nt)的干扰效果最好。
第四部分RNA干扰抑制EphA7基因表达对裸鼠肝癌移植瘤生长的影响
目的
通过建立裸鼠肝癌移植瘤模型,在活体动物体内观察siRNA阻断EphA7基因表达对肝癌细胞SMMC-7721生长的影响。
方法
裸鼠荷瘤模型采用左上肢皮下注射肝癌细胞的方法建立。将32只裸鼠随机分为4组,每组8只,根据皮下注射细胞的不同分为以下几组：SMMC-7721组(注射肝癌细胞SMMC-7721)；SMMC-7721/siEphA7组(注射转染干扰载体pRNA-siEphA7-3的肝癌细胞SMMC-7721);SMMC-7721/Vector组(注射转染空载体的肝癌细胞SMMC-7721);PBS组(不注射细胞,仅注入PBS做对照)。移植瘤模型成功建立后5周以颈椎脱臼法处死裸鼠,观察并比较各组肝癌移植瘤的大小及重量,应用Real-time PCR、免疫组织化学技术以及Western blot检测荷瘤组织中EphA7基因和蛋白的表达情况。
结果
1.注射细胞约9-12天后,除PBS组外其余各组均可在注射部位的发现肝癌移植瘤的形成,证明肝癌细胞SMMC-7721、转染干扰载体以及空载体的肝癌细胞SMMC-7721在裸鼠体内均有成瘤活性,可成功建立裸鼠肝癌移植瘤模型。
2.移植瘤模型建立后第35天,SMMC-7721组、SMMC-7721/siEphA7-3组和SMMC-7721/Vector组的移植瘤体积分别为2.19±1.18cm3、0.84±0.32cm3和2.33±0.97cm3,重量分别为1.78±0.58g、0.79±0.14g和1.83±0.72g。SMMC-7721组和SMMC-7721/Vector组的移植瘤体积和重量明显大于SMMC-7721/siEphA7-3组的移植瘤体积,差异有统计学意义(P<0.05),而SMMC-7721组和SMMC-7721/Vector组的移植瘤体积大小和重量差异无统计学意义(P>0.05)。转染干扰载体pRNA-siEphA7-3对裸鼠肝癌移植瘤的的抑瘤率为55%。
3.实时荧光定量PCR结果显示SMMC-7721/siEphA7-3组、SMMC-7721/Vector组和SMMC-7721组的裸鼠移植瘤组织中EphA7基因的表达分别是0.191 1±0.0204、0.3900±0.0863和0.4219±0.0595。SMMC-7721/siEphA7-3组中EphA7基因的表达明显低于SMMC-7721组和SMMC-7721/Vector组,差异有统计学意义(P<0.05),而SMMC-7721组和SMMC-7721/Vector组之间EphA7基因的表达差异无统计学意义(P>0.05)
4.免疫组化分析结果显示SMMC-7721/siEphA7-3组EphA7蛋白阳性染色的细胞较少,染色较浅,呈浅黄色。而SMMC-7721组和SMMC-7721/Vector组的EphA7蛋白阳性染色细胞较多,染色较深,呈棕黄色。Western blot分析显示,SMMC-7721/siEphA7-3组、SMMC-7721/Vector组和SMMC-7721组的裸鼠移植瘤组织中EphA7蛋白的表达分别是0.2044±0.0153、0.4582±0.0631和0.4816±0.0607,SMMC-7721/siEphA7-3组中EphA7蛋白的表达明显低于SMMC-7721组和SMMC-7721/Vector组,差异有统计学意义(P<0.05),而SMMC-7721组和SMMC-7721/Vector组之间EphA7蛋白的表达差异无统计学意义(P>0.05)
结论
利用siRNA抑制肝癌细胞SMMC-7721中EphA7的表达,可减缓肝癌细胞SMMC-7721在活体动物体内的生长。
Primary hepatocellular carcinoma is one of the malignant tumors with the highest incidence and mortality in the world, which ranking sixth in cancer incidence and third in mortality. Primary hepatocellular carcinoma seriously affects the health of people's lives because of its high recurrence rate, metastasis rate and poor prognosis after surgery. China is an area with a high incidence of liver cancer, more than 300,000 new cases each year, accounting for more than half of the world. In recent years, as the concern and attention about liver cancer increasing and along with the gradually intensive researches, the etiology and risk factors of liver cancer has been definite basically. The pathological changes involved in the development and progression of liver cancer has basically clear. Furthermore, the early screening method for the high-risk patients has been continuously improved. But the survival time of the patients with liver cancer have not been improved significantly. This is mainly due to the obscurity of the condition of the patients with liver cancer, resulting in the difficulty in diagnosis. So, patients are mostly advanced liver cancer and most of them can only receive palliative surgery, chemotherapy, radiotherapy and other therapies. Only a few patients can be cure by surgical resection. Although liver transplantation is a new alternative method of treating liver cancer, its development is limited by the shortage of donor. The patients with unresectable liver cancer can only be treated with the other treatment, such as transcatheter arterial chemoembolization, percutaneous ethanol injection, radiofrequency ablation, microwave coagulation and hyperthermia, etc. But the recurrence of liver cancer often occurs after these treatments and the long-term effects of the treatments are not satisfactory. Therefore, to study the mechanism of metastasis and recurrence and explore appropriate treatment measures of liver cancer are still the focus of current cancer research.
With regard to new treatment of liver cancer is complex treatment that combined with a variety of treatment methods. Studies has confirmed that the combined therapy have obvious advantages in the treatment effect compared with monotherapy. In this regard, gene therapy can be considered as a potential auxiliary treatment. To this day, clinical trials have been completed showed that the side effects of gene therapy were at an acceptable range in most cases. As the mechanism of gene therapy is different from the traditional treatment, so the rational combination of gene therapy and traditional treatment can achieve synergistic therapeutic effect. In addition, the improved traditional treatment such as TACE and PEI can deliver the gene therapy vectors into the interior of the liver cancer, thereby increasing the effective dose and minimize side-effects of non-target cells. Today, people use these gene tools to study the factors known or suspected that play a role in tumor formation, to explore the new regulation factor in the process of tumor formation, thus providing new options for the treatment of the disease.
Eph receptor family is the largest sub-tribe of tyrosine kinase family, which involved in many important physiological processes such as embryonic development, cell growth, tissue molding and signal transduction in the nervous system, etc. As a key member of the family, EphA7 gene located on chromosome 6q16.1, close to the breaking point. EphA7 gene is widely distributed in the human body with high homology. EphA7 receptor can bind with its Ephrin ligand and form a a bi-directional signal transduction, which involved in the early vertebrate embryo development, axon guidance and many important physiological processes. In the development of central nervous system, the formation of brain connections, neural crest cells orientation and the formation process of motor nerve axon, EphA7 gene plays a key role.
In the past, most studies of EphA7 in the past focused on the physical context. In recent years, the role of EphA7 gene in the development of cancer had also caught the attention of researchers. Many studies indicate that abnormal expression of EphA7 was found in lung cancer, colon cancer, gastric cancer, prostatic carcinoma, pleomorphic malignant glioma and other human malignancies. In addition, there is evidence that EphA7 play an important role in tumor angiogenesis, tumor cell proliferation, invasion and metastasis. But overall, the research of EphA7 in tumor is still in the exploration of information and knowledge accumulated phase. At present, few studies about EphA7 have reported at home and abroad and many of them are only initial screening, failed to develop a systematic study. Furthermore, the studies were usually based on a small number of clinical specimens, which may affect the reliability of the results and still need further studies to confirm.
Therefore, detect the expression of EphA7 in human hepatocellular carcinoma and explore the effect of EphA7 gnen on biological behavior of hepatocellular carcinoma cells, which would help us to understand the mechanism of EphA7 in the genesis and development of hepatocellular carcinoma, as well as guide the clinical work.
In this study, by means of detecting the expression of EphA7 in human primary liver cancer tissues, adjacent liver cancer tissues and normal liver tissues and analyzing he relations with its clinical pathological factors, we intend exploring the relativity and clinical significance of it. After that,the siRNA eukaryotic expression vector targeting EphA7 gene was build and transfected the hepatoma cells. The effects of RNA interference targeting EphA7 gene on the biological behavior of hepatoma cell line in vitro were observed. Then, the nude mice xenograft model was established by injection of this transfected hepatoma cells in order detect the growth of hepatoma cells after the expression of EphA7 gene expression is blocked by siRNA. This study is divided into the following four parts:
Part I The expression and clinical significance of EphA7 gene and protein expression in hepatocellular carcinoma
Objective
To investigate the expression of EphA7 gene and protein in primary hepatocellular carcinoma and analyze the relation with the clinical pathological features of it in order to explore its clinical significance.
Methods
40 cases of HCC tissues and 40 cases of adjacent liver tissues and 10 cases of normal liver tissues were collected. Among the HCC tissues,23 cases were male and 17 cases were female. The range of age is from 41 to 58 and the average age is 48.3. With tumor grade,16 cases were gradeⅠ～Ⅱ,24 cases were gradeⅢ～Ⅳ. With clinical stage,26 patients were stageⅠ～Ⅱand 14 cases were stageⅢ. RT-PCR, immunohistochemistry, Real-time fluorescence quantitative PCR and Western blot were used to detect the expression of EphA7 mRNA and protein in the specimens mentioned above respectively. The, relation between the expression o f EphA7 and the clinical pathological features of HCC were analyzed too.
Results
1. The expression of EphA7 mRNA was detected in 40 cases of HCC tissues their adjacent liver tissues and 10 normal liver tissues. The expression level of EphA7 mRNA were 20.0711±32.0232,4.5184±9.4738 and 4.1764±4.7193 (F= 5.399, P<0.05). Statistical analysis showed that the expression of EphA7 mRNA was significantly higher than that in the adjacent liver tissues (P=0.015) and normal liver tissues (P=0.013), while there was no significant difference between the adjacent liver tissues and normal tissues (P=0.998).
2. The expression of EphA7 mRNA was unrelated to the age, tumor size, clinical stage, the levels of AFP and other clinical pathological parameters (P>0.05) except the degree of tumor differentiation, portal vein tumor thrombus and lymph node metastasis (P<0.05). The expression of EphA7 mRNA was significantly higher in patients with low degree of differentiation, portal vein tumor thrombosis and lymph node metastasis than those with the higher degree of differentiation (P=0.030), without portal vein tumor thrombosis (P=0.017) and lymph node metastasis (P=0.013).
3. The expression of EphA7 protein was mainly located in the cytoplasm of liver cells and hepatoma cells, as well as endothelial cells of vessels in fiber septa. The expression of EphA7 protein was strongly positive and over stain in hepatoma cells. While in the adjacent liver tissues and normal liver tissues, it was general lighter. Western blot analysis showed that the expression of EphA7 protein in HCC tissues, adjacent liver tissues and normal liver tissues were 0.5752±0.2590, 0.4019±0.2234 and 0.3169±0.1594 (F=7.826, P<0.05). The expression of EphA7 protein in HCC tissues was significantly higher than that in the adjacent liver tissues (P=0.001) and normal liver tissues (P=0.003), while there was no significant difference between the adjacent liver tissues and normal tissues (P= 0.309).
4. The expression of EphA7 protein was unrelated to the age, tumor size, clinical stage and other clinical pathological parameters (P> 0.05) except the tumor differentiation, portal vein tumor thrombus, lymph node metastasis and the level of AFP (P<0.05). The expression of EphA7 protein was significantly higher in patients with low degree of differentiation, portal vein tumor thrombosis, lymph node metastasis and lower AFP level than those with the higher higher degree of differentiation (P=0.001), without portal vein tumor thrombosis (P=0.008) and lymph node metastasis (P=0.033) and higher AFP levels (P=0.013).
Conclusion
EphA7 is involved not only in the growth and development of normal liver cell but also with the malignant transformation of liver cells. It may play an important role in malignant transformation, invasion and metastasis and other biological behavior of liver cancer.
Part II Construction and Identification of interference vector pRNA-siEphA7
Objective
To construct the eukaryotic expression vector pRNA-siEphA7 for the tyrosine kinase receptor EphA7 gene
Methods
According to the nucleotide sequence of the human EphA7 gene in Genbank and the principles of siRNA design,3 segment sequences were chosen:638-656nt, 713-731nt and 1456-1474nt. All of the three sequences were combined with the corresponding plasmid vector pRNA-u6.1/Neo restriction recognition sites and transcription terminator at the end of them. By this way, we got the double-stranded siRNA for EphA7. After annealing, endonuclease reaction and reconstitution in vivo, the siRNA expressing vectors pRNA-siEphA7 targeting EphA7 gene was constructed. The three vectors:pRNA-siEphA7-1,2 and 3 were transfected into E. coli DH5a. After cloning and selection, PCR and gene sequencing analysis was used to verify the accuracy of recombinant vector.
Results
1. The sense and antisense oligonucleotide can form the double-stranded after annealing process. The DNA fragments were electrophoresed in 1.5% agarose gel after purification and recovery and analyzed in the UVP image acquisition and analysis system. A clear band around l00bp was found and which is coincided with the 118bp oligonucleotide designed originally.
2. By using the empty vector pRNA-u6.1/Neo as control, PCR technique was used to identify whether the vector was recombined with the double-stranded siRNA by using certified primer P1 and P2. We finally found the amplified fragment about 310bp of the positive clones which proved that the pRNAT-U6.1/Neo vector was inserted successfully.
3. The positive recombinant plasmids were sequenced and the sequence of the inserted oligonucleotide is proved to be correct, which confirmed that the recombinant plasmid was constructed successfully.
Conclusion
The siRNA expressing vectors targeting EphA7 gene:pRNA-siEphA7-1,2 and 3 were constructed. After cloning, screening and identification, it was confirmed that the inserted oligonucleotide is correct and the plasmids were constructed successfully.
Part III Effects of RNA interference targeting EphA7 gene on the biological behavior of SMMC-7721 hepatoma cell line in vito
Objective
To investigate the changes of proliferation activity, cell cycle and invasive ability of SMMC-7721 hepatoma cell line after transfected with recombinant vector in vivo. To select the most effective recombinant vector pRNA-siEphA7.
Methods
By using the SMMC-7721 cells transfected with empty vector as control, the vectors:pRNA-siEphA7-1,2 and 3 were transfected into SMMC-7721 hepatoma cells by using liposome. G418 screening method was used to obtain the stable transfection SMMC-7721/siEphA7 hepatoma cells.Then, Real-time PCR and Western blot were used to detect the changes of EphA7 gene and protein in the stable transfection SMMC-7721 cells. MTT assay was used to detect the proliferative activity of the stable transfection SMMC-7721 cells. The changes of cell cycle after transfection was detected by flow cytometry assay. The changes of invasion and migration abilities after transfection were measured by Transwell chamber invasion assay.
Results
1. Green fluorescence could be seen from the SMMC-7721 cells that transfected with vectors pRNA-siEphA7-1,2,3 and transfected with empty vector through the inverted fluorescence microscope, which proved that the transfection is well. Green fluorescence could not be observed from the SMMC-7721 cells without transfection.
2. Real-time fluorescence quantitative PCR results showed that the expression of EphA7 gene in the SMMC-7721 cells that transfected with interference vector pRNA-siEphA7-1,2,3, empty vector and non-transfected SMMC-7721 cells were as follows:0.1911±0.0153,0.1488±0.0199,0.0874±0.0123,0.4293±0.0533 and 0.4311±0.0410. The statistical analysis showed that the expression of EphA7 gene in the SMMC-7721 cells that transfected with recombinant interference vector were significantly lower than that in empty vector transfected group and non-transfected group, the difference was statistically significant (P <0.05). In the three groups that transfected with interference vector, the expression of EphA7 gene in group that transfected with pRNA-siEphA7-3 was the lowest. The difference compared with the other two groups was statistically significant (P<0.05). The expression of EphA7 gene had no significance between the two groups that transfected with pRNA-siEphA7-1 and 2 (P> 0.05), as well as between the groups that transfected with empty vector and non-transfected group (P> 0.05).
3. Western blot results showed that the expression of EphA7 protein in the SMMC-7721 cells that transfected with interference vector pRNA-siEphA7-1,2 and 3, as well as empty vector and non-transfected SMMC-7721 cells were as follows:0.2786±0.0392,0.3011±0.0372,0.2142±0.0147,0.5822±0.0291 and 0.5832±0.0154. The statistical analysis showed that the expression of EphA7 protein in the SMMC-7721 cells that transfected with interference vectors were significantly lower than that in empty vector transfected group and non-transfected group, the difference was statistically significant (P<0.05). In the three groups that transfected with interference vector, the expression of EphA7 protein in group that transfected with pRNA-siEphA7-3 was the lowest. The difference compared with the other two groups was statistically significant (P<0.05). The expression of EphA7 protein had no significance between the two groups that transfected with pRNA-siEphA7-1 and 2 (P> 0.05), as well as between the groups that transfected with empty vector and non-transfected group (P> 0.05).
4. MTT assay showed that the speed of cell proliferation among the five groups had no significant difference on the first day (P> 0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with three different interference vectors was slower than that of empty vector transfected group and non-transfected group on the second day (P<0.05). But among the three different interference vectors transfected SMMC-7721 cells, it had no significant difference (P> 0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with pRNA-siEphA7-3 was slower than that of the pRNA-siEphA7-1 and 2 transfected SMMC-7721 cells since the third day and continued to the seventh day (P<0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with pRNA-siEphA7-1 was slower than that of the pRNA-siEphA7-2 transfected SMMC-7721 cells since the fifth day and continued to the seventh day (P<0.05). The speed of cell proliferation between empty vector transfected group and non-transfection group had no statistical difference all the time(P> 0.05).
5. Flow cytometry results showed that among the SMMC-7721 cells that transfected with three different interference vectors, as well as empty vector transfected group and non-transfected group, the distribution of the proportion of cells in G1, G2 and S phase are basically similar. The differences among them had not statistically significant (P> 0.05).
6. Transwell chamber invasion assay showed that the SMMC-7721 cells that transfected with pRNA-siEphA7-1,2 and 3 passed through the artificial basement membrane were:23.6±3.65,27±5.57 and 13.4±3.43. The number of the SMMC-7721 cells that transfected with pRNA-siEphA7-3 passed through the membrane was lower than that of the pRNA-siEphA7-1 and 2 (P<0.05). But between the pRNA-siEphA7-1 and 2 transfected SMMC-7721 cells, it had no significant difference (P> 0.05). The number of SMMC-7721 cells that transfected with pRNA-siEphA7-1,2 and 3 passed through the membrane were lower than those of the empty vector transfected group (68.6±6.50) and non-transfected group (72±9.30), the difference was statistically significant (P <0.05). Between the empty vector transfected group and non-transfected group, it had no significant difference (P> 0.05).
Conclusion
The expression of EphA7 gene and protein in SMMC-7721 was significantly inhibited after transfected with interference vector pRNA-siEphA7-1,2 and 3. The down regulation of EphA7 can reduce the proliferation activity, weaken the invasive ability in vitro of SMMC-7721 cell, but bring little change in the cell cycle. Among the three interference vectors, the interference effect of pRNA-siEphA7-3 (1456-1474nt) is best.
Part IV Effects of RNA interference targeting EphA7 gene on the growth of transplanted tumor in nude mice
Objective
To investigate the growth of SMMC-7721 cell after the expression of EphA7 gene expression is blocked by siRNA in nude mice transplanted tumor model.
Methods
The nude mice tumor model was established by injection of liver cells in the subcutaneous layer of left upper limb. The 32 nude mice were randomLy divided into four groups. According to the different cells injected, the nude mice were divided into the following groups:SMMC-7721 group (SMMC-7721 cells were injected); SMMC-7721/siEphA7-3 group (SMMC-7721 cells transfected with interference vector pRNA-siEphA7-3 were injected); SMMC-7721/Vector group (SMMC-7721 cells transfeceted with empty vector were injected); PBS group (PBS was injected). After five weeks, the nude mice were executed and the size and weight of transplanted tumor was compared. Real-time PCR, immunohistochemistry and Western blot were used to detecte the expression of EphA7 gene and protein in tumor tissues.
Results
1. About 9-12 days after the injection of cells, the formation of transplanted tumor can be observed at the injection site in addition to PBS group. It was proved that SMMC-7721 cells, SMMC-7721 cells transfected with interference vector and empty vector had the tumorigenic activity in nude mice. The nude mice liver tumor model could be established successfully by using these cells.
2.35 days after the model established the tumor volume of SMMC-7721 group, SMMC-7721/siEphA7-3 group and SMMC-7721/Vector group were 2.19±1.18cm3,0.84±0.32cm3 and 2.33±0.97cm3 respectively, the weight of the three groups were 1.78±0.58g,0.79±0.14g and 1.83±0.72g respectively. The tumor volume and weight of SMMC-7721 group and SMMC-7721/Vector group were significantly larger than those of SMMC-7721/siEphA7-3 group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P>0.05). The inhibition rate of transplanted tumor was 55%.
3. Real-time fluorescence quantitative PCR showed that the expression of EphA7 gene of SMMC-7721/siEphA7-3 group, SMMC-7721/Vector group and SMMC-7721 group were 0.1911±0.0204,0.3900±0.0863 and 0.4219±0.0595. The expression of EphA7 gene of SMMC-7721/siEphA7-3 group was lower than that of SMMC-7721 group and SMMC-7721/Vector group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P> 0.05).
4. Immunohistochemistry showed that in SMMC-7721/siEphA7-3 group, the cells of positive staining of EphA7 protein was less and the staining was lighter. But in SMMC-7721 group and SMMC-7721/Vector group, those were more and deeper. Western blot showed that the expression of EphA7 protein of SMMC-7721/siEphA7-3 group, SMMC-7721/Vector group and SMMC-7721 group were 0.2044±0.0153,0.4582±0.0631 and 0.4816±0.0607. The expression of EphA7 protein of SMMC-7721/siEphA7-3 group was lower than those of SMMC-7721 group and SMMC-7721/Vector group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P> 0.05).
Conclusion
Using siRNA to suppress the expression of EphA7 gene and its protein of SMMC-7721 hepatoma cell line, the growth of SMMC-7721 cells in vivo could be slowed.
目前,关于肝癌新的治疗理念是以外科治疗为主同时联合应用多种方法的综合治疗。研究证实,综合治疗较单一治疗在治疗效果上优势明显。在这方面,基因治疗可被视为一个潜在的辅助治疗措施,迄今为止,已经完成的临床实验均证实,基因治疗的副作用在大多数情况下处于可以接受的范围内。由于基因治疗的作用机制与传统的治疗不同,因此将基因治疗方法和传统治疗方案的合理组合可以达到协同治疗效应。此外,改良的传统的治疗方法如TACE和PEI可以将基因治疗的载体送入肝癌内部,从而增加有效剂量并尽可能减少非靶细胞的副作用。RNA干扰技术是研究功能基因组学最有前途的工具,目前被广泛的应用在基因研究和基因治疗中。如今,人们利用这种技术研究已知或可疑的因素在肿瘤形成中发挥的作用,来探索肿瘤形成过程中新的调节因子,从而为疾病的治疗提供新的选择。
Eph(erythropoietin producing hepatoma cell line)家族是酪氨酸蛋白激酶受体中的最大亚族,参与了胚胎发育、细胞生长、组织塑形、神经系统信号传递等重要的生理过程。EphA7作为该家族的重要一员,定位于染色体6q16.1靠近断裂点的位置,其基因同源性较高且在人体内分布广泛。EphA7受体及其配体Ephrin结合后可形成双向信号传导,参与脊椎动物胚胎的早期发育和轴突导向等许多重要的生理过程,其在中枢神经系统发育、大脑连接处的形成、神经嵴细胞导向以及运动神经轴突形成过程中发挥着关键作用。以往对EphA7的研究多集中在生理方面,近几年来,其在肿瘤中所起的作用也被人们日益关注如在肺癌、结肠癌、胃癌、前列腺癌、多形性恶性胶质瘤等多种人类恶性肿瘤中均发现EphA7的异常表达。此外,有证据显示EphA7在肿瘤血管的形成、肿瘤细胞的增殖、侵袭和转移过程中发挥着重要作用。但总的来说,对EphA7在肿瘤中的研究尚处于资料和认识的探索、积累阶段。目前,国内外有关EphA7在肝癌中的研究报道很少,仅做了初步筛查,未能形成系统性研究,且基于较少数量的临床标本,有可能会影响结果的可靠性,仍需进一步的研究加以证实。因此,对EphA7在人类原发性肝癌中表达的情况进行检测,并从基因水平探索其对肝癌细胞生物学行为的影响,对于了解EphA7在肝癌发生发展中的作用机制、指导临床工作具有重要的意义。
本研究对EphA7在人类原发性肝癌、癌旁肝组织及正常肝组织中的表达情况进行检测,并分析其与原发性肝细胞癌临床病理学因素之间的关系,对两者的相关性及临床意义作初步探讨。在此基础上,构建针对EphA7基因的siRNA真核表达载体并转染肝癌细胞SMMC-7721,观察RNA干扰抑制EphA7的表达对肝癌细胞SMMC-7721生长、增殖和侵袭等生物学行为的影响。然后将转染后的肝癌细胞在裸鼠中建立移植瘤模型,验证EphA7的下调对肝癌细胞在活体动物体内生长等的影响,奠定利用该基因治疗肝癌的实验基础,为了解EphA7在肝癌发生发展中的作用机制、靶向EphA7基因治疗肝癌提供理论依据。本研究分为以下四个部分：
第一部分EphA7基因和蛋白在肝癌中的表达及临床意义
目的
观察EphA7基因和蛋白在原发性肝细胞癌中的表达,分析EphA7的表达与肝癌临床病理特征的关系并探讨其临床意义。
方法
收集40例肝癌组织及其相应的癌旁肝组织和10例正常肝脏组织标本。40例肝癌患者中男23例,女17例。年龄41-58岁,平均年龄48.3岁。Ⅰ-Ⅱ级16例,Ⅲ-Ⅳ级的24例。Ⅰ-Ⅱ期26例,Ⅲ期14例。分别采用RT-PCR、实时荧光定量PCR、免疫组织化学和Western blot方法检测上述标本中EphA7基因和蛋白的表达情况,并分析其于原发性肝癌临床病理因素的关系。
结果
1.40例肝癌组织及相应的癌旁肝组织和10例正常肝脏组织中均有EphA7基因的表达,其表达量分别为20.0711±32.0232、4.5184±9.4738和4.1764±4.7193(F=5.399,P<0.05)。统计分析显示肝癌组织中EphA7基因的表达量显著高于癌旁肝组织(P=0.015)和正常肝组织(P=0.013),而在旁癌肝组织和正常肝组织中EphA7基因的表达差异无统计学意义(P=0.998)。
2. EphA7 mRNA的表达与肝癌患者的年龄、肿瘤大小、临床分期和甲胎蛋白水平等各项临床病理指标均无关(P>0.05),而与肿瘤的分化程度、门静脉癌栓和淋巴结转移有关(P<0.05)。分化程度低的肝癌患者EphA7 mRNA的表达明显高于分化程度高者(P=0.030),有门静脉癌栓的患者EphA7 mRNA表达明显高于无门静脉癌栓者(P=0.017),有淋巴结转移者EphA7 mRNA表达明显高于无淋巴结转移者(P=0.013)。
3. EphA7蛋白的阳性表达主要定位于肝细胞和肝癌细胞的胞浆,以及纤维间隔内血管壁的内皮细胞。其中肝癌细胞中EphA7蛋白的阳性表达较强,染色较深。癌旁肝组织及正常肝脏组织虽也可见到EphA7蛋白的阳性表达,但颜色普遍较浅。Western blot分析发现EphA7蛋白在肝癌组织、癌旁肝组织和正常组织中的表达量分别为0.5752±0.2590、0.4019±0.2234和0.3169±0.1594(P=7.826,P<0.05)。肝癌组织组织中EphA7蛋白的表达量显著高于癌旁肝组织(P=0.001)和正常肝组织(P=0.003),而在癌旁肝组织和正常肝组织中的其表达差异无统计学意义(P=0.309)。
4.肝癌组织中EphA7蛋白的表达与肝癌患者的年龄、肿瘤大小、临床分期等临床病理指标均无关(P>0.05),而与肿瘤的分化程度、有门静脉癌栓、淋巴结转移和AFP水平有关(P<0.05)。分化程度低的肝癌患者EphA7蛋白的表达明显高于分化程度高者(P=0.001),有门静脉癌栓的患者EphA7蛋白的表达明显高于无门静脉癌栓者(P=0.008),有淋巴结转移者EphA7蛋白的表达明显高于无淋巴结转移者(P=0.033),AFP水平高者EphA7蛋白的表达显著高于AFP水平低者(P=0.013)
结论
EphA7不仅参与了正常肝脏细胞的生长发育过程,还与肝脏细胞的恶性转化密切相关。其可能在肝癌的恶性转化、侵袭和转移等生物学行为中发挥重要作用。
第二部分pRNA-siEphA7干扰表达载体的构建与鉴定
目的
成功构建针对酪氨酸蛋白激酶受体EphA7基因的RNA干扰真核表达载体pRNA-siEphA7。
方法
根据Genbank上人类EphA7基因的核苷酸序列及siRNA的设计原则,选择3段序列：638-656nt、713-731nt和1456-1474nt作为干扰片段,所有寡核苷酸末端均加上与质粒载体pRNA-U6.1/Neo对应的酶切识别位点及转录终止子,人工合成针对EphA7的发夹样结构siRNA互补双链寡核苷酸。经过退火、质粒线性化以及体外重组等过程,构建抑制EphA7基因表达的干扰载体pRNA-siEphA7-1、2和3。将干扰载体转染入感受态大肠杆菌DH5α后进行克隆和筛选,PCR鉴定及基因测序验证干扰载体的准确性。
结果
1.新合成的正义及反义寡核苷酸,经退火处理形成双链互补结构。经DNA片段纯化及回收后,在1.5%的琼脂糖凝胶上进行电泳,在UVP凝胶成像分析仪上对照Marker进行验证,可见100bp附近的清晰条带,与设计的寡核苷酸大小118bp相吻合。
2.以空载体pRNA-U6.1/Neo为对照,使用插入鉴定引物P1和P2进行PCR扩增筛选鉴定,得到310bp左右的扩增片段的阳性克隆,证明已经插入pRNA-U6.1/Neo载体。
3.对3个重组质粒进行测序,结果证实插入的寡核苷酸序列正确,进一步证实重组质粒成功构建。
结论
构建了针对EphA7基因表达的干扰载体pRNA-siEphA7-1、2和3,经克隆、筛选及鉴定,证实其插入的寡核苷酸序列正确,进一步验证了其准确性并证实干扰载体pRNA-siEphA7构建成功。
第三部分RNA干扰抑制EphA7基因表达对肝癌细胞株SMMC-7721生物学特性的影响
目的
观察转染干扰载体pRNA-siEphA7对肝癌细胞SMMC-7721的增殖活性、细胞周期以及体外侵袭能力等生物学行为的影响,筛选出抑制效果最强的干扰表达载体。
方法
以转染空载体和未转染的肝癌细胞SMMC-7721作为对照,将构建好的3组干扰载体pRNA-siEphA7-1、2和3转染至肝癌细胞SMMC-7721,通过G418筛选的方法建立稳定转染的肝癌细胞SMMC-7721/siEphA7。利用Real-time PCR和Western blot技术检测转染干扰载体前后肝癌细胞SMMC-7721中EphA7基因和蛋白的变化,MTT法测定转染干扰载体前后肝癌细胞SMMC-7721的增殖活性的改变,流式细胞技术分析转染干扰载体前后肝癌细胞SMMC-7721细胞周期的变化,Transwell小室侵袭实验检测转染干扰载体前后对肝癌细胞SMMC-7721体外侵袭能力的影响。
结果
1.通过荧光倒置显微镜,可发现转染干扰载体pRNA-siEphA7-1、2和3、转染空载体的肝癌细胞SMMC-7721中均可见清晰的绿色荧光,证明其转染成功。而未转染的肝癌细胞SMMC-7721则不见荧光。
2.实时荧光定量PCR结果显示,转染干扰载体pRNA-siEphA7-1、2和3、转染空载体和未转染的的肝癌细胞SMMC-7721中EphA7基因的相对表达量分别为：0.1911±0.0153、0.1488±0.0199、0.0874±0.0123、0.4293±0.0533、0.4311±0.0410。经统计分析显示三组转染干扰载体的肝癌细胞中EphA7基因的表达明显低于空载体转染组和未转染组,差异有统计学意义(P<0.05),在三组转染干扰载体的肝癌细胞中,转染pRNA-siEphA7-3的肝癌细胞中EphA7基因的表达最低,与其他两组相比差异有统计学意义(P<0.05)。转染pRNA-siEphA7-1和2的两组肝癌细胞中EphA7基因的表达差异无统计学意义(P>0.05)。转染空载体和未转染载体的肝癌细胞中EphA7基因的表达差异无统计学意义(P>0.05)
3. Western blot结果显示转染干扰载体pRNA-siEphA7-1、2和3、转染空载体和未转染的的肝癌细胞SMMC-7721中EphA7蛋白的相对表达量分别为：0.2786±0.0392、0.3011±0.0372、0.2142±0.0147、0.5822±0.0291和0.5832±0.0154。三组转染干扰载体的肝癌细胞中EphA7蛋白的表达低于空载体转染组和未转染组,差异有统计学意义(P<0.05)。其中转染pRNA-siEphA7-3后对肝癌细胞中EphA7蛋白表达抑制最为明显,显著低于另外两个干扰载体转染组,差异有统计学意义(P<0.05)。转染pRNA-siEphA7-1和2的两组肝癌细胞之间,以及转染空载体和未转染的肝癌细胞之间EphA7蛋白的表达差异无统计学意义(P>0.05)
4.经MTT法检测转染干扰载体pRNA-siEphA7-1、2和3、转染空载体及未转染的肝癌细胞SMMC-7721的体外增殖能力并绘制生长曲线,发现第1天各组之间细胞增殖速度无明显差异(P>0.05)。自第2天开始,转染干扰载体的三组肝癌细胞增殖速度明显低于空载体转染组和未转染组(P<0.05),但转染干扰载体的三组肝癌细胞之间增殖速度无明显差异(P>0.05)。自第3天开始,转染pRNA-siEphA7-3的肝癌细胞增殖速度开始低于转染干扰载体pRNA-siEphA7-1和2的肝癌细胞,并持续至第7天(P<0.05)。自第5天开始,转染干扰载体pRNA-siEphA7-1的肝癌细胞增殖速度开始低于转染pRNA-siEphA7-2的肝癌细胞,并持续至第7天(P<0.05)。而空载体转染组和未转染组的肝癌细胞之间增殖速度始终没有统计学差异(P>0.05)。
5.流式细胞检测结果发现转染干扰载体pRNA-siEphA7-1、2和3、转染空载体和未转染载体的肝癌细胞SMMC-7721在G1期、G2期和S期的细胞分布比例基本相近,差异无统计学意义(P>0.05)。
6.Transwell小室侵袭实验结果发现转染干扰载体pRNA-siEphA7-1、2和3的肝癌细胞SMMC-7721穿过人工基底膜的细胞分别是23.6±3.65、27±5.57和13.4±3.43.pRNA-siEphA7-3转染组的肝癌细胞穿膜细胞数明显低于pRNA-siEphA7-1和2转染组的穿膜细胞数,差异有统计学意义(P<0.05)。而pRNA-siEphA7-1和2转染组的肝癌细胞穿膜细胞计数无统计学差异(P>0.05)。转染干扰载体的三组肝癌细胞穿膜细胞计数明显低于空载体转染组(68.6±6.50)和未转染组(72±9.30),差异有统计学意义(P<0.05)。空载体转染组和未转染组相比,其穿膜细胞计数差异无统计学意义(P>0.05)。
结论
转染干扰载体pRNA-siEphA7-1.2和3后,肝癌细胞SMMC-7721中EphA7基因及蛋白的表达明显受到抑制。EphA7的下调使肝癌细胞SMMC-7721的增殖活性明显降低、体外侵袭能力减弱,但细胞周期的变化不大。3组干扰载体中以pRNA-siEphA7-3(1456-1474nt)的干扰效果最好。
第四部分RNA干扰抑制EphA7基因表达对裸鼠肝癌移植瘤生长的影响
目的
通过建立裸鼠肝癌移植瘤模型,在活体动物体内观察siRNA阻断EphA7基因表达对肝癌细胞SMMC-7721生长的影响。
方法
裸鼠荷瘤模型采用左上肢皮下注射肝癌细胞的方法建立。将32只裸鼠随机分为4组,每组8只,根据皮下注射细胞的不同分为以下几组：SMMC-7721组(注射肝癌细胞SMMC-7721)；SMMC-7721/siEphA7组(注射转染干扰载体pRNA-siEphA7-3的肝癌细胞SMMC-7721);SMMC-7721/Vector组(注射转染空载体的肝癌细胞SMMC-7721);PBS组(不注射细胞,仅注入PBS做对照)。移植瘤模型成功建立后5周以颈椎脱臼法处死裸鼠,观察并比较各组肝癌移植瘤的大小及重量,应用Real-time PCR、免疫组织化学技术以及Western blot检测荷瘤组织中EphA7基因和蛋白的表达情况。
结果
1.注射细胞约9-12天后,除PBS组外其余各组均可在注射部位的发现肝癌移植瘤的形成,证明肝癌细胞SMMC-7721、转染干扰载体以及空载体的肝癌细胞SMMC-7721在裸鼠体内均有成瘤活性,可成功建立裸鼠肝癌移植瘤模型。
2.移植瘤模型建立后第35天,SMMC-7721组、SMMC-7721/siEphA7-3组和SMMC-7721/Vector组的移植瘤体积分别为2.19±1.18cm3、0.84±0.32cm3和2.33±0.97cm3,重量分别为1.78±0.58g、0.79±0.14g和1.83±0.72g。SMMC-7721组和SMMC-7721/Vector组的移植瘤体积和重量明显大于SMMC-7721/siEphA7-3组的移植瘤体积,差异有统计学意义(P<0.05),而SMMC-7721组和SMMC-7721/Vector组的移植瘤体积大小和重量差异无统计学意义(P>0.05)。转染干扰载体pRNA-siEphA7-3对裸鼠肝癌移植瘤的的抑瘤率为55%。
3.实时荧光定量PCR结果显示SMMC-7721/siEphA7-3组、SMMC-7721/Vector组和SMMC-7721组的裸鼠移植瘤组织中EphA7基因的表达分别是0.191 1±0.0204、0.3900±0.0863和0.4219±0.0595。SMMC-7721/siEphA7-3组中EphA7基因的表达明显低于SMMC-7721组和SMMC-7721/Vector组,差异有统计学意义(P<0.05),而SMMC-7721组和SMMC-7721/Vector组之间EphA7基因的表达差异无统计学意义(P>0.05)
4.免疫组化分析结果显示SMMC-7721/siEphA7-3组EphA7蛋白阳性染色的细胞较少,染色较浅,呈浅黄色。而SMMC-7721组和SMMC-7721/Vector组的EphA7蛋白阳性染色细胞较多,染色较深,呈棕黄色。Western blot分析显示,SMMC-7721/siEphA7-3组、SMMC-7721/Vector组和SMMC-7721组的裸鼠移植瘤组织中EphA7蛋白的表达分别是0.2044±0.0153、0.4582±0.0631和0.4816±0.0607,SMMC-7721/siEphA7-3组中EphA7蛋白的表达明显低于SMMC-7721组和SMMC-7721/Vector组,差异有统计学意义(P<0.05),而SMMC-7721组和SMMC-7721/Vector组之间EphA7蛋白的表达差异无统计学意义(P>0.05)
结论
利用siRNA抑制肝癌细胞SMMC-7721中EphA7的表达,可减缓肝癌细胞SMMC-7721在活体动物体内的生长。
Primary hepatocellular carcinoma is one of the malignant tumors with the highest incidence and mortality in the world, which ranking sixth in cancer incidence and third in mortality. Primary hepatocellular carcinoma seriously affects the health of people's lives because of its high recurrence rate, metastasis rate and poor prognosis after surgery. China is an area with a high incidence of liver cancer, more than 300,000 new cases each year, accounting for more than half of the world. In recent years, as the concern and attention about liver cancer increasing and along with the gradually intensive researches, the etiology and risk factors of liver cancer has been definite basically. The pathological changes involved in the development and progression of liver cancer has basically clear. Furthermore, the early screening method for the high-risk patients has been continuously improved. But the survival time of the patients with liver cancer have not been improved significantly. This is mainly due to the obscurity of the condition of the patients with liver cancer, resulting in the difficulty in diagnosis. So, patients are mostly advanced liver cancer and most of them can only receive palliative surgery, chemotherapy, radiotherapy and other therapies. Only a few patients can be cure by surgical resection. Although liver transplantation is a new alternative method of treating liver cancer, its development is limited by the shortage of donor. The patients with unresectable liver cancer can only be treated with the other treatment, such as transcatheter arterial chemoembolization, percutaneous ethanol injection, radiofrequency ablation, microwave coagulation and hyperthermia, etc. But the recurrence of liver cancer often occurs after these treatments and the long-term effects of the treatments are not satisfactory. Therefore, to study the mechanism of metastasis and recurrence and explore appropriate treatment measures of liver cancer are still the focus of current cancer research.
With regard to new treatment of liver cancer is complex treatment that combined with a variety of treatment methods. Studies has confirmed that the combined therapy have obvious advantages in the treatment effect compared with monotherapy. In this regard, gene therapy can be considered as a potential auxiliary treatment. To this day, clinical trials have been completed showed that the side effects of gene therapy were at an acceptable range in most cases. As the mechanism of gene therapy is different from the traditional treatment, so the rational combination of gene therapy and traditional treatment can achieve synergistic therapeutic effect. In addition, the improved traditional treatment such as TACE and PEI can deliver the gene therapy vectors into the interior of the liver cancer, thereby increasing the effective dose and minimize side-effects of non-target cells. Today, people use these gene tools to study the factors known or suspected that play a role in tumor formation, to explore the new regulation factor in the process of tumor formation, thus providing new options for the treatment of the disease.
Eph receptor family is the largest sub-tribe of tyrosine kinase family, which involved in many important physiological processes such as embryonic development, cell growth, tissue molding and signal transduction in the nervous system, etc. As a key member of the family, EphA7 gene located on chromosome 6q16.1, close to the breaking point. EphA7 gene is widely distributed in the human body with high homology. EphA7 receptor can bind with its Ephrin ligand and form a a bi-directional signal transduction, which involved in the early vertebrate embryo development, axon guidance and many important physiological processes. In the development of central nervous system, the formation of brain connections, neural crest cells orientation and the formation process of motor nerve axon, EphA7 gene plays a key role.
In the past, most studies of EphA7 in the past focused on the physical context. In recent years, the role of EphA7 gene in the development of cancer had also caught the attention of researchers. Many studies indicate that abnormal expression of EphA7 was found in lung cancer, colon cancer, gastric cancer, prostatic carcinoma, pleomorphic malignant glioma and other human malignancies. In addition, there is evidence that EphA7 play an important role in tumor angiogenesis, tumor cell proliferation, invasion and metastasis. But overall, the research of EphA7 in tumor is still in the exploration of information and knowledge accumulated phase. At present, few studies about EphA7 have reported at home and abroad and many of them are only initial screening, failed to develop a systematic study. Furthermore, the studies were usually based on a small number of clinical specimens, which may affect the reliability of the results and still need further studies to confirm.
Therefore, detect the expression of EphA7 in human hepatocellular carcinoma and explore the effect of EphA7 gnen on biological behavior of hepatocellular carcinoma cells, which would help us to understand the mechanism of EphA7 in the genesis and development of hepatocellular carcinoma, as well as guide the clinical work.
In this study, by means of detecting the expression of EphA7 in human primary liver cancer tissues, adjacent liver cancer tissues and normal liver tissues and analyzing he relations with its clinical pathological factors, we intend exploring the relativity and clinical significance of it. After that,the siRNA eukaryotic expression vector targeting EphA7 gene was build and transfected the hepatoma cells. The effects of RNA interference targeting EphA7 gene on the biological behavior of hepatoma cell line in vitro were observed. Then, the nude mice xenograft model was established by injection of this transfected hepatoma cells in order detect the growth of hepatoma cells after the expression of EphA7 gene expression is blocked by siRNA. This study is divided into the following four parts:
Part I The expression and clinical significance of EphA7 gene and protein expression in hepatocellular carcinoma
Objective
To investigate the expression of EphA7 gene and protein in primary hepatocellular carcinoma and analyze the relation with the clinical pathological features of it in order to explore its clinical significance.
Methods
40 cases of HCC tissues and 40 cases of adjacent liver tissues and 10 cases of normal liver tissues were collected. Among the HCC tissues,23 cases were male and 17 cases were female. The range of age is from 41 to 58 and the average age is 48.3. With tumor grade,16 cases were gradeⅠ～Ⅱ,24 cases were gradeⅢ～Ⅳ. With clinical stage,26 patients were stageⅠ～Ⅱand 14 cases were stageⅢ. RT-PCR, immunohistochemistry, Real-time fluorescence quantitative PCR and Western blot were used to detect the expression of EphA7 mRNA and protein in the specimens mentioned above respectively. The, relation between the expression o f EphA7 and the clinical pathological features of HCC were analyzed too.
Results
1. The expression of EphA7 mRNA was detected in 40 cases of HCC tissues their adjacent liver tissues and 10 normal liver tissues. The expression level of EphA7 mRNA were 20.0711±32.0232,4.5184±9.4738 and 4.1764±4.7193 (F= 5.399, P<0.05). Statistical analysis showed that the expression of EphA7 mRNA was significantly higher than that in the adjacent liver tissues (P=0.015) and normal liver tissues (P=0.013), while there was no significant difference between the adjacent liver tissues and normal tissues (P=0.998).
2. The expression of EphA7 mRNA was unrelated to the age, tumor size, clinical stage, the levels of AFP and other clinical pathological parameters (P>0.05) except the degree of tumor differentiation, portal vein tumor thrombus and lymph node metastasis (P<0.05). The expression of EphA7 mRNA was significantly higher in patients with low degree of differentiation, portal vein tumor thrombosis and lymph node metastasis than those with the higher degree of differentiation (P=0.030), without portal vein tumor thrombosis (P=0.017) and lymph node metastasis (P=0.013).
3. The expression of EphA7 protein was mainly located in the cytoplasm of liver cells and hepatoma cells, as well as endothelial cells of vessels in fiber septa. The expression of EphA7 protein was strongly positive and over stain in hepatoma cells. While in the adjacent liver tissues and normal liver tissues, it was general lighter. Western blot analysis showed that the expression of EphA7 protein in HCC tissues, adjacent liver tissues and normal liver tissues were 0.5752±0.2590, 0.4019±0.2234 and 0.3169±0.1594 (F=7.826, P<0.05). The expression of EphA7 protein in HCC tissues was significantly higher than that in the adjacent liver tissues (P=0.001) and normal liver tissues (P=0.003), while there was no significant difference between the adjacent liver tissues and normal tissues (P= 0.309).
4. The expression of EphA7 protein was unrelated to the age, tumor size, clinical stage and other clinical pathological parameters (P> 0.05) except the tumor differentiation, portal vein tumor thrombus, lymph node metastasis and the level of AFP (P<0.05). The expression of EphA7 protein was significantly higher in patients with low degree of differentiation, portal vein tumor thrombosis, lymph node metastasis and lower AFP level than those with the higher higher degree of differentiation (P=0.001), without portal vein tumor thrombosis (P=0.008) and lymph node metastasis (P=0.033) and higher AFP levels (P=0.013).
Conclusion
EphA7 is involved not only in the growth and development of normal liver cell but also with the malignant transformation of liver cells. It may play an important role in malignant transformation, invasion and metastasis and other biological behavior of liver cancer.
Part II Construction and Identification of interference vector pRNA-siEphA7
Objective
To construct the eukaryotic expression vector pRNA-siEphA7 for the tyrosine kinase receptor EphA7 gene
Methods
According to the nucleotide sequence of the human EphA7 gene in Genbank and the principles of siRNA design,3 segment sequences were chosen:638-656nt, 713-731nt and 1456-1474nt. All of the three sequences were combined with the corresponding plasmid vector pRNA-u6.1/Neo restriction recognition sites and transcription terminator at the end of them. By this way, we got the double-stranded siRNA for EphA7. After annealing, endonuclease reaction and reconstitution in vivo, the siRNA expressing vectors pRNA-siEphA7 targeting EphA7 gene was constructed. The three vectors:pRNA-siEphA7-1,2 and 3 were transfected into E. coli DH5a. After cloning and selection, PCR and gene sequencing analysis was used to verify the accuracy of recombinant vector.
Results
1. The sense and antisense oligonucleotide can form the double-stranded after annealing process. The DNA fragments were electrophoresed in 1.5% agarose gel after purification and recovery and analyzed in the UVP image acquisition and analysis system. A clear band around l00bp was found and which is coincided with the 118bp oligonucleotide designed originally.
2. By using the empty vector pRNA-u6.1/Neo as control, PCR technique was used to identify whether the vector was recombined with the double-stranded siRNA by using certified primer P1 and P2. We finally found the amplified fragment about 310bp of the positive clones which proved that the pRNAT-U6.1/Neo vector was inserted successfully.
3. The positive recombinant plasmids were sequenced and the sequence of the inserted oligonucleotide is proved to be correct, which confirmed that the recombinant plasmid was constructed successfully.
Conclusion
The siRNA expressing vectors targeting EphA7 gene:pRNA-siEphA7-1,2 and 3 were constructed. After cloning, screening and identification, it was confirmed that the inserted oligonucleotide is correct and the plasmids were constructed successfully.
Part III Effects of RNA interference targeting EphA7 gene on the biological behavior of SMMC-7721 hepatoma cell line in vito
Objective
To investigate the changes of proliferation activity, cell cycle and invasive ability of SMMC-7721 hepatoma cell line after transfected with recombinant vector in vivo. To select the most effective recombinant vector pRNA-siEphA7.
Methods
By using the SMMC-7721 cells transfected with empty vector as control, the vectors:pRNA-siEphA7-1,2 and 3 were transfected into SMMC-7721 hepatoma cells by using liposome. G418 screening method was used to obtain the stable transfection SMMC-7721/siEphA7 hepatoma cells.Then, Real-time PCR and Western blot were used to detect the changes of EphA7 gene and protein in the stable transfection SMMC-7721 cells. MTT assay was used to detect the proliferative activity of the stable transfection SMMC-7721 cells. The changes of cell cycle after transfection was detected by flow cytometry assay. The changes of invasion and migration abilities after transfection were measured by Transwell chamber invasion assay.
Results
1. Green fluorescence could be seen from the SMMC-7721 cells that transfected with vectors pRNA-siEphA7-1,2,3 and transfected with empty vector through the inverted fluorescence microscope, which proved that the transfection is well. Green fluorescence could not be observed from the SMMC-7721 cells without transfection.
2. Real-time fluorescence quantitative PCR results showed that the expression of EphA7 gene in the SMMC-7721 cells that transfected with interference vector pRNA-siEphA7-1,2,3, empty vector and non-transfected SMMC-7721 cells were as follows:0.1911±0.0153,0.1488±0.0199,0.0874±0.0123,0.4293±0.0533 and 0.4311±0.0410. The statistical analysis showed that the expression of EphA7 gene in the SMMC-7721 cells that transfected with recombinant interference vector were significantly lower than that in empty vector transfected group and non-transfected group, the difference was statistically significant (P <0.05). In the three groups that transfected with interference vector, the expression of EphA7 gene in group that transfected with pRNA-siEphA7-3 was the lowest. The difference compared with the other two groups was statistically significant (P<0.05). The expression of EphA7 gene had no significance between the two groups that transfected with pRNA-siEphA7-1 and 2 (P> 0.05), as well as between the groups that transfected with empty vector and non-transfected group (P> 0.05).
3. Western blot results showed that the expression of EphA7 protein in the SMMC-7721 cells that transfected with interference vector pRNA-siEphA7-1,2 and 3, as well as empty vector and non-transfected SMMC-7721 cells were as follows:0.2786±0.0392,0.3011±0.0372,0.2142±0.0147,0.5822±0.0291 and 0.5832±0.0154. The statistical analysis showed that the expression of EphA7 protein in the SMMC-7721 cells that transfected with interference vectors were significantly lower than that in empty vector transfected group and non-transfected group, the difference was statistically significant (P<0.05). In the three groups that transfected with interference vector, the expression of EphA7 protein in group that transfected with pRNA-siEphA7-3 was the lowest. The difference compared with the other two groups was statistically significant (P<0.05). The expression of EphA7 protein had no significance between the two groups that transfected with pRNA-siEphA7-1 and 2 (P> 0.05), as well as between the groups that transfected with empty vector and non-transfected group (P> 0.05).
4. MTT assay showed that the speed of cell proliferation among the five groups had no significant difference on the first day (P> 0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with three different interference vectors was slower than that of empty vector transfected group and non-transfected group on the second day (P<0.05). But among the three different interference vectors transfected SMMC-7721 cells, it had no significant difference (P> 0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with pRNA-siEphA7-3 was slower than that of the pRNA-siEphA7-1 and 2 transfected SMMC-7721 cells since the third day and continued to the seventh day (P<0.05). The speed of cell proliferation of SMMC-7721 cells that transfected with pRNA-siEphA7-1 was slower than that of the pRNA-siEphA7-2 transfected SMMC-7721 cells since the fifth day and continued to the seventh day (P<0.05). The speed of cell proliferation between empty vector transfected group and non-transfection group had no statistical difference all the time(P> 0.05).
5. Flow cytometry results showed that among the SMMC-7721 cells that transfected with three different interference vectors, as well as empty vector transfected group and non-transfected group, the distribution of the proportion of cells in G1, G2 and S phase are basically similar. The differences among them had not statistically significant (P> 0.05).
6. Transwell chamber invasion assay showed that the SMMC-7721 cells that transfected with pRNA-siEphA7-1,2 and 3 passed through the artificial basement membrane were:23.6±3.65,27±5.57 and 13.4±3.43. The number of the SMMC-7721 cells that transfected with pRNA-siEphA7-3 passed through the membrane was lower than that of the pRNA-siEphA7-1 and 2 (P<0.05). But between the pRNA-siEphA7-1 and 2 transfected SMMC-7721 cells, it had no significant difference (P> 0.05). The number of SMMC-7721 cells that transfected with pRNA-siEphA7-1,2 and 3 passed through the membrane were lower than those of the empty vector transfected group (68.6±6.50) and non-transfected group (72±9.30), the difference was statistically significant (P <0.05). Between the empty vector transfected group and non-transfected group, it had no significant difference (P> 0.05).
Conclusion
The expression of EphA7 gene and protein in SMMC-7721 was significantly inhibited after transfected with interference vector pRNA-siEphA7-1,2 and 3. The down regulation of EphA7 can reduce the proliferation activity, weaken the invasive ability in vitro of SMMC-7721 cell, but bring little change in the cell cycle. Among the three interference vectors, the interference effect of pRNA-siEphA7-3 (1456-1474nt) is best.
Part IV Effects of RNA interference targeting EphA7 gene on the growth of transplanted tumor in nude mice
Objective
To investigate the growth of SMMC-7721 cell after the expression of EphA7 gene expression is blocked by siRNA in nude mice transplanted tumor model.
Methods
The nude mice tumor model was established by injection of liver cells in the subcutaneous layer of left upper limb. The 32 nude mice were randomLy divided into four groups. According to the different cells injected, the nude mice were divided into the following groups:SMMC-7721 group (SMMC-7721 cells were injected); SMMC-7721/siEphA7-3 group (SMMC-7721 cells transfected with interference vector pRNA-siEphA7-3 were injected); SMMC-7721/Vector group (SMMC-7721 cells transfeceted with empty vector were injected); PBS group (PBS was injected). After five weeks, the nude mice were executed and the size and weight of transplanted tumor was compared. Real-time PCR, immunohistochemistry and Western blot were used to detecte the expression of EphA7 gene and protein in tumor tissues.
Results
1. About 9-12 days after the injection of cells, the formation of transplanted tumor can be observed at the injection site in addition to PBS group. It was proved that SMMC-7721 cells, SMMC-7721 cells transfected with interference vector and empty vector had the tumorigenic activity in nude mice. The nude mice liver tumor model could be established successfully by using these cells.
2.35 days after the model established the tumor volume of SMMC-7721 group, SMMC-7721/siEphA7-3 group and SMMC-7721/Vector group were 2.19±1.18cm3,0.84±0.32cm3 and 2.33±0.97cm3 respectively, the weight of the three groups were 1.78±0.58g,0.79±0.14g and 1.83±0.72g respectively. The tumor volume and weight of SMMC-7721 group and SMMC-7721/Vector group were significantly larger than those of SMMC-7721/siEphA7-3 group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P>0.05). The inhibition rate of transplanted tumor was 55%.
3. Real-time fluorescence quantitative PCR showed that the expression of EphA7 gene of SMMC-7721/siEphA7-3 group, SMMC-7721/Vector group and SMMC-7721 group were 0.1911±0.0204,0.3900±0.0863 and 0.4219±0.0595. The expression of EphA7 gene of SMMC-7721/siEphA7-3 group was lower than that of SMMC-7721 group and SMMC-7721/Vector group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P> 0.05).
4. Immunohistochemistry showed that in SMMC-7721/siEphA7-3 group, the cells of positive staining of EphA7 protein was less and the staining was lighter. But in SMMC-7721 group and SMMC-7721/Vector group, those were more and deeper. Western blot showed that the expression of EphA7 protein of SMMC-7721/siEphA7-3 group, SMMC-7721/Vector group and SMMC-7721 group were 0.2044±0.0153,0.4582±0.0631 and 0.4816±0.0607. The expression of EphA7 protein of SMMC-7721/siEphA7-3 group was lower than those of SMMC-7721 group and SMMC-7721/Vector group, the difference had statistically significant (P<0.05). While between the SMMC-7721 group and SMMC-7721/Vector group, it had no significant difference (P> 0.05).
Conclusion
Using siRNA to suppress the expression of EphA7 gene and its protein of SMMC-7721 hepatoma cell line, the growth of SMMC-7721 cells in vivo could be slowed.
引文
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