RNA干扰抑制人乳腺癌MCF-7细胞S100A4基因表达的实验研究
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摘要
[研究目的和意义]
乳腺癌是女性乳腺最常见的恶性肿瘤,亦是女性最常见的恶性肿瘤之一。我国虽属乳腺癌的低发区,但近年乳腺癌的发病率同样呈上升趋势,不仅居女性恶性肿瘤的首位而且发病高峰提前,虽然针对乳腺癌的综合治疗已使其五年生存率提高,但是乳腺癌发生远处转移仍是影响其预后的重要因素。乳腺癌的基因治疗是继手术、放疗、化疗和内分泌治疗之后发展的一种新的治疗手段,是一种很有前景的生物治疗手段,目前已有一些研究成果应用于临床,并取得了显著的疗效。例如：美国食品及药物管理局(FDA)已于1998年10月正式批准将针对Her-2受体,经过高纯度重组DNA衍生的单克隆抗体Trastuzumab(商品名Herceptin, Genentech/罗氏公司)用于临床治疗Her-2/neu过度表达的转移性乳腺癌,这为乳腺癌的治疗带来了新的曙光。乳腺癌的基因治疗虽然取得了初步的成功,但还有许多问题仍待解决,其中最主要问题之一是可用于临床治疗的新基因太少。因此,寻找新的靶点基因显得尤为重要。S100A4蛋白是S100蛋白家族中的一员与其家族中另外14个成员的编码基因集中于染色体1q21,这一区域是与多种恶性肿瘤发生有关的不稳定区,因此S100A4蛋白的编码基因有其独特的表达形式。目前研究表明,S100A4蛋白参与细胞内外的信号传导,细胞周期调节,并且可以参与细胞增殖分化、细胞黏附、细胞运动等多种生命活动与包括乳腺癌在内的多种恶性肿瘤的发生、侵袭以及转移等病理过程密切相关。而且S100A4蛋白几乎表达于所有肿瘤细胞,在人体中,迄今只发现S100A4蛋白在单核细胞、巨噬细胞、多核粒细胞、角化细胞、郎罕细胞及汗腺细胞中有不同程度的表达；在正常肺、肾脏,乳腺、甲状腺、胰腺及结肠的组织细胞中没有表达,因此是S100A4蛋白肿瘤基因治疗的理想靶点。国内外的诸多研究皆已证实对于包括乳腺癌在内的多种恶性肿瘤,S100A4蛋白是一种可靠的预测预后和淋巴结转移的因子。在基因治疗方面,Shi Y等[1]运用RNA干扰技术沉默甲状腺未分化癌细胞系(ARO)中的S100A4的表达,发现癌细胞增殖降低了46%±7.6%,并且明显抑制癌细胞转移,而提高了癌细胞对多西紫杉醇的敏感性。Saleem M等[2]对高转移性前列腺癌细胞系(PC-3)进行针对S100A4的RNA干扰研究,也发现沉默S100A4后,可以明显抑制癌细胞增殖、侵袭和转移。除了上述研究以外,近几年来对于其他肿瘤例如：胰腺癌、前列腺癌、食道癌等的相关研究也得出了相似的结论,即：运用RNA干扰技术沉默癌细胞中S100A4基因的表达,可以抑制癌细胞增殖以及转移。
在国内外研究成果的基础上,本课题运用RNA干扰技术,以S100A4为目的基因体外合成S100A4-shRNA (short hairpin RNA)模板链,并连接载体pGenesil-1.1与S100A4-shRNA构建重组质粒,应用脂质体Lipofectamine 2000介导进行细胞转染,通过S100A4-shRNA沉默S100A4基因表达后,观察MCF-7细胞在增殖、凋亡,以及侵袭力和迁移力方面的变化。本课题可以为乳腺癌的基因治疗提供新的着眼点,具有一定的科研意义和临床实用价值。
[统计方法]
所有数据采用SPSS 13.0统计软件进行统计学分析,数据均以X±S表示,多组间比较采用单因素方差分析(one-way ANOVA):首先进行方差齐性检验,若各组间反应变量为方差齐性,则组间比较采用LSD法；若方差不齐采用近似方差分析Welch法和Brown-Forsythe法进行校正,若方差仍为不齐,则组间比较采用Dunnett's T3法。MTT实验另采用重复测量资料方差分析明确分组与时间点之间是否存在交互作用。P<0.05,为差异有统计学意义。
[研究方法和结果]
第一章靶向S100A4的重组质粒的构建
[目的]合成两条重组质粒做为S100A4-shRNA的表达载体转染入MCF-7细胞,使S100A4-shRNA沉默S100A4基因的表达。
[方法]分别设计并合成两条针对S100A4的shRNA序列,获得含有针对S100A4的RNA干扰序列片段,并将其插入到含有绿色荧光蛋白(EGFP)和稳定筛选标记的质粒载体pGenesil-1.1中,构建重组质粒pGenesil-1.1-S100A4-shR-NA-1、pGenesil-1.1-S100A4-shRNA-2,经酶切及测序鉴定正确后大量扩增。
[结果]使用质粒提取试剂盒提取重组质粒,经SacⅠ酶切后,行1%琼脂糖电泳鉴定。在2个插入的目的基因片段里分别设计了一个SacⅠ酶切位点,而质粒pGenesil-1.1中本身也含有一个SacⅠ酶切位点,因此如果连接正确,重组质粒就能被SacⅠ酶切出1条约916 bp的DNA小条带。酶切鉴定证明2条重组质粒均符合设计要求,再经上海英骏生物技术有限公司测序,证明序列完全正确。
第二章RNA干扰抑制MCF-7细胞S100A4基因表达的体内实验研究
2.1免疫组化实验检测MCF-7细胞中S100A4蛋白的表达
[目的]检测MCF-7细胞中S100A4蛋白的表达情况。
[方法]本实验分为两组,即：空白对照组和实验组。将苔盘兰染色活力>95%的MCF-7细胞以1x106/孔种于6孔板中的无菌盖玻片,使用免疫组化Envision法,检测S100A4蛋白在MCF-7细胞中的表达。
[结果]人乳腺癌MCF-7细胞中S100A4蛋白存在明显表达。
2.2 MCF-7细胞转染实验
[目的]检测经脂质体介导重组质粒转染入MCF-7细胞情况。
[方法]转染24后,荧光显微镜下检测经脂质体介导重组质粒转染入MCF-7细胞情况。
[结果]经脂质体介导重组质粒成功转染入MCF-7细胞。
2.3 QRT-PCR法检测转染后S100A4基因表达的变化
[目的]检测转染48h后,MCF-7细胞中S100A4基因的表达情况,并筛选出沉默S100A4表达效率更高的一条重组质粒。
[方法]本实验分为四组：空白对照组、阴性对照组、S100A4-shRNA-1组；S100A4-shRNA-2组,后三组分别转染阴性对照质粒、重组质粒pGenesil-1.1-S100A4-shRNA-1、重组质粒pGenesil-1.1-S100A4-shRNA-2,通过QRT-PCR法,以GAPDH为内参基因,对于转染48h后S100A4基因在各组中的表达进行检测。
[结果]QRT-PCR检测结果显示,转染48h后,三组间S100A4mRNA表达量差异具有统计学意义(F= 67.854, P=0.000), S100A4-shRNA-1组和S100A4-shR-NA-2组的S100A4mRNA表达量低于阴性对照组和空白对照组,差异均有统计学意义(P=0.000,P=0.000；P=0.000,P=0.000)；而S100A4-shRNA-1组中S100A4-mRNA表达量低于S100A4-shRNA-2组,差异具有显著性(P=0.017)。因此选用重组质粒pGenesil-1.1-S100A4-shRNA-1进行后续实验。
2.4 Westernblot法检测转染后S100A4蛋白表达的变化
[目的]检测转染48h后,MCF-7细胞中S100A4蛋白的表达情况。
[方法]实验分为3组,即实验组、阴性对照组、空白对照组。前两组分别转染重组质粒pGenesil-1.1-S100A4-shRNA-1以及阴性对照质粒。本实验通过West-ern blot法对于转染48h后,S100A4蛋白在各组中的表达进行检测。
[结果]Western blot法检测结果显示,与空白对照组和阴性对照组相比,实验组组细胞S100A4蛋白表达明显减弱,Tanon系统分析显示其蛋白条带灰度值较前两者分别降低57.21%和54.82%。
2.5 MTT法检测转染后细胞增殖的变化
[目的]检测转染后MCF-7细胞增殖的变化情况。
[方法]实验分组同上,将实验组、阴性对照组和空白对照组细胞以1x104个/孔密度种于96孔板,分别于转染24h、48h和72 h后每孔加入MTT(5mg/ml)20μl,置于CO2培养箱中培育4h,终止反应后弃去上清液。每孔加入DMSO 200μl,振荡10 min,在酶标仪上于490 nm波长下读取吸光度(OD)值,绘制各组细胞生长曲线,各组实验重复3次。
[结果]MTT法检测MCF-7细胞增殖水平显示,三组间在24h、48h、72h各时间点OD值差异均具有统计学意义(F=13.401,P=0.006；F=132.689,P=0.000；F=3150.772,P=0.000),分组与时间点之间存在交互作用(F=68.466,P=0.000)。实验组细胞在24h、48h、72h各时间点OD值均低于阴性对照组和空白对照组,差异有统计学意义(P=0.006,P=0.003；P=0.015,P=0.015；P=0.000,P=0.000),阴性对照组和空白对照组各时间点OD值差异均无统计学意义(P=0.571,P=0.287,P=0.211)。
2.6 Annexin V-FITC/PI双染色法检测转染后细胞凋亡率的变化
[目的]检测转染24h后,MCF-7细胞凋亡的变化情况。
[方法]实验分组同上,以1x106个/孔接种各组细胞于6孔板。每组设3个复孔。转染后24h,收集上清液和细胞,调整细胞密度为3x105个/mL,加入2μl Annexi-nV-FITC和5μlPI,应用流式细胞仪测定各组细胞凋亡情况。每组实验重复3次。结果判定：正常细胞为AnnexinV-FITC(-)/PI(-),早期凋亡细胞为AnnexinV-FI-TC(+)-/PI(-),晚期凋亡细胞为AnnexinV-FITC(+)/PI(+),坏死细胞为Annexin-V-FITC(-)/PI(+)。
[结果]转染24h后,三组间晚期凋亡细胞率差异具有统计学意义(F=5.895,P=0.038),实验组晚期凋亡细胞率较阴性对照组、空白对照组升高,差异具有显著性(P=0.036,P=0.019),而后两组差异无统计学意义(P=0.636)。
2.7稳定转染细胞株的建立及其形态观察
[目的]建立稳定转染细胞株观察其形态学的变化。
[方法]首先确定G418筛选稳定转染株细胞的浓度,将苔盘蓝计数细胞活力>95%的MCF-7细胞以1x106/孔种于6孔细胞培养板。细胞贴壁后分别换成含有0、100、200、300、400、500、600、700、800、900、1000ug/ml G418的10%FBS的RPMI1640培养液培养,每隔3天换液,对比观察正常细胞和G418筛选组细胞的生长情况,取一周70%死亡,二周100%死亡的最低G418浓度为筛选稳定转染株细胞的浓度(该实验确定G418筛选浓度为500ug/ml)。实验分为3组即：实验对照组、阴性对照组、空白对照组,前两组分别转染实验用重组质粒以及阴性对照质粒,将转染24h后的MCF-7细胞按照1：10传代。并设置正常细胞对照孔2孔。细胞贴壁后换含有500ug/ml G418的10%FBS的RPMI1640培养液,每隔3天换液,2周后观察正常细胞死亡率100%,转染细胞30%存活。换含有350ug/mlG418的10%FBS的RPMI1640培养液,1周后出现单个细胞。荧光显微镜下观察将培养板盖上发荧光,光学显微镜下为活细胞的MCF-7细胞标记。消化收集调整细胞浓度<=1个/100ul,100ul/孔种96孔板,扩大培养,2周后成细胞集落,5周后长至50%。待细胞长满后传至24孔板,逐渐扩大培养至6孔板和25cm2培养瓶。分别命名实验组稳定转染株细胞为MCF-7-S100A4细胞,阴性对照组稳定转染株细胞为MCF-7-HK细胞。使用免疫组化Envision法观察MCF-7-S100A4细胞、MCF-7-HK细胞以及MCF-7细胞在形态、大小等生物学特性中的区别。
[结果]筛选出稳定转染株细胞后,通过免疫组化检测发现,MCF-7细胞和MCF-7-HK细胞同样呈不规则形状、大小不一、核大而深染、呈圆形或椭圆形,细胞极性消失、排列紊乱；MCF-7-S100A4细胞在大小、形态以及生长方式上与以上细胞无明显差别。
2.8划痕实验检测稳定转染株细胞的迁移力
[目的]检测稳定转染株细胞迁移力的变化。
[方法]运用划痕实验检测稳定转染株细胞迁移力的变化,实验分为3组,即：实验组(MCF-7-S100A4细胞组)、阴性对照组(MCF-7-HK细胞组)以及空白对照组,取处于对数生长期的各组细胞以1x106个/孔接种于6孔板中培养。在单层细胞表面用100μl移液器枪头垂直划痕,PBS液轻洗2次,在倒置显微镜下观察0、6、24和48 h后划痕中细胞迁移情况,并拍照。
[结果]体外划痕实验结果显示,实验组中MCF-7-S100A4细胞的移动速度明显慢于,空白对照组中MCF-7细胞以及阴性对照组中MCF-7-HK细胞；接种后48h,MCF-7细胞以及MCF-7-HK细胞划痕已经基本长满,而MCF-7-S100A4细胞划痕尚未长满。
2.9 Transwell小室法检测稳定转染株细胞侵袭力
[目的]检测稳定转染株细胞侵袭力的变化。
[方法]首先实验分为3组,即：实验组(MCF-7-S100A4细胞组)、阴性对照组(MCF-7-HK细胞组)以及空白对照组,通过Transwell小室法对于稳定转染株细胞侵袭力的变化进行检测。
[结果]侵袭实验结果显示,三组间差异具有统计学意义(F=826.583,P=0.000),其中实验组穿膜细胞数低于阴性对照组和空白对照组,差异有统计学意义(P=0.000,P=0.000),而阴性对照组与空白对照组相比差异无统计学意义(P=0.128)。
第三章RNA干扰抑制MCF-7细胞S100A4基因表达的体内实验研究
3.1稳定转染株细胞在裸鼠体内的成瘤实验
[目的]通过将稳定转染株细胞细胞接种于裸鼠背部,检测裸鼠背部移植瘤成瘤情况。
[方法]本实验分组分为3组,即：阴性对照组(MCF-7-HK细胞组)、实验组(MCF-7-S100A4细胞组)、空白对照组,每组裸鼠10只。取上述各组细胞分别消化制成单细胞悬液,调整细胞密度为1×107个/mL,每只裸鼠颈背部皮下注射0.4ml。种植肿瘤细胞后,每天观察裸鼠饮食、精神及活动状况,每隔2d测定肿瘤大小,并测量肿瘤最长径(a)及其垂直方向最大横径(b),按公式V(mm3)=axb2/2计算肿瘤体积,并绘制移植瘤的生长曲线图并计算抑瘤率。观察35 d后,脱颈处死裸鼠,测量各组裸鼠移植瘤的质量,对结果进行统计分析。
[结果]体内实验部分中细胞接种裸鼠35d后,空白对照组及阴性对照组小鼠的平均肿瘤体积超过900mm3,成瘤率为100%；实验组小鼠于观察2-3周后,仅可在皮下触及极小体积肿瘤(约12mm3),且随着观察天数增加肿瘤不再生长,反而有缩小迹象(<12mm3),成瘤率为90%。肿瘤细胞接种35d后,处死动物,分离皮下瘤称重。实验结果显示,三组间肿瘤体积和质量差异具有统计学意义,(F=102201.907,P=0.000；F=8859.118,P=0.000)。实验组裸鼠的肿瘤质量和体积低于阴性对照组和空白对照组,差异具有显著性(P=0.000,P=0.000；P=0.000,P=0.000),而2个对照组裸鼠肿瘤之间差异无统计学意义(P=0.991,P=0.062)。使用公式计算抑瘤率(%)=[(空白对照组平均瘤重-治疗组平均瘤重)/空白对照组平均瘤重]×100%,可得抑瘤率为98.64%。
3.2 QRT-PCR法检测裸鼠背部移植瘤中S100A4基因的表达
[目的]检测各组裸鼠背部移植瘤中S100A4基因的表达。
[方法]本实验分组同上,每组随意选取4只成瘤裸鼠,取背部移植瘤组织进行实验,另取裸鼠背部正常组织作为对照。通过QRT-PCR的方法,采用Sybrgreen染料法,以GAPDH为内参基因,对于S100A4基因在各组裸鼠背部移植瘤组织中的表达量进行检测。
[结果]QRT-PCR检测结果显示,三组间S100A4mRNA表达量差异具有统计学意义(F=83.598,P=0.000),实验组中S100A4mRNA表达量低于阴性对照组和空白对照组,差异均有统计学意义(P=0.000,P=0.000),而后两组的S100A4mRNA表达量差异无统计学意义(P=0.915).
3.3 Westernblot法检测裸鼠背部移植瘤中S100A4蛋白的表达
[目的]检测各组裸鼠背部移植瘤中S100A4蛋白的表达。
[方法]实验分组同上,每组随意选取4只成瘤裸鼠,取背部移植瘤组织进行实验,另取裸鼠背部正常组织作为对照。通过Western blot法检测各组裸鼠背部移植瘤中S100A4蛋白的表达。
[结果]Tanon系统分析显示三组间S100A4蛋白表达量差异具有统计学意义(F=90.297,P=0.000),其中空白对照组和阴性对照组中的S100A4蛋白表达量高于实验组组差异均有统计学意义(P=0.000；P=0.000),而前两者差异无明显统计学意义(P=0.643).
[研究结论]
本研究以乳腺癌MCF-7细胞作为研究模型,以构建携带针对S100A4的特异干涉序列的S100A4-shRNA作为进行RNA干涉的工具,沉默S100A4基因的表达。根据实验结果,本研究阐明了以下几点：
1)通过S100A4-shRNA沉默S100A4,能有效抑制MCF-7细胞中S100A4的表达。
2)通过S100A4-shRNA沉默S100A4,能有效抑制MCF-7细胞增殖并促进凋亡的发生。
3)通过S100A4-shRNA沉默S100A4,能有效抑制MCF-7细胞迁移和侵袭力。
4)通过S100A4-shRNA沉默S100A4,能有效抑制MCF-7细胞在裸鼠体内成瘤的能力。
鉴于以上结果,得出以下结论,我们构建的携带S100A4特异干涉序列的S100A4-shRNA能有效沉默S100A4基因,抑制MCF-7细胞增殖以及侵袭和迁移能力,并促进其凋亡。这证明了S100A4蛋白参与细胞增殖分化、细胞运动等多种生命活动,并与乳腺癌MCF-7细胞的增殖、侵袭以及转移等病理过程密切相关。
[Objective and significance]
Breast cancer is the most commonly seen malignant carcinoma of female breasts, and also one of the malignant carcinomas with the highest occurrence among females. Though our country is a region with lower occurrence rate of breast cancer, the incidence rate is increasing in recent years. It not only ranks at the first among female malignant carcinomas, and with an earlier peak of morbidity. Though the five year survival is improved because of comprehensive therapy targeting to breast cancer, distant metastasis is still the important prognostic factor for breast cancer. The genetic therapy against breast cancer is a new therapeutic measure following surgical approach, radiotherapy, chemotherapy and endocrine therapy, and it is a biological therapeutic method with good prospective. At present, some research achievements have been applied to clinical practice, with significant therapeutic effects obtained. For example:in Oct.,1998, FDA had formally approved to administer Trastuzumab (trade name, Herceptin, Genentech/Roche), the monoclonal antibody derived from high purity recombinant DNA and targeting Her-2 receptor, to treat metastatic breast cancer with over expression of Her-2/neu, which brought the first morning light to therapy against breast cancer. Though primary success has been obtained in genetic therapy against breast cancer, there are still many problems, and one of them is that, there are only few genes which can be applied into clinical therapy. Therefore, it is especially important to find a new target gene. S100A4 protein is a member of S100 protein family, and its coding genes are localized at chromosome 1q21 with other 14 members, and this region is the unstable region correlated with the occurrence of many malignant carcinomas. Therefore, coding gene of S100A4 protein has its special expression styles. It is indicated in the current study that, S100A4 protein participates in the signal transduction in and out of cells, cell cycle regulation, and it is involved in many life activities including cell proliferation and differentiation, cell adhesion, cell migration, etc., and it is deeply related with the pathological process of many malignant carcinomas including breast cancer, including genesis, infiltration and metastasis, etc. Moreover, S100A4 protein is nearly expressed in all carcinoma cells. In human body, S100A4 expression of varied degrees is only observed in monocyte, macrophage, polymorphonuclear granulocyte, keratinocyte, Langhans'cell and sweat gland cell till now. In normal cells of lung, kidney, breast cancer, thyroid, pancreas and colon, S100A4 is not expressed, so S100A4 protein is the ideal target for genetic therapy against carcinomas. Many studies in China and other countries have confirmed that, for many malignant carcinomas including breast cancer, S100A4 protein is a stable factor to predicting prognosis and lymphatic metastasis. In the aspect of genetic therapy, Shi Y et al applied RNA interference to silence the S100A4 expression in undifferentiated thyroid carcinoma cell line (ARO), and found that the proliferation of carcinoma cell was decreased by 46%+7.6%, migration of carcinoma cells was also inhibited obviously, and sensitivity of carcinoma cells to taxotere was improved. Saleem M et al performed RNA interference study targe-ting S100A4 in highly metastatic prostate carcinoma cell line (PC-3), and found that after S100A4 was silenced, proliferation, infiltration and metastasis of carcinoma cells were significantly inhibited. Besides of the abovementioned studies, similar conclusion has been obtained in related studies on other carcinomas, including pancr-eatic carcinoma, prostate carcinoma and esophageal cancer, etc., i.e., RNA interfere-nce is applied to silence expression of S100A4 gene, and proliferation and metastasis of carcinoma cells can be inhibited.
Based on the study achievements in China and other countries, RNA interference was adopted in this study to synthesize S100A4-shRNA template strand with S100A4 as the target gene, and construct recombinant plasmid by linking two carriers of pGenesil-1.1 and S100A4shRNA. Liposome Lipofectamine 2000 was applied to me-diate cell transfect, and S100A4-shRNA was used to silence S100A4 gene expression, to observe the proliferation and apoptosis of MCF-7 cell, and changes of infiltrating and migrating capacity. Our study can provide a new approach for genetic therapy against breast cancer, and it has certain scientific significance and clinical practice.
[Statistical methods]
Statistical analysis was performed by SPSS13.0 software.All data were presente-d as mean±standard deviation.one-way ANOVA was used to determine the statistical significance,first of all,doing the test of Homogeneity of Variances among the group-s. If the equal variances assumed, LSD was used to determine the statistical significa-nce. If the equal variances not assumed, Dunnett's T3 was used to determine the stati-stical significance.Repeated measures ANOVA was used to test the cross correlation between factor and groups in MTT.P<0.05 was considered statistically significant.
[Methods and Results]
Chapter 1 Construction of recombinant plasmid for target S100A4
[Objective] To synthesize two recombinant plasmids as the expression vector of S100A4-shRNA to transfect into MCF-7 cells, and silence the expression of S100A4 gene.
[Methods] Two shRNA sequences targeting S100A4 were designed and synthesized to obtain RNA interference sequence segment for S100A4, which was inserted into the stably screened and labeled plasmid carrier pGenesil-1.1 containing green fluorescent protein (EGFP), then recombinant plasmids pGenesil-1.1-S100A4-shRNA-1 and pGenesil-1.1-S100A4-shR.NA-2 were constructed, and amplified in great amount after enzyme digested and sequenced.
[Results] After the recombinant plasmid extracted by using extraction kit was excised by Sac I enzyme, it was identified by using 1% agarose electrophoresis. A Sac I enzyme digestion site was designed in two target gene segments, and there was also a Sac I enzyme digestion site in plasmid pGenesil-1.1. Therefore, if the linkage was correct, a 916 bp DNA strap was excised from recombinant plasmid by Sac I enzyme. It was confirmed by enzyme excision that,2 recombinant plasmids conform-ed to design requirements, and the sequencing results obtained by Shanghai Invitroge-n Biological Technology Co., Ltd. confirmed that the sequence was correct.
Chapter 2 In vivo experimental study on S100A4 gene expression in MCF-7 cell inhibited by RNA interference
2.1 Expression of S100A4 protein in MCF-7 cell detected by immunohis-tochemical method
[Objective] To detect the expression of S100A4 protein in MCF-7 cell.
[Methods] The experiment had two groups including blank control group and test group. Inoculate MCF-7 cells with trypan blue staining activity>95% in sterile cover glass of 6-well plate at 1×106/well, Immunohistochemical SP method was applied to detect the expression of S100A4 protein in MCF-7 cell.
[Results] Obvious expression of S100A4 protein was observed in human breast cancer MCF-7 cell.
2.2 MCF-7 cell transfect test
[Objective] To detect the transfect of liposome mediated recombinant plasmid into MCF-7 cell.
[Methods] After transfect for 24h, the transfect of liposome mediated recombine-nt plasmid into MCF-7 cell was detected by using fluorescence microscope.
[Results] Liposome mediated recombinant plasmid was successfully transfected into MCF-7 cells.
2.3 Changes of S100A4 gene expression after transfect by QRT-PCR method
[Objective] To detect S100A4 gene expression in MCF-7 cell 48h after transfect, and screen a recombinant plasmid with a higher S100A4 expression rate.
[Methods] The experiment had four groups:blank control group, negative control group, S100A4-shRNA-1 group and S100A4-shRNA-2 group. The latter three groups were respectively transfected by negative control plasmid, recombinant plasmid pGenesil-1.1-S100A4-shRNA-1, recombinant plasmid pGenesil-1.1-S100A4-shRNA-2, and the expression of S100A4 gene in different groups were detected by QRT-PCR method 48h after transfect, with GAPDH as the internal reference gene.
[Results] Results of QRT-PCR indicated that,48h after transfect, there are stastic-al significance among the three groups (F= 67.854, P=0.000).The expression amount of S100A4 mRNA in S100A4-shRNA-1 group and S100A4-shRNA-2 group was lower than those in negative control and blank control groups(P=0.000, P=0.000; P=0.000, P=0.000); expression amount of S100A4mRNA in S100A4-shRNA-1 group was lower than that in S100A4-shRNA-2 group (P=0.017). Thus, recombinant plasmid pGenesil-1.1-S100A4-shRNA-1 was used to perform the subsequent experi-ment.
2.4 Changes of S100A4 protein expression detected by Westernblot method
[Objective] To detect the expression of S100A4 protein in MCF-7 cells 48h after transfect.
[Methods] The experiment had three groups, including test group, negative control group and blank control group. The first two groups were transfected by recombinant plasmids of pGenesil-1.1-S100A4-shR.NA-1 and negative control plasm-id. Western blot method was applied to detect the expression of S100A4 proteins in different groups 48h after transfect.
[Results] Results of Western blot indicated that, compared to blank control group and negative control group, expression of S100A4 protein was obviously weakened, and Tanon systemic analysis indicated that, its protein strap gray scale was decreased by 57.21% and 54.82% compared to those of the first two groups.
2.5 Changes of cell proliferation after transfect by using MTT method
[Objective] To detect the changes of MCF-7 cell proliferation after transfect.
[Methods] The experimental materials were grouped as abovementioned, and cells of test group, negative control group and blank control group were inoculated into a 96-well plate at 1×104 cells/well, MTT (5 mg/ml) 20μl was added into each well after the cells were transfected for 24,48 and 72 h. Then the plate was placed into a CO2 incubator for 4h, and the supernatant was discarded after the reaction was stopped. DMSO 200μl was added into each well, then the plate was shaken for 10min, and optical density (OD) value was read at 490 nm wavelength to draw the growth curves. The procedure was repeated for three times.
[Results] Experimental results indicated that, there are stastical significance among the three groups at 24,48 and 72h(F=13.401, P=0.006; F=132.689, P= 0.000; F=3150.772, P=0.000).OD value of test group was lower than those of negative control and blank control group at 24,48 and 72h (P=0.006, P=0.003; P=0.015, P=0.015; P=0.000, P=0.000), while difference of OD value was insignificant betwe-en negative control and blank control groups at all time points (P=0.571; P=0.287; P= 0.211).
2.6 Annexin V-FITC/PI double staining method was used to detect the changes of apoptosis rate after transfect.
[Objective] To detect the changes of MCF-7 cell apoptosis 24 h after transfect.
[Methods] The experimental materials were grouped as abovementioned, and cells were inoculated into a 6-well plate at 1×106 cells/plate. Triplicate wells were set for each group. Supernatant and cells were collected 24h after transfect. The cell density was adjusted to 3×105/mL, and then 2μl Annexin V-FITC and 5μl PI were added to determine the apoptosis of cells in different groups by using flow cytometry. The procedure was repeated for 3 times. Result judgment:normal cell was Annexin V-FITC (-)/PI (-), viable apoptotic cell was Annexin V-FITC (+)-/PI (-), non-viable apoptotic cell was Annexin V-FITC (+)/PI (+), and necrotic cell was Annexin V-FITC (-)/PI (+).
[Results] Experimental results indicated that,24h after transfect, there are stastic-al significance among the three groups in the apoptosis rate of non-viable apoptotic cell (F= 5.895, P=0.038).the apoptosis rate of non-viable apoptotic cell in test group was higher than those of negative control and blank control groups (P=0.036, P=0.019), but no statistical difference was observed between the latter two groups (P=0.636).
2.7 Construction and morphological observation of stable transfected cell strain
[Objective] To construct stable transfected cell strain and observe their changes.
[Methods] Firstly, use G418 to screen the concentration for stable transfect cell, and inoculate MCF-7 cells with cell activity>95% counted by trypan blue into a 6-well plate at 1×106 cells/well. After cells adhered to well wall, RPMI1640 solution containing 0,100,200,300,400,500,600,700,800,900 and 1000ug/ml G41810% FBS were used to culture the cells. The solution was replaced for every 3 days, growth of cells was observed between normal cells and G418 screened cells. G418 concentration with 70% death in one week and 100% death in the second week was selected to screen the concentration of stable transfect cell (in this experiment, G418 screening concentration was 500 ug/ml). The experiment had three groups including test control group, negative control group and blank control group. The first two groups were transfected with recombinant plasmid and negative control plasmid, and then MCF-7 cell was 1:10 passaged 24 h after transfect. Two control wells of normal cells were also set. After cells adhered to well wall,10% FBS RPMI1640 solution containing 500ug/ml G418 was adopted, and the solution was changed for every 3 days. After 2 weeks, normal cells had a death rate of 100%, and 30% transfected cells were survived. Then 10% FBS RPMI1640 solution containing 350ug/ml G418 was changed, and single cells appeared 1 week later. Under the fluorescent microscope, living MCF-7 cells with fluorescence were labeled. The cells were digested and collected to adjust the solution concentration<=1 cell/100ul, and cells were inoculated into a 96-well plate at 100ul/well. Then expand the culture, the cells formed cell cluster 2 weeks later. After 5 weeks,50% wells were covered. When cells covered 24 wells, transfer the cells gradually to a 6-well plate and a 25cm2 culture flask. Respectively name the stable transfect cell as MCF-7-S100A4 cell in test group, MCF-7-HK cell in negative control group. Immunohistochemical SP method was used to observe the differences of morphologies and size, etc. between MCF-7-S100-A4 cells, MCF-7-HK cells and MCF-7 cells.
[Results]After stable transfect cells were screened, it was found by immunohisto-chemical method that, MCF-7 cell and MCF-7-HK cell also had irregular morpholo-gies, varied sizes, big nuclei deeply staining, in round of oval shapes. The cell had no cell polarity, with confused arrangement. There was no significant difference between MCF-7-S100A4 cells and the above cells in cell size, morphology and living styles.
2.8 Migrating capacity of stable transfect cells by using scratch adhesion test
[Objective] To detect the changes of migrating capacity of stable transfect cells.
[Methods] Scratch adhesion test was used to detect the changes of migrating capacity of stable transfect cells. The experiment had three groups including test group (MCF-7-S100A4 cell group), negative control group (MCF-7-HK cell group) and blank control group. Cells at logarithmic phase were collected and inoculated into a 6-well plate at 1×106 cells/well. On single cell layer surface, scratch with 100μl transferpettor probe vertically, and flush with PBS for 2 times, then observe the migrating cells under a microscope for scratches made at 0,6,24 and 48 h, then take the images.
[Results] Results of scratch adhesion test indicated that, the migrating rate of MCF-7-S100A4 in test group was obviously smaller than those of MCF-7 cell in blank control group and MCF-7-HK cell in negative control group. After the cells were inoculated for 48 h, MCF-7 cells and MCF-7-HK cells covered the scratches, and MCF-7-S100A4 cells had not spread over the scratch.
2.9 Transwell chamber method was applied to determine the infiltrating capacity of stable transfect cells.
[Objective] To determine the changes of infiltrating capacity of stable transfect cells.
[Methods] The materials of the experiment were grouped as abovementioned, and Transwell chamber method was applied to determine the changes of infiltrating capacity of stable transfect cells.
[Results] Results of in vitro infiltrating experiment indicated that, there are stasti-cal significance among the three groups (F= 826.583, P=0.000), and the stastical dif-ference was observed between test group and the other two groups (P=0.000, P= 0.000), while no statistical difference was observed between negative and blank cont-rol groups (P=0.128). The results of the above experiment indicated that, after S100-A4 was silenced, the infiltrating and migrating capacities of MCF-7 cells were signif-icantly decreased.
Chapter 3 In vivo study on expression of S100A4 gene in MCF-7 cell inhibited by RNA interference
3.1 Tumorigenicity experiment on stable transfect cell in naked mice
[Objective] To determine the tumorigenicity of transplanted tumor by inoculating stable transfect cell into the back of naked mice.
[Methods] The experiment had three groups including negative control group (MCF-7-HK cell group),test group (MCF-7-S100A4 cell group) and blank control group, with 10 mice in each group. Take cells from the above groups and prepare single cell suspension, adjust the cell density to 1×107/mL, and 0.4 ml solution was injected subcutaneously into the back of naked mice. After the carcinoma cells were implanted, the diet, mental activities and movements were observed, tumor size was determined for every 2 days, and the maximal diameter (a) and maximal vertical transverse diameter of the tumor were determined. The tumor volume was calculated following V (mm3)=axb2/2, and growth curve was drawn to calculate the tumor inhibition rate. After observed for 35 d, the naked mice were decapitated, then the transplanted tumor was weighed, and the results were analyzed.
[Results]In experiment in vivo, after the naked mice were inoculated for 35 d, the average tumor volume was more than 900 mm3 in mice of blank and negative control groups, with a tumor formation rate of 100%. After observed for 2-3 weeks on mice of test group, tiny tumor (about 12mm3) could only observed touched subcutaneously, and the tumors would not growth as times went by, or they even shrunk (<12 mm3), and the tumor formation rate was 90%. After tumor cell was inoculated for 35d, the animals were executed, and subcutaneous tumors were isolated and weighed. Experimental results indicated that, there are stastical significance among the three groups in tumor mass and volume of naked mice (F= 102201.907, P=0.000; F= 8859.118, P=0.000).tumor mass and volume of naked mice in test group were significantly lower than those of negative and blank control groups (P=0.000, P=0.000; P= 0.000, P=0.000). while no significant difference was observed between two control groups(P=0.991, P=0.062). The following formula was used to calculate tumor inhibition rate (%)= [(average tumor weight in blank control group-average tumor weight in test group)/average tumor weight in blank control group]×100%, and the inhibition rate was 98.64%.
3.2 Expression of S100A4 gene in transplanted carcinoma determined by QRT-PCR method
[Objective] To determine the expression of S100A4 gene in transplanted carcinoma.
[Methods] Animals in this experiment were groups as abovementioned, with 4 naked mice with carcinomas randomized in each group. The transplanted carcinoma tissues were collected to perform the experiment, and normal tissues were also taken as controls. By using QRT-PCR and Sybrgreen staining method, GAPDH was adopted as the internal reference gene, and expression amount of S100A4 gene was determined in tissues of transplanted carcinoma on back of naked mice.
[Results] Results of QRT-PCR indicated that, there are stastical significance among the three groups (F=83.598, P=0.000) expression amount of S100A4 mRNA in test group was far lower than those of negative and blank control groups (P=0.000, P=0.000), while no significant difference was observed between the two control groups (P=0.915)。
3.3 Expression of S100A4 protein in transplanted carcinoma on back of naked mice determined by Westernblot
[Objective] To determine the expression of S100A4 protein in transplanted carcinoma on back of naked mice.
[Methods] Animals in this experiment were groups as abovementioned, with 4 naked mice with carcinomas randomized in each group. The transplanted carcinoma tissues were collected to perform the experiment, and normal tissues were also taken as controls. And Western blotting method was adopted to determine the expression of S100A4 protein in transplanted carcinoma on back of naked mice.
[Results] Results of Western blot indicated that, there are stastical significance among the three groups (F= 90.297, P=0.000).expression of S100A4 protein was significantly higher in blank and negative control groups compared to test group (P=0.000, P= 0.000), while no significant difference was observed between control groups(P=0.643).
[Conclusion]
In this study, breast cancer MCF-7 cell was adopted as the study model to construct S100A4-shRNA with specific interfering sequence S100A4 as the tool to perform RNA interference, and the expression of S100A4 gene was silenced. Accor-ding to the results, the study explained the following points:
1) Silencing S100A by using S100A4-shRNA can effectively inhibit expression S100A4 in MCF-7 cells.
2) Silencing S100A by using S100A4-shRNA can effectively inhibit MCF-7 proliferation and promote apoptosis.
3) Silencing S100A by using S100A4-shRNA can effectively inhibit cell migration and infiltration of MCF-7 cells.
4) Silencing S100A by using S100A4-shRNA can effectively inhibit the Tumorigenicity of MCF-7 cells in naked mice.
The following results are obtained according to the above results:the construc-ted S100A4-shRNA carrying S100A4 special interference sequence can effectively silence S100A4 gene, inhibit proliferation, infiltration and migration of MCF-7 cells, and promote their apoptosis. These results indicate that S100A4 protein participates in cell proliferation, differentiation and cell migration, and it is closely correlated with proliferation, infiltration and migration of breast cancer MCF-7 cells.
乳腺癌是女性乳腺最常见的恶性肿瘤,亦是女性最常见的恶性肿瘤之一。我国虽属乳腺癌的低发区,但近年乳腺癌的发病率同样呈上升趋势,不仅居女性恶性肿瘤的首位而且发病高峰提前,虽然针对乳腺癌的综合治疗已使其五年生存率提高,但是乳腺癌发生远处转移仍是影响其预后的重要因素。乳腺癌的基因治疗是继手术、放疗、化疗和内分泌治疗之后发展的一种新的治疗手段,是一种很有前景的生物治疗手段,目前已有一些研究成果应用于临床,并取得了显著的疗效。例如：美国食品及药物管理局(FDA)已于1998年10月正式批准将针对Her-2受体,经过高纯度重组DNA衍生的单克隆抗体Trastuzumab(商品名Herceptin, Genentech/罗氏公司)用于临床治疗Her-2/neu过度表达的转移性乳腺癌,这为乳腺癌的治疗带来了新的曙光。乳腺癌的基因治疗虽然取得了初步的成功,但还有许多问题仍待解决,其中最主要问题之一是可用于临床治疗的新基因太少。因此,寻找新的靶点基因显得尤为重要。S100A4蛋白是S100蛋白家族中的一员与其家族中另外14个成员的编码基因集中于染色体1q21,这一区域是与多种恶性肿瘤发生有关的不稳定区,因此S100A4蛋白的编码基因有其独特的表达形式。目前研究表明,S100A4蛋白参与细胞内外的信号传导,细胞周期调节,并且可以参与细胞增殖分化、细胞黏附、细胞运动等多种生命活动与包括乳腺癌在内的多种恶性肿瘤的发生、侵袭以及转移等病理过程密切相关。而且S100A4蛋白几乎表达于所有肿瘤细胞,在人体中,迄今只发现S100A4蛋白在单核细胞、巨噬细胞、多核粒细胞、角化细胞、郎罕细胞及汗腺细胞中有不同程度的表达；在正常肺、肾脏,乳腺、甲状腺、胰腺及结肠的组织细胞中没有表达,因此是S100A4蛋白肿瘤基因治疗的理想靶点。国内外的诸多研究皆已证实对于包括乳腺癌在内的多种恶性肿瘤,S100A4蛋白是一种可靠的预测预后和淋巴结转移的因子。在基因治疗方面,Shi Y等[1]运用RNA干扰技术沉默甲状腺未分化癌细胞系(ARO)中的S100A4的表达,发现癌细胞增殖降低了46%±7.6%,并且明显抑制癌细胞转移,而提高了癌细胞对多西紫杉醇的敏感性。Saleem M等[2]对高转移性前列腺癌细胞系(PC-3)进行针对S100A4的RNA干扰研究,也发现沉默S100A4后,可以明显抑制癌细胞增殖、侵袭和转移。除了上述研究以外,近几年来对于其他肿瘤例如：胰腺癌、前列腺癌、食道癌等的相关研究也得出了相似的结论,即：运用RNA干扰技术沉默癌细胞中S100A4基因的表达,可以抑制癌细胞增殖以及转移。
在国内外研究成果的基础上,本课题运用RNA干扰技术,以S100A4为目的基因体外合成S100A4-shRNA (short hairpin RNA)模板链,并连接载体pGenesil-1.1与S100A4-shRNA构建重组质粒,应用脂质体Lipofectamine 2000介导进行细胞转染,通过S100A4-shRNA沉默S100A4基因表达后,观察MCF-7细胞在增殖、凋亡,以及侵袭力和迁移力方面的变化。本课题可以为乳腺癌的基因治疗提供新的着眼点,具有一定的科研意义和临床实用价值。
[统计方法]
所有数据采用SPSS 13.0统计软件进行统计学分析,数据均以X±S表示,多组间比较采用单因素方差分析(one-way ANOVA):首先进行方差齐性检验,若各组间反应变量为方差齐性,则组间比较采用LSD法；若方差不齐采用近似方差分析Welch法和Brown-Forsythe法进行校正,若方差仍为不齐,则组间比较采用Dunnett's T3法。MTT实验另采用重复测量资料方差分析明确分组与时间点之间是否存在交互作用。P<0.05,为差异有统计学意义。
[研究方法和结果]
第一章靶向S100A4的重组质粒的构建
[目的]合成两条重组质粒做为S100A4-shRNA的表达载体转染入MCF-7细胞,使S100A4-shRNA沉默S100A4基因的表达。
[方法]分别设计并合成两条针对S100A4的shRNA序列,获得含有针对S100A4的RNA干扰序列片段,并将其插入到含有绿色荧光蛋白(EGFP)和稳定筛选标记的质粒载体pGenesil-1.1中,构建重组质粒pGenesil-1.1-S100A4-shR-NA-1、pGenesil-1.1-S100A4-shRNA-2,经酶切及测序鉴定正确后大量扩增。
[结果]使用质粒提取试剂盒提取重组质粒,经SacⅠ酶切后,行1%琼脂糖电泳鉴定。在2个插入的目的基因片段里分别设计了一个SacⅠ酶切位点,而质粒pGenesil-1.1中本身也含有一个SacⅠ酶切位点,因此如果连接正确,重组质粒就能被SacⅠ酶切出1条约916 bp的DNA小条带。酶切鉴定证明2条重组质粒均符合设计要求,再经上海英骏生物技术有限公司测序,证明序列完全正确。
第二章RNA干扰抑制MCF-7细胞S100A4基因表达的体内实验研究
2.1免疫组化实验检测MCF-7细胞中S100A4蛋白的表达
[目的]检测MCF-7细胞中S100A4蛋白的表达情况。
[方法]本实验分为两组,即：空白对照组和实验组。将苔盘兰染色活力>95%的MCF-7细胞以1x106/孔种于6孔板中的无菌盖玻片,使用免疫组化Envision法,检测S100A4蛋白在MCF-7细胞中的表达。
[结果]人乳腺癌MCF-7细胞中S100A4蛋白存在明显表达。
2.2 MCF-7细胞转染实验
[目的]检测经脂质体介导重组质粒转染入MCF-7细胞情况。
[方法]转染24后,荧光显微镜下检测经脂质体介导重组质粒转染入MCF-7细胞情况。
[结果]经脂质体介导重组质粒成功转染入MCF-7细胞。
2.3 QRT-PCR法检测转染后S100A4基因表达的变化
[目的]检测转染48h后,MCF-7细胞中S100A4基因的表达情况,并筛选出沉默S100A4表达效率更高的一条重组质粒。
[方法]本实验分为四组：空白对照组、阴性对照组、S100A4-shRNA-1组；S100A4-shRNA-2组,后三组分别转染阴性对照质粒、重组质粒pGenesil-1.1-S100A4-shRNA-1、重组质粒pGenesil-1.1-S100A4-shRNA-2,通过QRT-PCR法,以GAPDH为内参基因,对于转染48h后S100A4基因在各组中的表达进行检测。
[结果]QRT-PCR检测结果显示,转染48h后,三组间S100A4mRNA表达量差异具有统计学意义(F= 67.854, P=0.000), S100A4-shRNA-1组和S100A4-shR-NA-2组的S100A4mRNA表达量低于阴性对照组和空白对照组,差异均有统计学意义(P=0.000,P=0.000；P=0.000,P=0.000)；而S100A4-shRNA-1组中S100A4-mRNA表达量低于S100A4-shRNA-2组,差异具有显著性(P=0.017)。因此选用重组质粒pGenesil-1.1-S100A4-shRNA-1进行后续实验。
2.4 Westernblot法检测转染后S100A4蛋白表达的变化
[目的]检测转染48h后,MCF-7细胞中S100A4蛋白的表达情况。
[方法]实验分为3组,即实验组、阴性对照组、空白对照组。前两组分别转染重组质粒pGenesil-1.1-S100A4-shRNA-1以及阴性对照质粒。本实验通过West-ern blot法对于转染48h后,S100A4蛋白在各组中的表达进行检测。
[结果]Western blot法检测结果显示,与空白对照组和阴性对照组相比,实验组组细胞S100A4蛋白表达明显减弱,Tanon系统分析显示其蛋白条带灰度值较前两者分别降低57.21%和54.82%。
2.5 MTT法检测转染后细胞增殖的变化
[目的]检测转染后MCF-7细胞增殖的变化情况。
[方法]实验分组同上,将实验组、阴性对照组和空白对照组细胞以1x104个/孔密度种于96孔板,分别于转染24h、48h和72 h后每孔加入MTT(5mg/ml)20μl,置于CO2培养箱中培育4h,终止反应后弃去上清液。每孔加入DMSO 200μl,振荡10 min,在酶标仪上于490 nm波长下读取吸光度(OD)值,绘制各组细胞生长曲线,各组实验重复3次。
[结果]MTT法检测MCF-7细胞增殖水平显示,三组间在24h、48h、72h各时间点OD值差异均具有统计学意义(F=13.401,P=0.006；F=132.689,P=0.000；F=3150.772,P=0.000),分组与时间点之间存在交互作用(F=68.466,P=0.000)。实验组细胞在24h、48h、72h各时间点OD值均低于阴性对照组和空白对照组,差异有统计学意义(P=0.006,P=0.003；P=0.015,P=0.015；P=0.000,P=0.000),阴性对照组和空白对照组各时间点OD值差异均无统计学意义(P=0.571,P=0.287,P=0.211)。
2.6 Annexin V-FITC/PI双染色法检测转染后细胞凋亡率的变化
[目的]检测转染24h后,MCF-7细胞凋亡的变化情况。
[方法]实验分组同上,以1x106个/孔接种各组细胞于6孔板。每组设3个复孔。转染后24h,收集上清液和细胞,调整细胞密度为3x105个/mL,加入2μl Annexi-nV-FITC和5μlPI,应用流式细胞仪测定各组细胞凋亡情况。每组实验重复3次。结果判定：正常细胞为AnnexinV-FITC(-)/PI(-),早期凋亡细胞为AnnexinV-FI-TC(+)-/PI(-),晚期凋亡细胞为AnnexinV-FITC(+)/PI(+),坏死细胞为Annexin-V-FITC(-)/PI(+)。
[结果]转染24h后,三组间晚期凋亡细胞率差异具有统计学意义(F=5.895,P=0.038),实验组晚期凋亡细胞率较阴性对照组、空白对照组升高,差异具有显著性(P=0.036,P=0.019),而后两组差异无统计学意义(P=0.636)。
2.7稳定转染细胞株的建立及其形态观察
[目的]建立稳定转染细胞株观察其形态学的变化。
[方法]首先确定G418筛选稳定转染株细胞的浓度,将苔盘蓝计数细胞活力>95%的MCF-7细胞以1x106/孔种于6孔细胞培养板。细胞贴壁后分别换成含有0、100、200、300、400、500、600、700、800、900、1000ug/ml G418的10%FBS的RPMI1640培养液培养,每隔3天换液,对比观察正常细胞和G418筛选组细胞的生长情况,取一周70%死亡,二周100%死亡的最低G418浓度为筛选稳定转染株细胞的浓度(该实验确定G418筛选浓度为500ug/ml)。实验分为3组即：实验对照组、阴性对照组、空白对照组,前两组分别转染实验用重组质粒以及阴性对照质粒,将转染24h后的MCF-7细胞按照1：10传代。并设置正常细胞对照孔2孔。细胞贴壁后换含有500ug/ml G418的10%FBS的RPMI1640培养液,每隔3天换液,2周后观察正常细胞死亡率100%,转染细胞30%存活。换含有350ug/mlG418的10%FBS的RPMI1640培养液,1周后出现单个细胞。荧光显微镜下观察将培养板盖上发荧光,光学显微镜下为活细胞的MCF-7细胞标记。消化收集调整细胞浓度<=1个/100ul,100ul/孔种96孔板,扩大培养,2周后成细胞集落,5周后长至50%。待细胞长满后传至24孔板,逐渐扩大培养至6孔板和25cm2培养瓶。分别命名实验组稳定转染株细胞为MCF-7-S100A4细胞,阴性对照组稳定转染株细胞为MCF-7-HK细胞。使用免疫组化Envision法观察MCF-7-S100A4细胞、MCF-7-HK细胞以及MCF-7细胞在形态、大小等生物学特性中的区别。
[结果]筛选出稳定转染株细胞后,通过免疫组化检测发现,MCF-7细胞和MCF-7-HK细胞同样呈不规则形状、大小不一、核大而深染、呈圆形或椭圆形,细胞极性消失、排列紊乱；MCF-7-S100A4细胞在大小、形态以及生长方式上与以上细胞无明显差别。
2.8划痕实验检测稳定转染株细胞的迁移力
[目的]检测稳定转染株细胞迁移力的变化。
[方法]运用划痕实验检测稳定转染株细胞迁移力的变化,实验分为3组,即：实验组(MCF-7-S100A4细胞组)、阴性对照组(MCF-7-HK细胞组)以及空白对照组,取处于对数生长期的各组细胞以1x106个/孔接种于6孔板中培养。在单层细胞表面用100μl移液器枪头垂直划痕,PBS液轻洗2次,在倒置显微镜下观察0、6、24和48 h后划痕中细胞迁移情况,并拍照。
[结果]体外划痕实验结果显示,实验组中MCF-7-S100A4细胞的移动速度明显慢于,空白对照组中MCF-7细胞以及阴性对照组中MCF-7-HK细胞；接种后48h,MCF-7细胞以及MCF-7-HK细胞划痕已经基本长满,而MCF-7-S100A4细胞划痕尚未长满。
2.9 Transwell小室法检测稳定转染株细胞侵袭力
[目的]检测稳定转染株细胞侵袭力的变化。
[方法]首先实验分为3组,即：实验组(MCF-7-S100A4细胞组)、阴性对照组(MCF-7-HK细胞组)以及空白对照组,通过Transwell小室法对于稳定转染株细胞侵袭力的变化进行检测。
[结果]侵袭实验结果显示,三组间差异具有统计学意义(F=826.583,P=0.000),其中实验组穿膜细胞数低于阴性对照组和空白对照组,差异有统计学意义(P=0.000,P=0.000),而阴性对照组与空白对照组相比差异无统计学意义(P=0.128)。
第三章RNA干扰抑制MCF-7细胞S100A4基因表达的体内实验研究
3.1稳定转染株细胞在裸鼠体内的成瘤实验
[目的]通过将稳定转染株细胞细胞接种于裸鼠背部,检测裸鼠背部移植瘤成瘤情况。
[方法]本实验分组分为3组,即：阴性对照组(MCF-7-HK细胞组)、实验组(MCF-7-S100A4细胞组)、空白对照组,每组裸鼠10只。取上述各组细胞分别消化制成单细胞悬液,调整细胞密度为1×107个/mL,每只裸鼠颈背部皮下注射0.4ml。种植肿瘤细胞后,每天观察裸鼠饮食、精神及活动状况,每隔2d测定肿瘤大小,并测量肿瘤最长径(a)及其垂直方向最大横径(b),按公式V(mm3)=axb2/2计算肿瘤体积,并绘制移植瘤的生长曲线图并计算抑瘤率。观察35 d后,脱颈处死裸鼠,测量各组裸鼠移植瘤的质量,对结果进行统计分析。
[结果]体内实验部分中细胞接种裸鼠35d后,空白对照组及阴性对照组小鼠的平均肿瘤体积超过900mm3,成瘤率为100%；实验组小鼠于观察2-3周后,仅可在皮下触及极小体积肿瘤(约12mm3),且随着观察天数增加肿瘤不再生长,反而有缩小迹象(<12mm3),成瘤率为90%。肿瘤细胞接种35d后,处死动物,分离皮下瘤称重。实验结果显示,三组间肿瘤体积和质量差异具有统计学意义,(F=102201.907,P=0.000；F=8859.118,P=0.000)。实验组裸鼠的肿瘤质量和体积低于阴性对照组和空白对照组,差异具有显著性(P=0.000,P=0.000；P=0.000,P=0.000),而2个对照组裸鼠肿瘤之间差异无统计学意义(P=0.991,P=0.062)。使用公式计算抑瘤率(%)=[(空白对照组平均瘤重-治疗组平均瘤重)/空白对照组平均瘤重]×100%,可得抑瘤率为98.64%。
3.2 QRT-PCR法检测裸鼠背部移植瘤中S100A4基因的表达
[目的]检测各组裸鼠背部移植瘤中S100A4基因的表达。
[方法]本实验分组同上,每组随意选取4只成瘤裸鼠,取背部移植瘤组织进行实验,另取裸鼠背部正常组织作为对照。通过QRT-PCR的方法,采用Sybrgreen染料法,以GAPDH为内参基因,对于S100A4基因在各组裸鼠背部移植瘤组织中的表达量进行检测。
[结果]QRT-PCR检测结果显示,三组间S100A4mRNA表达量差异具有统计学意义(F=83.598,P=0.000),实验组中S100A4mRNA表达量低于阴性对照组和空白对照组,差异均有统计学意义(P=0.000,P=0.000),而后两组的S100A4mRNA表达量差异无统计学意义(P=0.915).
3.3 Westernblot法检测裸鼠背部移植瘤中S100A4蛋白的表达
[目的]检测各组裸鼠背部移植瘤中S100A4蛋白的表达。
[方法]实验分组同上,每组随意选取4只成瘤裸鼠,取背部移植瘤组织进行实验,另取裸鼠背部正常组织作为对照。通过Western blot法检测各组裸鼠背部移植瘤中S100A4蛋白的表达。
[结果]Tanon系统分析显示三组间S100A4蛋白表达量差异具有统计学意义(F=90.297,P=0.000),其中空白对照组和阴性对照组中的S100A4蛋白表达量高于实验组组差异均有统计学意义(P=0.000；P=0.000),而前两者差异无明显统计学意义(P=0.643).
[研究结论]
本研究以乳腺癌MCF-7细胞作为研究模型,以构建携带针对S100A4的特异干涉序列的S100A4-shRNA作为进行RNA干涉的工具,沉默S100A4基因的表达。根据实验结果,本研究阐明了以下几点：
1)通过S100A4-shRNA沉默S100A4,能有效抑制MCF-7细胞中S100A4的表达。
2)通过S100A4-shRNA沉默S100A4,能有效抑制MCF-7细胞增殖并促进凋亡的发生。
3)通过S100A4-shRNA沉默S100A4,能有效抑制MCF-7细胞迁移和侵袭力。
4)通过S100A4-shRNA沉默S100A4,能有效抑制MCF-7细胞在裸鼠体内成瘤的能力。
鉴于以上结果,得出以下结论,我们构建的携带S100A4特异干涉序列的S100A4-shRNA能有效沉默S100A4基因,抑制MCF-7细胞增殖以及侵袭和迁移能力,并促进其凋亡。这证明了S100A4蛋白参与细胞增殖分化、细胞运动等多种生命活动,并与乳腺癌MCF-7细胞的增殖、侵袭以及转移等病理过程密切相关。
[Objective and significance]
Breast cancer is the most commonly seen malignant carcinoma of female breasts, and also one of the malignant carcinomas with the highest occurrence among females. Though our country is a region with lower occurrence rate of breast cancer, the incidence rate is increasing in recent years. It not only ranks at the first among female malignant carcinomas, and with an earlier peak of morbidity. Though the five year survival is improved because of comprehensive therapy targeting to breast cancer, distant metastasis is still the important prognostic factor for breast cancer. The genetic therapy against breast cancer is a new therapeutic measure following surgical approach, radiotherapy, chemotherapy and endocrine therapy, and it is a biological therapeutic method with good prospective. At present, some research achievements have been applied to clinical practice, with significant therapeutic effects obtained. For example:in Oct.,1998, FDA had formally approved to administer Trastuzumab (trade name, Herceptin, Genentech/Roche), the monoclonal antibody derived from high purity recombinant DNA and targeting Her-2 receptor, to treat metastatic breast cancer with over expression of Her-2/neu, which brought the first morning light to therapy against breast cancer. Though primary success has been obtained in genetic therapy against breast cancer, there are still many problems, and one of them is that, there are only few genes which can be applied into clinical therapy. Therefore, it is especially important to find a new target gene. S100A4 protein is a member of S100 protein family, and its coding genes are localized at chromosome 1q21 with other 14 members, and this region is the unstable region correlated with the occurrence of many malignant carcinomas. Therefore, coding gene of S100A4 protein has its special expression styles. It is indicated in the current study that, S100A4 protein participates in the signal transduction in and out of cells, cell cycle regulation, and it is involved in many life activities including cell proliferation and differentiation, cell adhesion, cell migration, etc., and it is deeply related with the pathological process of many malignant carcinomas including breast cancer, including genesis, infiltration and metastasis, etc. Moreover, S100A4 protein is nearly expressed in all carcinoma cells. In human body, S100A4 expression of varied degrees is only observed in monocyte, macrophage, polymorphonuclear granulocyte, keratinocyte, Langhans'cell and sweat gland cell till now. In normal cells of lung, kidney, breast cancer, thyroid, pancreas and colon, S100A4 is not expressed, so S100A4 protein is the ideal target for genetic therapy against carcinomas. Many studies in China and other countries have confirmed that, for many malignant carcinomas including breast cancer, S100A4 protein is a stable factor to predicting prognosis and lymphatic metastasis. In the aspect of genetic therapy, Shi Y et al applied RNA interference to silence the S100A4 expression in undifferentiated thyroid carcinoma cell line (ARO), and found that the proliferation of carcinoma cell was decreased by 46%+7.6%, migration of carcinoma cells was also inhibited obviously, and sensitivity of carcinoma cells to taxotere was improved. Saleem M et al performed RNA interference study targe-ting S100A4 in highly metastatic prostate carcinoma cell line (PC-3), and found that after S100A4 was silenced, proliferation, infiltration and metastasis of carcinoma cells were significantly inhibited. Besides of the abovementioned studies, similar conclusion has been obtained in related studies on other carcinomas, including pancr-eatic carcinoma, prostate carcinoma and esophageal cancer, etc., i.e., RNA interfere-nce is applied to silence expression of S100A4 gene, and proliferation and metastasis of carcinoma cells can be inhibited.
Based on the study achievements in China and other countries, RNA interference was adopted in this study to synthesize S100A4-shRNA template strand with S100A4 as the target gene, and construct recombinant plasmid by linking two carriers of pGenesil-1.1 and S100A4shRNA. Liposome Lipofectamine 2000 was applied to me-diate cell transfect, and S100A4-shRNA was used to silence S100A4 gene expression, to observe the proliferation and apoptosis of MCF-7 cell, and changes of infiltrating and migrating capacity. Our study can provide a new approach for genetic therapy against breast cancer, and it has certain scientific significance and clinical practice.
[Statistical methods]
Statistical analysis was performed by SPSS13.0 software.All data were presente-d as mean±standard deviation.one-way ANOVA was used to determine the statistical significance,first of all,doing the test of Homogeneity of Variances among the group-s. If the equal variances assumed, LSD was used to determine the statistical significa-nce. If the equal variances not assumed, Dunnett's T3 was used to determine the stati-stical significance.Repeated measures ANOVA was used to test the cross correlation between factor and groups in MTT.P<0.05 was considered statistically significant.
[Methods and Results]
Chapter 1 Construction of recombinant plasmid for target S100A4
[Objective] To synthesize two recombinant plasmids as the expression vector of S100A4-shRNA to transfect into MCF-7 cells, and silence the expression of S100A4 gene.
[Methods] Two shRNA sequences targeting S100A4 were designed and synthesized to obtain RNA interference sequence segment for S100A4, which was inserted into the stably screened and labeled plasmid carrier pGenesil-1.1 containing green fluorescent protein (EGFP), then recombinant plasmids pGenesil-1.1-S100A4-shRNA-1 and pGenesil-1.1-S100A4-shR.NA-2 were constructed, and amplified in great amount after enzyme digested and sequenced.
[Results] After the recombinant plasmid extracted by using extraction kit was excised by Sac I enzyme, it was identified by using 1% agarose electrophoresis. A Sac I enzyme digestion site was designed in two target gene segments, and there was also a Sac I enzyme digestion site in plasmid pGenesil-1.1. Therefore, if the linkage was correct, a 916 bp DNA strap was excised from recombinant plasmid by Sac I enzyme. It was confirmed by enzyme excision that,2 recombinant plasmids conform-ed to design requirements, and the sequencing results obtained by Shanghai Invitroge-n Biological Technology Co., Ltd. confirmed that the sequence was correct.
Chapter 2 In vivo experimental study on S100A4 gene expression in MCF-7 cell inhibited by RNA interference
2.1 Expression of S100A4 protein in MCF-7 cell detected by immunohis-tochemical method
[Objective] To detect the expression of S100A4 protein in MCF-7 cell.
[Methods] The experiment had two groups including blank control group and test group. Inoculate MCF-7 cells with trypan blue staining activity>95% in sterile cover glass of 6-well plate at 1×106/well, Immunohistochemical SP method was applied to detect the expression of S100A4 protein in MCF-7 cell.
[Results] Obvious expression of S100A4 protein was observed in human breast cancer MCF-7 cell.
2.2 MCF-7 cell transfect test
[Objective] To detect the transfect of liposome mediated recombinant plasmid into MCF-7 cell.
[Methods] After transfect for 24h, the transfect of liposome mediated recombine-nt plasmid into MCF-7 cell was detected by using fluorescence microscope.
[Results] Liposome mediated recombinant plasmid was successfully transfected into MCF-7 cells.
2.3 Changes of S100A4 gene expression after transfect by QRT-PCR method
[Objective] To detect S100A4 gene expression in MCF-7 cell 48h after transfect, and screen a recombinant plasmid with a higher S100A4 expression rate.
[Methods] The experiment had four groups:blank control group, negative control group, S100A4-shRNA-1 group and S100A4-shRNA-2 group. The latter three groups were respectively transfected by negative control plasmid, recombinant plasmid pGenesil-1.1-S100A4-shRNA-1, recombinant plasmid pGenesil-1.1-S100A4-shRNA-2, and the expression of S100A4 gene in different groups were detected by QRT-PCR method 48h after transfect, with GAPDH as the internal reference gene.
[Results] Results of QRT-PCR indicated that,48h after transfect, there are stastic-al significance among the three groups (F= 67.854, P=0.000).The expression amount of S100A4 mRNA in S100A4-shRNA-1 group and S100A4-shRNA-2 group was lower than those in negative control and blank control groups(P=0.000, P=0.000; P=0.000, P=0.000); expression amount of S100A4mRNA in S100A4-shRNA-1 group was lower than that in S100A4-shRNA-2 group (P=0.017). Thus, recombinant plasmid pGenesil-1.1-S100A4-shRNA-1 was used to perform the subsequent experi-ment.
2.4 Changes of S100A4 protein expression detected by Westernblot method
[Objective] To detect the expression of S100A4 protein in MCF-7 cells 48h after transfect.
[Methods] The experiment had three groups, including test group, negative control group and blank control group. The first two groups were transfected by recombinant plasmids of pGenesil-1.1-S100A4-shR.NA-1 and negative control plasm-id. Western blot method was applied to detect the expression of S100A4 proteins in different groups 48h after transfect.
[Results] Results of Western blot indicated that, compared to blank control group and negative control group, expression of S100A4 protein was obviously weakened, and Tanon systemic analysis indicated that, its protein strap gray scale was decreased by 57.21% and 54.82% compared to those of the first two groups.
2.5 Changes of cell proliferation after transfect by using MTT method
[Objective] To detect the changes of MCF-7 cell proliferation after transfect.
[Methods] The experimental materials were grouped as abovementioned, and cells of test group, negative control group and blank control group were inoculated into a 96-well plate at 1×104 cells/well, MTT (5 mg/ml) 20μl was added into each well after the cells were transfected for 24,48 and 72 h. Then the plate was placed into a CO2 incubator for 4h, and the supernatant was discarded after the reaction was stopped. DMSO 200μl was added into each well, then the plate was shaken for 10min, and optical density (OD) value was read at 490 nm wavelength to draw the growth curves. The procedure was repeated for three times.
[Results] Experimental results indicated that, there are stastical significance among the three groups at 24,48 and 72h(F=13.401, P=0.006; F=132.689, P= 0.000; F=3150.772, P=0.000).OD value of test group was lower than those of negative control and blank control group at 24,48 and 72h (P=0.006, P=0.003; P=0.015, P=0.015; P=0.000, P=0.000), while difference of OD value was insignificant betwe-en negative control and blank control groups at all time points (P=0.571; P=0.287; P= 0.211).
2.6 Annexin V-FITC/PI double staining method was used to detect the changes of apoptosis rate after transfect.
[Objective] To detect the changes of MCF-7 cell apoptosis 24 h after transfect.
[Methods] The experimental materials were grouped as abovementioned, and cells were inoculated into a 6-well plate at 1×106 cells/plate. Triplicate wells were set for each group. Supernatant and cells were collected 24h after transfect. The cell density was adjusted to 3×105/mL, and then 2μl Annexin V-FITC and 5μl PI were added to determine the apoptosis of cells in different groups by using flow cytometry. The procedure was repeated for 3 times. Result judgment:normal cell was Annexin V-FITC (-)/PI (-), viable apoptotic cell was Annexin V-FITC (+)-/PI (-), non-viable apoptotic cell was Annexin V-FITC (+)/PI (+), and necrotic cell was Annexin V-FITC (-)/PI (+).
[Results] Experimental results indicated that,24h after transfect, there are stastic-al significance among the three groups in the apoptosis rate of non-viable apoptotic cell (F= 5.895, P=0.038).the apoptosis rate of non-viable apoptotic cell in test group was higher than those of negative control and blank control groups (P=0.036, P=0.019), but no statistical difference was observed between the latter two groups (P=0.636).
2.7 Construction and morphological observation of stable transfected cell strain
[Objective] To construct stable transfected cell strain and observe their changes.
[Methods] Firstly, use G418 to screen the concentration for stable transfect cell, and inoculate MCF-7 cells with cell activity>95% counted by trypan blue into a 6-well plate at 1×106 cells/well. After cells adhered to well wall, RPMI1640 solution containing 0,100,200,300,400,500,600,700,800,900 and 1000ug/ml G41810% FBS were used to culture the cells. The solution was replaced for every 3 days, growth of cells was observed between normal cells and G418 screened cells. G418 concentration with 70% death in one week and 100% death in the second week was selected to screen the concentration of stable transfect cell (in this experiment, G418 screening concentration was 500 ug/ml). The experiment had three groups including test control group, negative control group and blank control group. The first two groups were transfected with recombinant plasmid and negative control plasmid, and then MCF-7 cell was 1:10 passaged 24 h after transfect. Two control wells of normal cells were also set. After cells adhered to well wall,10% FBS RPMI1640 solution containing 500ug/ml G418 was adopted, and the solution was changed for every 3 days. After 2 weeks, normal cells had a death rate of 100%, and 30% transfected cells were survived. Then 10% FBS RPMI1640 solution containing 350ug/ml G418 was changed, and single cells appeared 1 week later. Under the fluorescent microscope, living MCF-7 cells with fluorescence were labeled. The cells were digested and collected to adjust the solution concentration<=1 cell/100ul, and cells were inoculated into a 96-well plate at 100ul/well. Then expand the culture, the cells formed cell cluster 2 weeks later. After 5 weeks,50% wells were covered. When cells covered 24 wells, transfer the cells gradually to a 6-well plate and a 25cm2 culture flask. Respectively name the stable transfect cell as MCF-7-S100A4 cell in test group, MCF-7-HK cell in negative control group. Immunohistochemical SP method was used to observe the differences of morphologies and size, etc. between MCF-7-S100-A4 cells, MCF-7-HK cells and MCF-7 cells.
[Results]After stable transfect cells were screened, it was found by immunohisto-chemical method that, MCF-7 cell and MCF-7-HK cell also had irregular morpholo-gies, varied sizes, big nuclei deeply staining, in round of oval shapes. The cell had no cell polarity, with confused arrangement. There was no significant difference between MCF-7-S100A4 cells and the above cells in cell size, morphology and living styles.
2.8 Migrating capacity of stable transfect cells by using scratch adhesion test
[Objective] To detect the changes of migrating capacity of stable transfect cells.
[Methods] Scratch adhesion test was used to detect the changes of migrating capacity of stable transfect cells. The experiment had three groups including test group (MCF-7-S100A4 cell group), negative control group (MCF-7-HK cell group) and blank control group. Cells at logarithmic phase were collected and inoculated into a 6-well plate at 1×106 cells/well. On single cell layer surface, scratch with 100μl transferpettor probe vertically, and flush with PBS for 2 times, then observe the migrating cells under a microscope for scratches made at 0,6,24 and 48 h, then take the images.
[Results] Results of scratch adhesion test indicated that, the migrating rate of MCF-7-S100A4 in test group was obviously smaller than those of MCF-7 cell in blank control group and MCF-7-HK cell in negative control group. After the cells were inoculated for 48 h, MCF-7 cells and MCF-7-HK cells covered the scratches, and MCF-7-S100A4 cells had not spread over the scratch.
2.9 Transwell chamber method was applied to determine the infiltrating capacity of stable transfect cells.
[Objective] To determine the changes of infiltrating capacity of stable transfect cells.
[Methods] The materials of the experiment were grouped as abovementioned, and Transwell chamber method was applied to determine the changes of infiltrating capacity of stable transfect cells.
[Results] Results of in vitro infiltrating experiment indicated that, there are stasti-cal significance among the three groups (F= 826.583, P=0.000), and the stastical dif-ference was observed between test group and the other two groups (P=0.000, P= 0.000), while no statistical difference was observed between negative and blank cont-rol groups (P=0.128). The results of the above experiment indicated that, after S100-A4 was silenced, the infiltrating and migrating capacities of MCF-7 cells were signif-icantly decreased.
Chapter 3 In vivo study on expression of S100A4 gene in MCF-7 cell inhibited by RNA interference
3.1 Tumorigenicity experiment on stable transfect cell in naked mice
[Objective] To determine the tumorigenicity of transplanted tumor by inoculating stable transfect cell into the back of naked mice.
[Methods] The experiment had three groups including negative control group (MCF-7-HK cell group),test group (MCF-7-S100A4 cell group) and blank control group, with 10 mice in each group. Take cells from the above groups and prepare single cell suspension, adjust the cell density to 1×107/mL, and 0.4 ml solution was injected subcutaneously into the back of naked mice. After the carcinoma cells were implanted, the diet, mental activities and movements were observed, tumor size was determined for every 2 days, and the maximal diameter (a) and maximal vertical transverse diameter of the tumor were determined. The tumor volume was calculated following V (mm3)=axb2/2, and growth curve was drawn to calculate the tumor inhibition rate. After observed for 35 d, the naked mice were decapitated, then the transplanted tumor was weighed, and the results were analyzed.
[Results]In experiment in vivo, after the naked mice were inoculated for 35 d, the average tumor volume was more than 900 mm3 in mice of blank and negative control groups, with a tumor formation rate of 100%. After observed for 2-3 weeks on mice of test group, tiny tumor (about 12mm3) could only observed touched subcutaneously, and the tumors would not growth as times went by, or they even shrunk (<12 mm3), and the tumor formation rate was 90%. After tumor cell was inoculated for 35d, the animals were executed, and subcutaneous tumors were isolated and weighed. Experimental results indicated that, there are stastical significance among the three groups in tumor mass and volume of naked mice (F= 102201.907, P=0.000; F= 8859.118, P=0.000).tumor mass and volume of naked mice in test group were significantly lower than those of negative and blank control groups (P=0.000, P=0.000; P= 0.000, P=0.000). while no significant difference was observed between two control groups(P=0.991, P=0.062). The following formula was used to calculate tumor inhibition rate (%)= [(average tumor weight in blank control group-average tumor weight in test group)/average tumor weight in blank control group]×100%, and the inhibition rate was 98.64%.
3.2 Expression of S100A4 gene in transplanted carcinoma determined by QRT-PCR method
[Objective] To determine the expression of S100A4 gene in transplanted carcinoma.
[Methods] Animals in this experiment were groups as abovementioned, with 4 naked mice with carcinomas randomized in each group. The transplanted carcinoma tissues were collected to perform the experiment, and normal tissues were also taken as controls. By using QRT-PCR and Sybrgreen staining method, GAPDH was adopted as the internal reference gene, and expression amount of S100A4 gene was determined in tissues of transplanted carcinoma on back of naked mice.
[Results] Results of QRT-PCR indicated that, there are stastical significance among the three groups (F=83.598, P=0.000) expression amount of S100A4 mRNA in test group was far lower than those of negative and blank control groups (P=0.000, P=0.000), while no significant difference was observed between the two control groups (P=0.915)。
3.3 Expression of S100A4 protein in transplanted carcinoma on back of naked mice determined by Westernblot
[Objective] To determine the expression of S100A4 protein in transplanted carcinoma on back of naked mice.
[Methods] Animals in this experiment were groups as abovementioned, with 4 naked mice with carcinomas randomized in each group. The transplanted carcinoma tissues were collected to perform the experiment, and normal tissues were also taken as controls. And Western blotting method was adopted to determine the expression of S100A4 protein in transplanted carcinoma on back of naked mice.
[Results] Results of Western blot indicated that, there are stastical significance among the three groups (F= 90.297, P=0.000).expression of S100A4 protein was significantly higher in blank and negative control groups compared to test group (P=0.000, P= 0.000), while no significant difference was observed between control groups(P=0.643).
[Conclusion]
In this study, breast cancer MCF-7 cell was adopted as the study model to construct S100A4-shRNA with specific interfering sequence S100A4 as the tool to perform RNA interference, and the expression of S100A4 gene was silenced. Accor-ding to the results, the study explained the following points:
1) Silencing S100A by using S100A4-shRNA can effectively inhibit expression S100A4 in MCF-7 cells.
2) Silencing S100A by using S100A4-shRNA can effectively inhibit MCF-7 proliferation and promote apoptosis.
3) Silencing S100A by using S100A4-shRNA can effectively inhibit cell migration and infiltration of MCF-7 cells.
4) Silencing S100A by using S100A4-shRNA can effectively inhibit the Tumorigenicity of MCF-7 cells in naked mice.
The following results are obtained according to the above results:the construc-ted S100A4-shRNA carrying S100A4 special interference sequence can effectively silence S100A4 gene, inhibit proliferation, infiltration and migration of MCF-7 cells, and promote their apoptosis. These results indicate that S100A4 protein participates in cell proliferation, differentiation and cell migration, and it is closely correlated with proliferation, infiltration and migration of breast cancer MCF-7 cells.
引文
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