【作者】 王璐；

【导师】 刘林嶓；

【作者基本信息】 郑州大学 ， 外科学， 2012， 博士

【摘要】 皮肤鳞状细胞癌(cutaneous squamous cell carcinoma, CSCC,简称皮肤鳞癌)是常见的皮肤恶性肿瘤之一,发病率在皮肤恶性肿瘤中位居第一位,其发病机制迄今为止仍不明确。尽管在Mohs手术(Mohs micrographic surgery)、化疗、放疗、生物治疗、免疫治疗等方面取得了长足进步,但是由于发病率逐年增长,而面积较大、年老体弱、特殊部位、手术禁忌症患者等不适于接受Mobs手术的越来越多,又由于CSCC是侵袭性癌、恶性程度高、耐药性、易发生淋巴结、远处转移和晚期可通过血液播散,导致CSCC患者治疗后复发甚至死亡。外部环境因素以及遗传因素,细胞内癌基因突变、抑癌基因失活、凋亡过程和信号转导通路的异常等多种致病因素综合作用导致CSCC的发生、发展,这一研究结果提示皮肤鳞癌的发生发展是一个多因素、多基因、多阶段的复杂过程,它发生、发展的重要机制是细胞增殖与凋亡失衡。Livin(ML/KIAP, BIRC7)是新近发现的IAPs (inhibitor of apoptosis proteins, IAPs)家族的新成员,直接结合天冬氨酸特异性半胱氨酸蛋白酶(cysteine-containing aspartate-specific proteases, Caspase),发挥较强的凋亡抑制功能,从而抑制细胞凋亡,促进肿瘤生长。Livin不仅在CSCC呈现高表达,并且在其它多种肿瘤组织中也呈现高表达,而Caspase-3和Smac(second mitochondria- derived activator of caspases, Smac)作为一种具有促凋亡功能的因子则往往低表达甚至缺失,这已被许多学者研究证实。Livin与Caspase-3和Smac这两种具有相反作用的因子通常呈负相关。RNAi (RNA interference, RNAi)技术沉默Livin基因表达,一方面抑制肿瘤细胞增殖和促进肿瘤细胞凋亡,另一方面增加肿瘤细胞对化疗的敏感性,表明Livin的高表达与肿瘤的发生、发展及预后相关。RNAi技术可高效、特异地下调目标基因的表达,是研究内源性基因功能和信号转导途径的强有力工具。本研究拟通过RNAi沉默Livin基因,建立沉默Livin基因表达的CSCC细胞株A431,在体内和体外通过一系列实验研究,观察Livin基因表达沉默后,CSCC细胞生长、增殖、凋亡、细胞周期、转移能力和克隆能力的变化情况；同时检测凋亡相关因子Caspase-3和Smac的表达水平的变化以及治疗皮肤鳞癌的化疗药物博来霉素(Bleomycin, BLM)耐药性的逆转并分析其可能机制,为皮肤鳞癌的治疗提供新的靶点和策略。课题共分四部分如下述。第一部分Livin和Smac基因在人皮肤鳞状细胞癌中的表达目的检测Livin和Smac在人CSCC组织、人CSCC细胞株A431和正常皮肤组织、人皮肤角质形成细胞株HaCaT中的表达,探讨Livin和Smac的表达及意义,以及Livin和Smac在CSCC组织中的表达与临床病理特征的关系。方法1.选择2009年1月至2011年8月整形外科手术切除并经病理学确诊的人皮肤鳞癌组织标本50例,正常皮肤组织标本20例。2.培养人皮肽鳞状细胞癌细胞株A431、人皮肤角质形成细胞株HaCaT。3.免疫组化SP法检测CSCC组织与正常皮肤组织中Livin和Smac蛋白表达,根据阳性细胞百分比和染色强弱综合判断两种蛋白的表达水平,并分析其相关性。4.采用Western blot、荧光定量RT-PCR检测Livin和Smac在人CSCC细胞株A431与人皮肤角质形成细胞株HaCaT的表达。5.分析Livin和Smac与临床病理因素的关系。结果1.Livin及Smac表达主要定位于CSCC细胞的细胞质,为棕黄色颗粒。50例cscc组织中Livin阳性表达率为70.0%(35/50),正常皮肤组织中无阳性表达,CSCC组织中Livin蛋白的阳性表达明显高于正常皮肤组织,差异有统计学意义(P<0.05);Smac蛋白阳性表达率为46.0%(23/50),在正常皮肤组织中Smac表达率为100%, Smac蛋白的阳性表达低于正常皮肤组织,差异有统计学意义(P<0.05)。2. Livin mRNA和Livin蛋白在人CSCC细胞株A431中的相对表达量分别为0.842+0.191和0.828+0.230,在人皮肤角质形成细胞株HaCaT中分别为0.249±0.023和0.175±0.020。Livin mRNA和Livin蛋白在人CSCC细胞株A431表达明显高于人皮肤角质形成细胞株HaCaT,差异有统计学意义(P<0.05);Smac mRNA和Smac蛋白在人CSCC细胞株A431中的相对表达量分别为0.328±0.038和0.231±0.032,在人皮肤角质形成细胞株HaCaT中分别为0.824±0.183和0.808±0.215。Smac mRNA和Smac蛋白表达低于人皮肤角质形成细胞株HaCaT,差异有统计学意义(P<0.05)。3.Livin和Smac蛋白的表达与CSCC的发病年龄、性别、肿瘤大小等差异均无统计学意义(P>0.05)；而与是否发生转移以及肿瘤的分化程度差异有统计学意义(P<0.05);Livin和Smac的表达从强到弱分别为：低分化、中分化、高分化,正常皮肤组织无表达和正常皮肤组织、高分化、中分化、低分化,且二者表达呈负相关。小结1. Livin和Smac基因在人CSCC组织和人CSCC细胞系中分别呈高表达和低表达,与肿瘤的转移以及分化程度相关。2. Livin和Smac在CSCC组织中的表达呈负相关。第二部分Livin基因siRNA真核表达载体的构建和鉴定目的构建人Livin基因Livin-siRNA真核表达载体并进行鉴定,探讨RNAi沉默靶基因的效果及其在靶细胞内稳定表达的情况,为研究Livin基因功能提供稳定的转染载体。方法1.设计2个针对Livin基因的siRNA靶片段,合成短发夹DNA单链,退火形成双链DNA片段。2.DNA片段与pSilencer2.1/U6载体连接和转化,经筛选并进行酶切和测序鉴定插入序列,得到正确的2个针对Livin基因的siRNA载体。3.细胞经培养后分组为沉默Livin 1组：转染pSilencer2.1/U6-si438;沉默Livin2组：转染pSilencer2.1/U6-si475;空载体组：转染pSilencer2.1/U6;空白对照组：未转染的A431细胞。4.制备shRNA-LipofectamineTM 2000复合物f稀释质粒pSilencer2.1/U6-si438 or pSilencer2.1/U6-si475 or pSilencer2.1/U6和稀释LipofectamineTM 2000轻轻混合孵育形成),按分组转染CSCC A431细胞。5.经G418筛选扩增后得到对应的稳定转染细胞株,采用Real-time-quantitative-RT-PCR和Western blot检测转染的A431细胞Livin mRNA和Livin蛋白的表达。结果1.据GenBank中Livin基因已知序列(No.NM-139317, No.NM-022161, BIRC7 baculoviral IAP repeat containing 7 [Homo sapiens], Also known as KIAP, LIVIN, MLIAP, RNF50, ML-IAP, CDS 1741070),利用GeneLinkTM公司的RNAi Explorer和Dharmacon公司的siDESIGN Center进行扫描,经BLAST同源性分析,选择确定19个碱基438-456(GTGGTTCCCCAGCTGTCAG),475-493(GGAAG AGACTTTGTCCACA)为2条针对Livin基因的干扰序列,合成两对DNA单链为编码短发夹RNA序列,在两端分别加上BamHI和HindⅢ内切酶残基,并命名为Livin438和Livin475,退火形成双链DNA片段(si438和si475)。2.DNA片段与pSilencer2.1/U6载体连接和转化后,经筛选并进行酶切和测序鉴定插入序列,证实得到的2个针对Livin基因的siRNA载体pSilencer2.1/ U6-si438和pSilencer2.1/U6-si475与设计的一致。3.沉默Livinl组f转染pSilencer2.1/U6-si438)、沉默Livin2组(转染pSilencer2.1/U6-si475)和空载体组(转染pSilencer2.1/U6)细胞经G418筛选,均得到稳定转染细胞株。4. Livin mRNA和Livin蛋白相对表达量在空载体组分别为0.826+0.205和0.813±0.189,在空白对照组分别为0.852+0.197和0.832+0.202。空载体组和空白对照比较,Livin mRNA和Livin蛋白表达水平无明显变化,差异无统计学意义(P>0.05);Livin mRNA和Livin蛋白相对表达量在沉默Livin 1组分别为0.253+0.031和0.233+0.024,在沉默Livin 2组分别为0.132+0.031和0.128+0.031。沉默Livin 1组、沉默Livin 2组细胞与2个对照组比较,Livin mRNA和Livin蛋白表达水平均显著降低,差异有统计学意义(P<0.05),基因沉默效应均达到70%以上；沉默Livin2组细胞Livin mRNA和Livin蛋白的相对表达量最低。小结1.成功构建2个针对Livin的siRNA载体,获得最佳干扰序列pSilencer2.1/ U6-si475载体及2株Livin表达沉默的皮肤鳞癌A431细胞株。2.通过RNAi技术可以沉默靶细胞Livin基因,转染入皮肤鳞癌细胞后可达到70%以上的基因沉默效应,可在转录水平及蛋白水平抑制目的基因Livin的表达。第三部分RNAi干扰Livin基因表达对皮肤鳞状细胞癌细胞株生物学功能的影响目的探讨RNAi干扰Livin基因表达对CSCC细胞株生物学功能的影响及可能机制,为进一步的临床试验提供科学的实验依据。方法1.体外实验分组为沉默Livin组：稳定转染pSilencer2.1/U6-siLivin的A431细胞,沉默Livin基因表达；空载体组：稳定转染空载体pSilencer2.1/U6的A431细胞；空白细胞组：未转染的A431细胞。2.采用荧光定量RT-PCR, Western blot检测Smac mRNA, Smac蛋白在各组细胞的表达水平；ELISA检测Caspase-3的活性。3.MTT法测定细胞的增值和耐药性影响。4.流式细胞仪检测细胞凋亡和周期变化。5.Transwell小室细胞迁移实验检测细胞的转移能力。6.克隆形成实验检测细胞的克隆能力。结果1.沉默Livin组f稳定转染pSilencer2.1/U6-siLivin的A431细胞)与空白细胞组(未转染的A431细胞)和空载体组(稳定转染空载体pSilencer2.1/U6的A431细胞)相比,Smac的mRNA、蛋白表达水平上升,分析结果差异有统计学意义(P<0.05);Caspase-3的活性显著升高,分析结果差异有统计学意义(P<0.05)。2.沉默Livin组与空载体组和空白细胞组相比,沉默Livin组细胞OD值显著降低,差异有统计学意义(p<0.05)；沉默Livin组联合BLM治疗组的细胞凋亡率显著高于空载体组和空白细胞组,差异有统计学意义(P<0.05)。3.沉默Livin组与空载体组和空白细胞组相比,沉默Livin组细胞凋亡率明显升高,差异有统计学意义(P<0.05)；沉默Livin组细胞Go／G1期细胞比率显著升高,分析结果差异有统计学意义(P<0.05),S和G2/M期比率显著降低,分析结果差异有统计学意义(P<0.05)。4.沉默Livin组与空载体组和空白细胞组相比,沉默Livin组细胞穿膜细胞数显著降低,差异有统计学意义(P<0.05)；沉默Livin组细胞克隆形成率显著降低,差异有统计学意义(P<0.05)。小结1. pSilencer2.1/U6-siLivin沉默Livin基因后,可明显上调体外培养的人CSCC A431细胞的促凋亡蛋白Smac的表达,增加凋亡相关因子Caspase-3活性,从而抑制CSCC细胞增殖及转移能力,诱导凋亡。2. pSilencer2.1/U6-siLivin沉默Livin基因后,增强A431细胞对于BLM化疗的敏感性,逆转BLM耐药性。第四部分RNAi沉默Livin基因对皮肤鳞状细胞癌A431细胞裸鼠移植瘤生长的影响目的探讨Livin特异性的siRNA在人CSCC细胞株A431裸鼠移植瘤中沉默Livin基因的表达情况及对移植瘤生长的抑制作用,为进一步临床试验提供基础。方法1.将人CSCC细胞株A431接种于裸鼠左腋下皮下,建立裸鼠皮下移植瘤模型。接种A431细胞后第8天将成瘤后的裸鼠随机分成空白对照组、空载体对照组、治疗组。2.分别向移植瘤空白对照组注射生理盐水(NS)、空载体对照组注射pSilencer2.1/U6质粒、治疗组注射重组质粒pSilencer2.1/U6-siLivin,检测观察肿瘤的生长情况,绘制肿瘤生长曲线,接种细胞后第33天处死裸鼠照相、剥取瘤组织称重,肿瘤组织适当处理后备用。3.H.E染色观察肿瘤组织的病理改变。4.应用荧光定量RT-PCR检测移植瘤组织中Livin和Smac mRNA表达,应用Western blot分别检测Livin蛋白、Smac蛋白和Caspase-3蛋白的表达水平。结果1.裸鼠接种A431细胞后第8天时成瘤,治疗组瘤体生长较缓慢,肿瘤的体积明显低于空白对照组和空载体对照组。2.治疗组瘤体的平均重量较轻(0.58+0.03)g与空白对照组(1.27+0.08)g和空载体对照组(1.24+0.05)g比较,差异有统计学意义(P<0.05)。3.治疗组肿瘤生长受到抑制,肿瘤细胞坏死、凋亡增加。4.治疗组与空白对照组和空载体对照组比较,Livin mRNA和蛋白表达水平显著降低,差异有统计学意义(P<0.05), Smac mRNA和蛋白表达水平显著升高,差异有统计学意义(P<0.05), Caspase-3蛋白表达水平亦显著升高。小结1.沉默Livin可以有效抑制皮肤鳞癌A431细胞在裸鼠成瘤实验中移植瘤的生长。2.沉默Livin基因后,Smac和Caspase-3蛋白表达水平显著升高,与体外实验结果一致。1.Livin和Smac基因在人皮肤鳞状细胞癌组织和A431中分别呈高表达和低表达,表达呈负相关,与肿瘤的转移以及分化程度相关。2.构建2个针对Livin的siRNA载体,筛选得到最佳干扰序列pSilencer2.1/ U6-si475载体和Livin表达沉默的皮肤鳞癌A431细胞株。3. pSilencer2.1/U6-siLivin沉默Livin基因后,可明显上调体外培养的人皮肤鳞癌细胞A431的促凋亡蛋白Smac的表达,增加凋亡相关因子Caspase-3活性,从而抑制皮肤鳞癌细胞增殖及转移能力,诱导凋亡。4.pSilencer2.1/U6-siLivin沉默Livin基因后,可增强A431细胞对于BLM化疗的敏感性,逆转BLM耐药性。5.体内体外实验结果均显示沉默Livin可以有效抑制皮肤鳞癌A431细胞增殖,诱导凋亡。更多还原

【Abstract】 Cutaneous squamous cell carcinoma (CSCC), as the most commonly occurring cancer in order of incidence in skin cancer, is one of the common skin cancer, and its mechanisms remain unknown up to date. Although it has made considerable progress in Mohs surgery (Mohs micrographic surgery),chemotherapy, radiotherapy, biotherapy, immunotherapy, etc, incidence of CSCC is still increasing, for some special CSCC cases which are not suitable to adopt Mohs micrographic surgery are becoming more and more, and the CSCC is an invasive carcinoma with high degree of malignancy, drug resistance, high incidence of lymph node metastasis, distant metastasis and late spread through the blood, which can lead to the CSCC patients relapse after treatment and even death.A lot of factors lead to the occurrence and development of CSCC, including the external environmental factors, genetic factors, and internal factors such as oncogene mutation, tumor suppressor gene inactivation, abnormal apoptosis and signal transduction pathway, This indicates that the occurrence and development of CSCC is a process with multifactor, polygene and multi-stage, and its important mechanism is cell proliferation and apoptosis imbalance.Livin, a in inhibitor of apoptosis proteins (IAPs) family, has strong inhibitor of apoptosis by direct binding to cysteine aspartate-specific protease (Caspase), so as to inhibits apoptosis and promotes the growth of cancer cells. Studies have shown that Livin was over-expression in many tumor cells including CSCC, while Caspase-3 and second mitochondria-derived activator of caspases (Smac), a factors of pro-apoptotic function, are often in low expression or even lack of. Livin and Caspase-3, Smac, factors of two opposite effects, are negatively related. RNA interference (RNAi) to silence expression of Livin can promote tumor cell apoptosis and inhibit tumor cell proliferation, and increasing the sensitivity of tumor cells to chemotherapeutic, which showing that it is related to the occurrence, development and prognosis of tumor in the over-expression of Livin.Concerning inhibiting target gene expression, RNA interference (RNAi) technology can be efficiently and specifically, and it is a powerful tool for the study of endogenous gene function and signal transduction pathways. In this study, CSCC cell lines with silencing of Livin were established by RNAi silencing Livin gene. Through a series of in vivo and in vitro experimental study, the changes of cell growth, proliferation, apoptosis, cell cycle, migration, cloning capability was observed after Livin gene expression being silenced. It was simultaneously detected the changes in the level of expression of caspase-3 and Smac, also the resistance reversal of the chemotherapy drug bleomycin (BLM) in the treating CSCC and its possible mechanism. These studies provided new targets and methods for therapy of CSCC. This study included four parts as follows. Part 1 Expression of Livin and Smac in cutaneous squamous cell carcinomaObjectiveTo detect the expression of Livin and Smac in human cutaneous squamous cell carcinoma tissues, human cutaneous squamous cell carcinoma cell line A431, normal skin tissue and human keratinocyte cell line HaCaT. To investigate the expression and significance of Livin and Smac, and analyze the correlation of expression of Livin and Smac, and clinical and pathological features in cutaneous squamous cell carcinoma tissues.Methods1. The pathologically-confirmed human cutaneous squamous cell carcinoma tissue specimens of 50 cases selected from Plastic Surgery from January 2009 to August 2011, and 20 cases of normal skin tissue specimens is also.2. Cultured human cutaneous squamous cell carcinoma cell line A431, human skin keratinocyte cell line HaCaT.3. Livin and Smac protein expression was detected in CSCC and normal skin specimens by SP immunohistochemical technique, the expression of these two proteins were comprehensive determined by the percentage of positive cells and the intensity of staining, and their correlation was analyzed.4. Western blot and Real-time-quantitive-RT-PCR were used to detect the gene expression of Livin and Smac in human cutaneous squamous cell carcinoma cell line A431 and human keratinocyte cell line HaCaT.5.To study correlation of Livin or Smac and tumor pathological characters.Results1. Livin and Smac were found in a brown pattern in the cytoplasm. The positive expression rate of Livin protein was 70.0%(35/50) in 50 CSCC tissues but in none of the normal skin tissues.The protein expression in CSCC was higher than those in normal skin tissue. The positive of Livin protein expression had significant differences between cutaneous squamous cell carcinoma and normal skin tissues (P<0.05). The positive expression rate of Smac was 46.0%(23/50) in 50 CSCC tissues and that of Smac was 100%, Smac protein showed lower expression in CSCC than normal skin tissues (P< 0.05).2. Both Livin mRNA and Livin protein correspondence expressions were consistently detected in 0.842±0.191 and 0.828±0.230 in human cutaneous squamous cell carcinoma cell line A431,but that of human keratinocyte cell line HaCaT were 0.249±0.023 and 0.175±0.020. The positive expression of Livin mRNA and Livin protein in human cutaneous squamous cell carcinoma cell line A431 were higher than those in human keratinocyte cell line HaCaT. The positive of Livin mRNA and Livin protein expression had significant differences between human cutaneous squamous cell carcinoma cell line A431 and human keratinocyte cell line HaCaT (P<0.05). Both Smac mRNA and Smac protein correspondence expressions were consistently detected in 0.328±0.038 and 0.231±0.032 in human cutaneous squamous cell carcinoma cell line A431,but that of human keratinocyte cell line HaCaT were 0.824±0.183 and 0.808±0.215.Smac mRNA and Smac protein showed lower expression in human cutaneous squamous cell carcinoma cell line A431 than human keratinocyte cell line HaCaT (P<0.05).3. The expression of Livin and Smac had not relationship with age, sex, the sizes of CSCC(P>0.05).The expression of Livin and Smac had relationship with the lymphatic metastasis and histologic differentiation of CSCC with a statistically significant difference (P<0.05).The positive expression rate of Livin protein was increased in CSCC from highly differentiated to poorly differentiated,and the positive expression rate of Smac was decreased in CSCC from highly differentiated to poorly differentiated.The expression of Livin protein was negatively related to that of Smac protein in CSCC.Summary1.The expression of Livin protein in CSCC was high, while the positive expression of Smac protein in CSCC was lower, and the expression of Livin and Smac had relationship with the lymphatic metastasis and histologic differentiation of CSCC.2. There were negatively related between Livin and Smac.Part 2 Construction and identification of Livin gene siRNA eukaryotic expression vectorsObjectiveThe Livin-siRNA eukaryotic expression vectors of human Livin gene was constructed and identified, so as to investigate the inhibitory effect of gene silencing mediated by Livin-siRNA and stable expression in targeting to cells, in order to provide stable transfection vectors for the study of the Livin gene function.Methods1. The two siRNA target sequences for Livin gene were designed, so as to synthesize hairpin-shaped single-stranded DNA, and to form double-stranded DNA after annealing.2. The double-stranded DNA with pSilencer2.1/U6 vector was connected, the recombinants were transformed and selected, and the inserted fragments were identified by sequence and enzyme cut. Two siRNA target sequences for Livin gene were obtained.3. The cultured cells were randomly allocated into silence Livin group 1 (transfection of pSilencer2.1/U6-si438), silence Livin group 2 (transfection of pSilencer2.1/U6-si475), empty vector group (transfection of pSilencer2.1/U6) and blank group (untransfected the A431 cells).4. We transfected Livin siRNA vectors into CSCC A431 cell line by shRNA-LipofectamineTM 2000.5. After G418 selection stably transfected cell lines were obtained. The mRNA and protein expression level of Livin was detected in the CSCC A431 cells transfected by Real-time-quantitative-RT-PCR and Western blot. Results1. According to human Livin known sequence(No.NM-022161,No.NM-139317)of GenBank, RNAi explorer of GeneLinkTM Company and siDESIGN center of Dharmacon Company were used to scan, two siRNA target sequences with 19bp were obtained by BLAST homological analysis confirmed,438-456 (GTGGTT-CCCCAGCTGTCAG),475-493 (GGAAGAGACTTTGTCCACA). We synthesized the two siRNA target sequences for Livin gene for two hairpin-shaped single-stranded DNA and at both ends with BamHⅠand HindⅢwithin the enzyme residues (named Livin438 and Livin475),and double-stranded DNA were formed after annealing(named si438 and si475).2.After the double-chain DNA (si438 and si475) was connected to pSilencer2.1/ U6 vector, the recombined plasmid were transformed and selected, and the inserted fragments were identified by DNA sequence analysis and after enzyme cut.Two siRNA target sequences for Livin gene (pSilencer2.1/U6-si438 and pSilencer2.1/ U6-si475) were obtained and were in consistency with the design.3. The stable transfected cell lines were obtained by G418 selection in silence Livin group 1 (transfection of pSilencer2.1/U6-si438), silence Livin group 2 (transfection of pSilencer2.1/U6-si475), empty vector group (transfection of pSilencer 2.1/U6).4. Both Livin mRNA and Livin protein correspondence expressions were consistently detected in 0.826±0.205 and 0.813±0.189 in empty vector groups, and that of blank group were 0.852±0.197 and 0.832±0.202.The Livin mRNA and Livin protein expression level did be not changed significantly between blank group and empty vector groups no a statistically significant difference (P>0.05). However, both Livin mRNA and Livin protein correspondence expressions were consistently detected in 0.253±0.031 and 0.233±0.024 in silence Livin group 1,and that of silence Livin group 2 were 0.132±0.031 and 0.128±0.031. The Livin mRNA and Livin protein expression level in silence Livin group 1 and silence Livin group 2 were significantly reduced in comparison with the two control groups with a statistically significant difference (P<0.05), the more than 70% the Livin gene silencing effect were achieved, and silence Livin group 2 showed the lowest Livin mRNA and Livin protein relative expression.Summary1. Two human Livin gene RNAi vector were successfully constructed. Optimal RNAi vector (pSilencer2.1/U6-si475) and two CSCC A431 cell lines of silencing of Livin were established successfully.2. Livin gene of target cells can be silenced by RNAi technology, The more than 70% the Livin gene silencing effect were achieved after the CSCC A431 cell were transfected into, and the expression of target sequences for Livin gene is inhibited in the transcription and protein levels.Part 3 Effects on biological behavior of cutaneous squamous cell carcinoma A431 cell lines silenced by reconstructed single siRNA vector targeting Livin genesObjectiveTo investigate the effect on biological behavior of cutaneous squamous cell carcinoma A431 cell lines silenced by reconstructed single siRNA vector targeting Livin genes and to study its possible mechanism, so as to provide the basis of scientific experiments for further clinical trials.Methods1.In vitro experiments are grouped into Livin group (stably transfected pSilencer2.1/U6-siLivin and silent Livin gene expression of A431 cells), empty vector group (stably transfected pSilencer2.1/U6 of A431 cells),blank group (untransfected the A431 cells).2.The mRNA and protein expression level of Smac were detected in the cells in each group by Real-Time-quantitative-PCR and Western blot, and activities of caspase-3 were detected by ELISA.3.The cell proliferation and resistance were detected by MTT. 4. The cell cycle and apoptosis were detected by flow cytometry.5. The ability to metastasis were detected by transwell hole.6. The clonogenic capacity were detected by Colony formation assay.Results1. Compared with the mRNA and protein expression level of Smac in blank group and empty vector group, the mRNA and protein expression level of Smac in silence Livin group was significantly increased with a statistically significant difference (P<0.05), and the activities of caspase-3 was increased significantly.2. MTT technique revealed that compared with the OD value in blank group and empty vector group, the OD value in silence Livin group was decreased significantly with a statistically significant difference (P<0.05), and compared with the cell apoptosis rate in blank group and empty vector group, the cell apoptosis rate in silence Livin group joint BLM treatment group was significantly increased (P<0.05).3. Compared with the rate of apoptosis in blank group and empty vector group, the rate of apoptosis in silence Livin group was increased significantly with a statistically significant difference (P<0.05), and compared with the cells G0/G1 phase percentage in blank group and empty vector group, the cells G0/G1 phase percentage in silence Livin group was significantly increased (P<0.05),and the cells S and G2/M phase percentage in silence Livin group was significantly decreased (P<0.05).4. Compared with the average numbers of the cell passed through matrigel in blank group and empty vector group, the average numbers of the cell passed through matrigel in silence Livin group was decreased significantly with a statistically significant difference (P<0.05), and cell colony formation rate was significantly lower(P<0.05).Summary1. Through upregulating the expressions of Smac and activities of caspase-3, Livin gene is silenced by pSilencer2.1/U6-siLivin wich can inhibit proliferation and ability to metastasis and induce apoptosis in A431 cells.2. After Livin gene is silenced by pSilenccr2.1/U6-siLivin, the sensitivity of A431 cells to BLM chemotherapy were enhanced, and the resistance of A431 cells to BLM could be reversed.Part 4 Effects of silencing Livin gene by RNA interference on xenograft growth of cutaneous squamous cell carcinoma A431 cells in nude mouseObjectiveTo investigate livin gene expression and growth inhibition of silencing Livin gene by RNA interference on xenograft of cutaneous squamous cell carcinoma A431 cells in nude mouse, so as to provide the basis for further clinical trials.Methods1. The nude mice tumor xenograft model was establish by injecting the human CSCC A431 cell lines into the left armpit subcutaneous tissue of the nude mice. All the nude mice were randomly divided into three groups in the eighth day after inoculation, which are the blank group, the empty vector group, the treatment group.2. The xenograft was injected normal saline (the blank group), empty pSilencer2.1/U6 vectors (the empty vector group), pSilencer2.1/U6-siLivin vectors (the treatment group). The tumor growth was observed and detected at the same time, and the curve of tumor growth was then described. The nude mice were putted to death and photoed on the 33th days, and tumors were measured, stripped and weighed.3. Pathological changes of tumor was observed with HE staining.4. Real-time-quantitative-RT-PCR were used to detect mRNA level of Livin and Smac in tumor tissues of nude mouse, Western blot were used to detect protein expression level of Livin, Smac and Caspase-3.Results1. Implanted tumors were observed apparently 8 days after the nude mice inoculated with A431 cells. The growth of the tumor in treatment group were significantly slower than in the empty vector group and the blank group,and tumor volume was significantly lower also.2. The average tumor weight in the treatment group were 0.58±0.03g, which were significantly lower than those of the empty vector group and the blank group (1.24±0.05g and 1.27±0.08g, P<0.05).3. The tumor growth was inhibited, while the necrosis and apoptosis were evident in the treatment group.4. Compared with the mRNA and protein expression level of Livin in blank group and the empty vector group, the mRNA and protein expression level of Livin in the treatment group was decreased significantly with a statistically significant difference (P<0.05), and compared with the mRNA and protein expression level of Smac in blank group and empty vector group, the mRNA and protein expression level of Smac in the treatment group was significantly increased (P<0.05), and the protein expression level of Caspase-3 increased significantly also.Summary1. The growth of A431 cell xenograft in nude mice were inhibited by silencing Livin gene.2. The protein expression level of Smac and Caspase-3 were increased significantly by silencing Livin gene, and experimental results was consistency in vitro and in vivo. 1. The expression of Livin protein in CSCC was high, while the positive expression of Smac protein in CSCC was lower. There were negatively related between Livin and Smac, and the expression of Livin and Smac had relationship with the lymphatic metastasis and histologic differentiation of CSCC.2. Two human Livin gene RNAi vector were successfully constructed. Optimal RNAi vector (pSilencer2.1/U6-si475) and two CSCC A431 cell lines of silencing of Livin were established successfully.3. Through upregulating the expressions of Smac and activities of caspase-3, Livin gene is silenced by pSilencer2.1/U6-siLivin wich can inhibit proliferation and ability to metastasis and induce apoptosis in A431 cells.4. After Livin gene is silenced by pSilencer2.1/U6-siLivin, the sensitivity of A431 cells to BLM chemotherapy were enhanced, and the resistance of A431 cells to BLM could be reversed.5. Livin gene is silenced wich can inhibit proliferation and induce apoptosis in A431 cells, and experimental results was consistency in vitro and in vivo.更多还原