驱动蛋白KIF4对胃癌细胞BGC增殖和卵巢癌细胞SK-OV-3迁移的影响
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摘要
研究目的：
研究驱动蛋白KIF4在胃癌组织的表达情况及对胃癌细胞BGC增殖的影响。同时研究KIF4对卵巢癌细胞SK-OV-3迁移的影响。为阐明KIF4在肿瘤发生发展中的作用奠定基础,同时也为探索新的肿瘤生物治疗方法提供帮助。
研究方法：
一、驱动蛋白KIF4在胃癌组织中的表达情况
1、免疫组织化学染色实验比较KIF4在胃癌组织和相应癌旁组织中的表达情况
本研究中23例原发性胃癌病人于2006-2008年间在山东大学齐鲁医院接受外科手术,手术之后,肿瘤样本和它相应的癌旁组织用石蜡包埋,室温储存。石蜡包埋的标本组织被切成4-5μm,放置至载玻片上。羊血清封闭后,抗KIF4的多克隆抗体以1：500稀释,加至载玻片上。免疫染色用晶美生物技术公司的Histostain-Plus试剂盒,过程参照说明书。阴性对照用PBS溶液代替一抗。每张标本用奥林帕斯IX81倒置显微镜随机拍取5张照片。照片的累积光密度用Image-Pro Plus6.0软件进行分析。
2、统计分析KIF4的表达量与临床资料的相关性
用SPSS11.0进行统计学数据分析。每一个实验的结果用平均值±标准差来描述。用双侧t检验来比较实验组和对照组。用列联表来分析KIF4的表达与胃癌病人的病理学变量的关系。
二、驱动蛋白KIF4对胃癌细胞BGC增殖的影响
1、筛选稳定表达GFP-KIF4的BGC细胞株(BGC-GFP-KIF4)及稳定表达GFP的BGC细胞株(BGC-GFP)
(1)真核表达载体pEGFP-KIF4的构建
PCR扩增产生的KIF4编码区域通过BglⅡ-SalⅠ双酶切位点克隆至真核表达载体pEGFP-C1。
(2)转染pEGFP-KIF4质粒或pEGFP质粒至BGC细胞
取生长良好的BGC细胞,按3×104／孔接种到24孔板,培养过夜。用脂质体2000将0.5μg pEGFP-KIF4或pEGFP质粒转染入细胞。2天后,细胞转移入10cm平皿,在含1mg／mlG418的完全培养基中培养,每隔3天换一次液。2到3周后,在倒置荧光显微镜下挑选出单个阳性克隆,继续在1mg／mlG418的完全培养基中培养。最后,稳定表达GFP-KIF4或GFP的BGC细胞被筛选出来,通过Western blot进行检测。
(3) Western blot检测GFP-KIF4在稳筛细胞株中的表达
离心收集细胞,用裂解液(50mmol/L Tris-HCl,150mmol/L NaCl,0.5% NP-40, 0.5%dexycholate-Na,0.1% SDS, protease inhibitor cocktail)裂解细胞。40μg总蛋白通过12%SDS—聚丙烯酰胺凝胶电泳分离,并转移至PVDF膜。兔抗KIF4的抗体1：1000稀释来检测内源性KIF4和表达的GFP-KIF4融合蛋白。鼠抗Tubulin的单克隆抗体1：1000倍稀释,来显示微管蛋白作为内参。随后加入HRP标记的羊抗兔／鼠IgG的二抗,最后用ECL发光系统显色。
2、免疫荧光染色显示过表达KIF4引起的细胞多核性
生长在盖玻片上的稳筛细胞株用冷甲醇室温固定5min,然后用含5%羊血清、0.3%TritonX-100的PBS封闭。一抗用鼠抗Tubulin的抗体(1：10000稀释),二抗用TexasRed交联的羊抗鼠的抗体(1：10000稀释),分别染色2h。DNA用DAPI(1μg/ml)染色5min。PBS洗后,用FluoroGuard抗淬灭剂将盖玻片固定在干净的载玻片上,指甲油封边。
3、细胞增殖计数及MTT检测过表达KIF4对BGC增殖的影响
将两种稳筛细胞株接种于24孔板中,每孔500μl细胞悬液中含5000个细胞,置培养箱中培养。在接下来的9天内,每隔一天计数细胞数量。根据数据绘制细胞生长曲线。
将两种稳筛细胞株接种于96孔板中,每孔200μl细胞悬液中含5000个细胞,置培养箱中培养。在接下来的9天内,每隔一天采用MTT法检测细胞增殖情况。每孔加入20μl 5mg／mlMTT,继续培养4个小时。然后去除细胞培养基,加入200μlDMSO,20min后,在酶标仪上570nm测量吸光度值。根据数据绘制曲线。
4、克隆形成实验检测过表达KIF4对BGC增殖的影响
在35mm平皿中铺入2ml含0.6%琼脂的培养基,室温下凝固作为下层胶。1×104个细胞加入1ml含0.3%琼脂的培养基中,作为上层胶。培养3周后,用0.05%结晶紫染色细胞30min。拍摄照片并用Image-Pro Plus6.0进行克隆计数。
5、裸鼠体内成瘤检测过表达KIF4对BGC成瘤性的影响
实验用的雌性BABL／c小鼠(6-8周大)从中国科学院上海实验动物中心购进。取4×106个BGC-GFP-KIF4或BGC-GFP细胞和100 gMatrigel接种到无胸腺小鼠(nu／nu)的左腋下。每组5只。肿瘤的大小每周用游标卡尺进行测量。肿瘤的体积通过下列公式计算：体积=(长×宽2)／2。7周后,脱颈法处死小鼠,从小鼠体内取出肿瘤组织测量体积和称重并照相。
6、统计分析
使用SPSS16.0软件进行统计学分析。对正态分布的配对数据进行t检验。P<0.05为差异有统计学意义。
三、驱动蛋白KIF4对卵巢癌SK-OV-3迁移能力的影响
1.筛选稳定低表达KIF4的SK-OV-3细胞株
(1) pGPU6/GFP/Neo-KIF4质粒及pGPU6/GFP/Neo-NC质粒的构建
于文献资料中检索到KIF4的siRNA序列,它是针对KIF4的19nt的RNA片段。序列为5'GCAGATTGAAAGCCTAGAG3'.将此片段补充设计为shDNA,并克隆入pGPU6/GFP/Neo载体,命名为pGPU6/GFP/Neo-KIF4。阴性对照干扰序列为5'GTTCTCCGAACGTGTCACGT 3'。将此片段补充设计为shDNA,并克隆入pGPU6／GFP／Neo载体,命名为pGPU6／GFP／Neo-NC。
(2)转染pGPU6/GFP/Neo-KIF4质粒及pGPU6/GFP/Neo-NC质粒至SK-OV-3细胞
人卵巢癌细胞SK-OV-3用含10%胎牛血清的RPMI-1640,于37℃、5%C02的条件下培养,每周传代2-3次。当SK-OV-3培养至对数生长期,用0.25%胰酶消化,按每孔3×104／400μL接种于24孔板,次日当细胞融合率达70%左右时,用Lipofectamine2000分别转染pGPU6/GFP/Neo-KIF4及pGPU6/GFP/Neo-NC。每孔质粒：脂质体=0.5μg：0.5μL。用无血清RPMI-1640培养基50μL稀释脂质体0.5μL,室温静置5min。无血清RPMI-1640培养基50μL稀释质粒0.5gg,室温静置5min。混合上述液体,室温静置20min。将混合液100μL加入24孔板细胞中,轻摇混匀。转染4-6h后更换成含10%胎牛血清的新鲜培养基,继续培养。48h后将孔板中的细胞消化,转入10cm平皿中,用含1mg／mLG418的培养基进行筛选,隔3d换液,待形成阳性单克隆后,用枪头吸取细胞培养。转染pGPU6/GFP/Neo-NC的阳性克隆命名为NC对照组,转染pGPU6/GFP/Neo-KIF4的阳性克隆分别命名为KIF4敲除组1、KIF4敲除组2。
(3) Western blot检测KIF4的敲除情况
每组收集1×106个细胞,用RIPA裂解液冰上裂解40min,4℃最高速离心后取上清。使用BCA蛋白浓度检测试剂盒进行蛋白浓度测定,取50μg总蛋白上样。10%SDS-聚丙烯酰胺凝胶、稳流30mA、1.5h,电泳分离蛋白质。400mA,2h,转移至PVDF膜。5%脱脂奶粉封闭1h,以兔抗KIF4抗体(1：1000)孵育2h,辣根过氧化物酶偶联的IgG二抗(1：10000)1h,用ECL发光系统检测,暗室内X胶片显影。
2、低表达KIF4对卵巢癌细胞SK-OV-3迁移能力的影响
(1)细胞迁移实验
将细胞分为4组：SK-OV-3组、NC对照组、KIF4敲除组1、KIF4敲除组2。每组设3个复孔,在8μm孔径的Transwell小室中进行实验。取对数生长期的细胞,用0.25%胰酶消化,无血清RPMI-1640洗涤细胞两次,计数1×105／100μL个细胞,种于上室。下室加600μL含10%胎牛血清的RPMI-1640培养基,37℃、5%CO2培养22h。取出上室,PBS洗涤小室上下部2次,甲醇固定10min。1%伊红染色5min。PBS洗涤后,用棉签擦去上室面的细胞。小心用刀片将膜从小室上切下,膜下室面朝上置于载玻片上,干燥后,中性树胶封固。显微镜下观察。每组3个复孔中的每个孔于显微镜下随机选取5个视野拍照。用Image-Pro Plus 6.0进行计数分析。
(2)统计学处理
使用SPSS16.0软件进行统计学分析。对正态分布的配对数据进行t检验。P<0.05为差异有统计学意义。
研究结果
一、驱动蛋白KIF4在胃癌组织中的表达情况
1、驱动蛋白KIF4在胃癌中表达降低
免疫组织化学检测了23例胃癌样本及相应的癌旁组织。KIF4的表达水平根据免疫组化染色实验分成8级。结果显示,23例中有13(56.5%)例呈现癌组织比癌旁组织低表达。应用Image-Pro Plus6.0软件,测量样本KIF4染色的累积光密度,发现所有癌旁组织的累积光密度比癌组织要高4倍。
2、KIF4在胃癌组织中表达降低与胃癌分化有相关性
我们接下来研究KIF4的表达和临床病理特征,临床档案资料之间的联系。结果显示KIF4的低表达和胃癌的低分化之间有显著相关性。在19例低分化的胃癌中,13例呈现出KIF4在癌组织中较癌旁组织中低表达。但是,4例中分化的肿瘤样本中,没有一例癌组织比癌旁组织KIF4低表达。所有结果表明,驱动蛋白KIF4在胃癌组织中低表达,且表达程度与肿瘤分化级别密切相关。
二、驱动蛋白KIF4对胃癌细胞BGC增殖的影响
1、成功筛选稳定表达GFP-KIF4的BGC细胞株(BGC-GFP-KIF4)及稳定表达GFP的BGC细胞株(BGC-GFP)
(1)成功构建融合表达载体pEGFP-KIF4
重组质粒双酶切后电泳,获得的目的基因片段与预期大小一致。DNA测序证明重组载体pEGFP-KIF4构建成功。
(2)转染pEGFP-KIF4质粒或pEGFP质粒至BGC细胞
转染质粒至细胞中,2天后,荧光比率可达约80%。转入10cm平皿培养2-3周后,挑取荧光较强的单克隆进行培养。
(3) Western blot检测GFP-KIF4或GFP在稳筛细胞中的表达
Western blot结果显示pEGFP-KIF4转染的BGC细胞中有明显的特异性条带,而转染空质粒和空细胞的对照组均未见条带,表明重组质粒可在BGC细胞中表达外源KIF4即GFP-KIF4。
2、免疫荧光染色显示过表达KIF4引起细胞多核性
在BGC-GFP-KIF4细胞中存在比BGC-GFP细胞中更多的多核细胞。在BGC-GFP-KIF4细胞中有14.5±0.82%的多核细胞,而在BGC-GFP中只有4.4±0.46%,与BGC细胞中相似。
3、细胞增殖计数及MTT表明过表达KIF4对BGC增殖有抑制作用
细胞增殖计数及MTT每隔一天检测一次,一共持续9天。结果显示,前3天内,两株细胞间没有显著的差别。但是从第5天开始,BGC-GFP-KIF4相对于BGC-GFP和BGC细胞来说,增殖显著抑制。而在BGC-GFP和BGC之间没有增殖效率的不同,说明在BGC细胞表达GFP不会影响细胞的增殖。
4、克隆形成实验表明过表达KIF4对BGC增殖有抑制作用
为了检验细胞在不贴壁情况下的生存能力,利用软琼脂克隆形成实验来检测两种细胞系。1×104个细胞种植在35mm的软琼脂培养基中。3周后,细胞用结晶紫染色后拍照。计数细胞克隆。在BGC-GFP中有平均143±9个细胞克隆,而在BGC-GFP-KIF4中只有30±9个细胞克隆。这些结果说明在细胞不贴壁的情况下,当在BGC中过表达KIF4,细胞增殖被显著抑制。
5、裸鼠体内成瘤表明过表达KIF4对BGC成瘤性的抑制作用
4×106个BGC-GFP-KIF4或BGC-GFP细胞接种到无胸腺裸鼠的左腋下(每组5只),每周测量记录肿瘤大小,测量7周。由BGC-GFP-KIF4衍生出的肿瘤比BGC-GFP衍生出的生长缓慢很多。7周后,所有小鼠处死,分离肿瘤。然后将肿瘤样本称重并拍照。BG-GFP-KIF4生长出的肿瘤的平均重量(0.9±0.24g)显著低于BGC-GFP生长出的肿瘤(3.0±1.17g),大约要轻3倍。这些结果都表明,BGC-GFP-KIF4在裸鼠形成肿瘤的能力相对于BGC-GFP来说要弱很多。表明在BGC细胞过表达KIF4可抑制体内肿瘤的形成。
三、驱动蛋白KIF4对卵巢癌SK-OV-3迁移能力的影响
1、筛选稳定低表达KIF4的SK-OV-3细胞株
(1) pGPU6/GFP/Neo-KIF4质粒及pGPU6/GFP/Neo-NC质粒的构建
重组质粒双酶切后电泳,获得的目的基因片段与预期大小一致。DNA测序证明重组载体pGPU6/GFP/Neo-KIF4及pGPU6/GFP/Neo-NC构建成功。
(2)细胞培养及转染
pGPU6/GFP/Neo-NC及pGPU6/GFP/Neo-KIF4中含有编码绿色荧光蛋白GFP的基因,经细胞瞬时转染和G418稳定筛选后,稳筛成功的细胞在荧光显微镜下观察可发出绿色荧光,绿色荧光比率均可达到92%以上。
(3)免疫印迹法检测KIF4的表达
免疫印迹结果显示,与转染pGPU6/GFP/Neo-NC的NC对照组相比,转染pGPU6/GFP/Neo-KIF4质粒的阳性稳筛细胞株能显著抑制KIF4蛋白表达。
2、细胞迁移实验
SK-OV-3组、NC对照组、KIF4敲除组1、KIF4敲除组2下室面细胞平均数分别为122、112、298、312。KIF4敲除组迁移到下室面的细胞数明显多于SK-OV-3组和NC对照组,可达大约3倍。
结论及意义：
1.本研究成功构建了一个过表达质粒pEGFP-KIF4和两个敲除质粒pGPU6/GFP/Neo-KIF4及pGPU6/GFP/Neo-NC,三种载体的构建为进一步研究KIF4的功能提供了必要的条件。
2.本研究表明驱动蛋白KIF4在胃癌中表达降低,且与胃癌分化有相关性。为胃癌发病机制的研究奠定了基础,也为下一步的细胞水平实验指明了方向。
3.本研究证明在胃癌细胞BGC中过表达KIF4可抑制BGC的增殖和体内成瘤,为进一步深入研究KIF4功能奠定了基础,同时也为发现胃癌治疗新靶点提供实验依据。
4.本研究证明在SK-OV-3中低表达KIF4可使细胞迁移能力增强,为进一步研究KIF4在细胞迁移中的作用奠定了基础。
Objective:
To identify the expression of kinesin KIF4 in gastric caner and explore its effects on cell proliferation of gastric cancer cells BGC. Furthermoer, to investigate its effect on migration of ovarian cancer cells SK-OV-3.
Method:
一、The research of KIF4 expression in gastric cancer tissue
1、The expression condition of kinesin KIF4 in human gastric carcinoma
23 patients with the primary gastric cancer included in this study were performed surgery in Qilu Hospital, Shandong University between 2006 and 2008. After surgery, tumor samples and their adjacent tissues from the patients were paraffin-embedded and stored at room temperature. These samples were approved by the local ethics committee to be used in the study. Their clinicalpathological characteristics were obtained from the Qilu Hospital.
For immunohistochemical staining assay, paraffin-embedded tumor samples were sectioned at a thickness of 4-5μm and placed on clean slides. After blocking with goat sera, polyclonal anti-KIF4 antibodies were added onto the slides by 1:500 dilution. The immunostaining was carried out using Histostain-Plus Kit following the manufacturer's instructions. Negative control was performed using PBS solution instead of primary antibodies. Each slide was photographed with a digital camera on an invert microscope of OlympusⅨ81. Five pictures on each slide were randomly taken. The integrated optical density (IOD) of pictures was measured using Image-Pro Plus 6.0 software.
2、The correlation between KIF4 expression and clinical data
Statistical analysis was done using SPSS 11.0 software:The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples. Contingency table was used to analyze the relationship between KIF4 expression and clinicopathologic variables in gastric cancer patients.
二、The influence of KIF4 on proliferation of gastric cancer cell BGC
1. Select the stable cell line which expressed GFP-KIF4 or GFP
(1) Construction of pEGFP-KIF4 expression plasmids
The cDNAs of full-length coding region of KIF4 was generated by PCR and subcloned into BglⅡ-Sal I sites of the mammalian expression vector pEGFP-C1.
(2) Transfect pEGFP-KIF4 or pEGFP to BGC cell
Cell transfection was performed using Lipofectamine 2000 following the manufacturer's instruction. BGC cells stably expressing GFP-KIF4 or GFP were generated using G418-selection methods. In brief,3×10~4 of BGC cells were plated into each well of 24-well plate and cultured overnight. Then 0.5μg of pEGFP-KIF4 or pEGFP plasmids was transfected into the cells using Lipofectamne 2000. Two days later, cells were split into a 10cm dish and cultured in complete medium contained 1 mg/ml of G418. After two to three weeks, positive cell colonies were picked up under an invert fluorescent microscope and then continued to culture with 1mg/ml of G418 selection medium. Finally, the BGC cells stably expressing GFP-KIF4 (BGC-GFP-KIF4 cells) or GFP (BGC-GFP cells) were grown up and lysed for Western Blot assays or fixed for immunofluorescent staining assays.
(3) Western blot analysis for KIF4 protein expression
Cells were collected by centrifugation and lysed in lysis buffer.40μg of cell extract was resolved on 12% polyacrylamide gels using minigel apparatus and transferred to PVDF membrane. Affinity-purified rabbit antibodies against KIF4 were diluted 1:1000 to detect endogenous KIF4 and over expressed GFP-KIF4 fusion protein. The anti-a-tubulin monoclonal antibody was used with 1:1000 dilution. Blots were then exposed to HRP-conjugated goat anti-rabbit lgG(1:10000 dilution) followed by development using ECL reagent.
2. Immunofluorescence staining show the polynucleation of BGC-GFP-KIF4
Cells grown on coverslips were fixed with cold methanol for 5 minutes at room temperature and then blocked with PBS contained 5% goat serum and 0.3% Triton X-100. The staining was performed using mouse anti-a-tubulin (1:10000 dilution) followed by Texas Red conjugated goat anti-mouse antibodies (1:10000 dilution) for 2 hours at room temperature respectively. Finally, DNA was stained with DAPI (1μg/ml) for 5 minutes. After washing with PBS, coverslips were amounted on clean slides with FluoroGuard Antifade Reagent and sealed with Cytoseal 60.
3. Cell proliferate counting and MTT assay investigate the influence on proliferation of BGC when overexpressing KIF4
5000 cells were seeded into each well of 24-well plates and cultured in complete RPMI-1640 medium. In the following 9 days, cells were counting every two day as routine. Then cell growth curves were drew according to the data. Each independent experiment was performed three times.
5000 cells were seeded into each well of 96-well plates and cultured in complete RPMI-1640 medium. In the following 9 days, cells were assayed every two day by MTT method as routine. Briefly,20μl of 5mg/ml MTT was added into each well and continued to culture for four hours. Then the cell culture medium was removed followed by adding another 200μl of DMSO.30 minutes later, the plates were placed on a micro-plate auto-reader. Optical density (OD) was read at 570 nm wavelength, then cell growth curves were drew according to the data. Each independent experiment was performed three times.
4. Soft-agar colony-formation assay investigate the influence on proliferation of BGC when overexpressing KIF4
For cell colony formation assay, 1×104 cells were added to 1 ml of growth medium with 0.3%agar and layered onto 2 ml of 0.6%agar beds in 35mm dishes. After three weeks culture in soft agar, cells were stained with 0.05% crystal violet for half an hour and photographed. Cell colonies were counted using Image-Pro Plus 6.0 software. Assays were conducted in triplicate in three independent experiments.
5. Xenograft assays in nude mice investigate the potentiality of tumor formation when overexpressing KIF4 in BGC
Female BABL/c mice (6-8 weeks old) were purchased from SLAC laboratory animal Co. Ltd. A total of 4×10~6 cells of BGC-GFP-KIF4 or BGC-GFP cells and 100μg of Matrigel per animal were inoculated into the left flank of athymic nude (nu/nu) mice (n=5 for each group). All procedures complied with the protocols approved by the Institutional Animal Care Committee. The situation of tumor growth was recorded every week with a caliper-like instrument. Tumor volume was calculated according to the formula:volume=(lengthxwidth2)/2. At the end of 7 weeks, mice were sacrificed by cervical dislocation. Tumor tissues were removed from mice and the volume and weight of tumor tissues were measured and photographed.
6. Statistical analysis
Statistical analysis was done using SPSS 11.0 software. The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples.
三、The research of KIF4 on ovarian cancer cells SK-OV-3 migration.
1. Select the stable cell line which low expressing KIF4
(1)Construction of pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC expression plasmids
The shRNA toward KIF4 was generated by synthesis and subcloned into the mammalian knockdown vector pGPU6/GFP/Neo.
(2) Transfect pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC to SK-OV-3 cell
Cell transfection was performed using Lipofectamine 2000 following the manufacturer's instruction. SK-OV-3 cells stably expressing pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC were generated using G418-selection methods. In brief,3×104 of SK-OV-3 cells were plated into each well of 24-well plate and cultured overnight. Then 0.5μg of pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC plasmids was transfected into the cells using Lipofectamne 2000. Two days later, cells were split into a 10cm dish and cultured in complete medium contained 1 mg/ml of G418. After two to three weeks, positive cell colonies were picked up under an invert fluorescent microscope and then continued to culture with 1 mg/ml of G418 selection medium. Finally, the SK-OV-3 cells stably expressing pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC were grown up and lysed for Western Blot assays or fixed for immunofluorescent staining assays.
(3) Western blot analysis for KIF4 protein expression
Cells were collected by centrifugation and lysed in lysis buffer.40μg of cell extract was resolved on 12%polyacrylamide gels using minigel apparatus and transferred to PVDF membrane. Affinity-purified rabbit antibodies against KIF4 were diluted 1:1000 to detect endogenous KIF4. The anti-α-tubulin monoclonal antibody was used with 1:1000 dilution. Blots were then exposed to HRP-conjugated goat anti-rabbit lgG(1:10000 dilution) followed by development using ECL reagent.
2、Transwell assay investigate the influence on migration of SK-OV-3 when knockdown KIF4
(1) Transwell assay
Migration of SK-OV-3 cells were measured by Transwell chambers with 8.0μm pore membranes.The cells were seeded into the top chamber of the Transwell with a concentration of 1×10~5/100μL in FBS free medium.The bottom chamber of the Transwell contained 600μL 10%FBS medium.Cells were allowed to migration for 22 h at 37℃,5% CO_2.Then the Transwell top surface of the membrance was cleared of adherent cells with a cotton swab.Cells that had migrated to the bottom side of the membrance were fixed in methanol for 10min,stained with eosin.The number of migrated cells,counted under a microscope at 200×magnification,is the sum of cells found in the bottom side of the membrance and the lower chamber.
(2) Statistical analysis
Statistical analysis was done using SPSS 11.0 software. The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples.
Result:
一、The research of KIF4 on expression in gastric cancer tissue
1、Kinesin KIF4 appears reduced expression in human gastric carcinoma
Kinesin KIF4 is previously report to play an intriguing role in tumorigenesis. To investigate the critical role of kinesin KIF4 in human gastric cancer, we examined 23 gastric carcinoma samples along with corresponding adjacent tissue samples using immunohistochemical analysis. Immunohistochemical staining on the paraffin-embedded samples was performed using anti-KIF4 polyclonal antibodies as previously published. The expression level of kinesin KIF4 in each specimen is classified into eight grades according to immunohistochemical staining assay following the instruction of previous described methods. The result come out that 13 of 23 (56.5%) samples appeared reduced expression of KIF4 in carcinoma compare with their corresponding adjacent tissues. Using Image-Pro Plus 6.0 software, we measured the integrated optical density (IOD) of KIF4 staining in all above samples and found 4-fold lower level of KIF4 expression in total carcinoma than that of adjacent tissues. Immunohistochemical analysis clearly showed KIF4 localized in cytoplasm and nucleus in gastric tissues which is consistent with previous reports in other human tissues or in vitro cultured cells.
2、KIF4 appears reduced expression in human gastric carcinoma and associates with the differentiation of gastric carcinoma.
We next studied the associativity between expression of KIF4 and clinicopathologic features as well as clinical profiles of these samples. Our results indicated a significant association of low expression of KIF4 with poor differentiation of clinical gastric carcinoma. Among nineteen poorly differentiated carcinoma samples, thirteen samples showed lower expression of KIF4 in carcinoma compare with corresponding adjacent tissues. However none of four moderately differentiated carcinoma samples took place lower level of KIF4 than their adjacent tissues. All these results suggest that kinesin KIF4 is lowly expressed in human gastric cancer and its expression level is closely correlated with the tumor differentiation grade.
二、The influence of KIF4 on proliferation of gastric cancer cell BGC
1. Successfully select the stable cell line which expressed GFP-KIF4 or GFP
(1) Construction of pEGFP-KIF4 expression plasmids
Enzymes digestion, PCR and DNA sequencing confirmed that the combinant vector, pEGFP-KIF4 successfully constructed.
(2) Transfect pEGFP-KIF4 or pEGFP to BGC cell
The stable cell lines which expressing GFP-KIF4 or GFP were selected. The ratio which cell expressing green fluorescence protein is about 93%.
(3) Western blot analysis for KIF4 protein expression
Western blot results showed that GFP-KIF4 protein could express in BGC stable cell line. GFP-KIF4 fusion protein was highly expressed in BGC-GFP-KIF4 cells and even higher than endogenous KIF4 protein in the cells as well as in BGC and BGC-GFP cells.
2. Immunofluorescence staining show that over expression of KIF4 in gastric cancer cells resulted in multinucleation.
Given that low expression of KIF4 in clinical gastric carcinoma specimens, the effect of over expression of KIF4 in human gastric cancer cells such as BGC cells would be interestingly to survey. To study the critical role of KIF4 in the growth of BGC cells, we stably expressed GFP-KIF4 fusion protein in BGC cells (designated as BGC-GFP-KIF4 cells) as well as GFP in BGC cells (designated as BGC-GFP cells). The Western Blotting assay showed that GFP-KIF4 fusion protein was highly expressed in BGC-GFP-KIF4 cells and even higher than endogenous KIF4 protein in the cells as well as in BGC and BGC-GFP cells. We then performed immunofluorescent staining on the cells to examine morphological differences between these cells. Consistent with previous reports in in vitro cultured cells, GFP-KIF4 predominantly localized to the nucleus in BGC-GFP-KIF4 cells. Strikingly, we found more multinucleation exist in BGC-GFP-KIF4 cells than in BGC-GFP cells. There were 14.5±0.82%of BGC-GFP-KIF4 cells that come out multinucleation. In contrast, only 4.4±0.46% of BGC-GFP cells become multinucleated similar to maternal BGC cells.
3. Cell proliferate counting and MTT assay investigate the influence on proliferation of BGC when overexpressing KIF4
Over expression of KIF4 in BGC cells resulted in multinucleation and then potentially affected cell proliferation in vitro. To assess inhibition effect of cell proliferation by over expression of KIF4 in gastric cancer cells,we carried out cell proliferate counting and MTT assay in BGC-GFP-KIF4 cells as well as BGC-GFP cells. Assay was performed every two day for 9 days and three independent experiments have been achieved. The result showed that no significant difference occurred in above cells within three days. However, the proliferation of BGC-GFP-KIF4 cells were gradually inhibited from the fifth day when compare with BGC-GFP as well as BGC cells. There was no difference of cell proliferation rate between BGC-GFP and BGC cells indicating that expression of GFP protein in BGC cells did not affect the ability of cell proliferation.
4. Soft-agar colony-formation assay investigate the influence on proliferation of BGC when overexpressing KIF4
To examine cell viability in anchorage-independent condition, we performed soft-agar colony-formation assay with BGC-GFP-KIF4 and BGC-GFP cells.104 cells were plated on the soft-agar medium in a 35 mm dish. Three weeks later, the cells were stained with crystal violet and photographed. The cell colonies were scored and results from three independent experiments were analyzed together. Average 143±9 cell colonies were formed in BGC-GFP cells but only 30±9 colonies in BGC-GFP-KIF4 cells. These results suggested that cell growth were significantly inhibited in anchorage-independent condition in BGC cells because of over expression of KIF4.
5. Xenograft assays in nude mice investigate the potentiality of tumor formation when overexpressing KIF4 in BGC
As described above, the growth rate of BGC-GFP-KIF4 cells was noticeably lower than that of BGC-GFP cells in vitro. We next determined the ability of these cells giving rise to tumor in nude mice.4×10~6 of BGC-GFP-KIF4 or BGC-GFP cells were inoculated into the left flank of athymic nude mice (n=5 for each group) and tumor size was recorded weekly for 7 weeks. The tumor derived from BGC-GFP-KIF4 cells grew much slowly compare with BGC-GFP cells during this period. Seven weeks later, all mice were killed and dissected to isolate the final xenografts. Then the tumor samples were weighed and photographed. The weight of tumors derived from BGC-GFP-KIF4 cells (0.9±0.24g) was significantly less than that from BGC-GFP cells (3.0±1.17g). It was about 3-fold lighter of tumors from BGC-GFP-KIF4 cells than that from BGC-GFP cells. These results indicated that the ability of BGC-GFP-KIF4 cells to form tumor in nude mice was much weaker than that of BGC-GFP cells suggesting that over expression of ectopic KIF4 protein in BGC cells can inhibit tumor formation in vivo.
三、The research of KIF4 on ovarian cancer cells SK-OV-3 migration.
1. Select the stable cell line which low expressing KIF4
(1)Construction of pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC expression plasmids
Enzymes digestion, PCR and DNA sequencing confirmed that the combinant vector, pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC successfully constructed.
(2) Transfect pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC to BGC cell
The stable cell lines which expressing GFP-KIF4 or GFP were selected. The ratio which cell expressing green fluorescence protein is about 92%.
(3) Western blot analysis for KIF4 protein expression
Western blot results showed that KIF4 protein was lowly expressed in SK-OV-3 cells which was transfected pGPU6/GFP/Neo-KIF4.
2、Transwell assay investigate the influence on migration of SK-OV-3 when knockdown KIF4
The migration of SK-OV-3 cells was determined by transwell chambers assay.Migratory ability of pGPU6/GFP/Neo-KIF4 transfected SK-OV-3 cells was increased significantly compared to that pGPU6/GFP/Neo-NC transfected and Mock SK-OV-3 cells(P<0.05).
Conclusions:
1. We successfully constructed one fusion expression vectors pEGFP-KIF4, two knockdown vectors pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC,which could produce a marked effect in vitro efficiently. This can provide requirement for further study of KIF4 function.
2. This study confirmed kinesin KIF4 appears reduced expression in human gastric carcinoma and associates with the differentiation of gastric carcinoma.This study provided necessary experimental data for further research on the function of KIF4.
3. In this study, both of our in vitro and in vivo studies showed an apparent inhibition of proliferation in BGC cells by way of over expression of KIF4. These results strongly imply that chromokinesin KIF4 functions as an inhibitor for the proliferation of human gastric cancer cells.
4. This study confirmed silencing KIF4 in human ovarian cancer cell SK-OV-3 increases its migration capability.
研究驱动蛋白KIF4在胃癌组织的表达情况及对胃癌细胞BGC增殖的影响。同时研究KIF4对卵巢癌细胞SK-OV-3迁移的影响。为阐明KIF4在肿瘤发生发展中的作用奠定基础,同时也为探索新的肿瘤生物治疗方法提供帮助。
研究方法：
一、驱动蛋白KIF4在胃癌组织中的表达情况
1、免疫组织化学染色实验比较KIF4在胃癌组织和相应癌旁组织中的表达情况
本研究中23例原发性胃癌病人于2006-2008年间在山东大学齐鲁医院接受外科手术,手术之后,肿瘤样本和它相应的癌旁组织用石蜡包埋,室温储存。石蜡包埋的标本组织被切成4-5μm,放置至载玻片上。羊血清封闭后,抗KIF4的多克隆抗体以1：500稀释,加至载玻片上。免疫染色用晶美生物技术公司的Histostain-Plus试剂盒,过程参照说明书。阴性对照用PBS溶液代替一抗。每张标本用奥林帕斯IX81倒置显微镜随机拍取5张照片。照片的累积光密度用Image-Pro Plus6.0软件进行分析。
2、统计分析KIF4的表达量与临床资料的相关性
用SPSS11.0进行统计学数据分析。每一个实验的结果用平均值±标准差来描述。用双侧t检验来比较实验组和对照组。用列联表来分析KIF4的表达与胃癌病人的病理学变量的关系。
二、驱动蛋白KIF4对胃癌细胞BGC增殖的影响
1、筛选稳定表达GFP-KIF4的BGC细胞株(BGC-GFP-KIF4)及稳定表达GFP的BGC细胞株(BGC-GFP)
(1)真核表达载体pEGFP-KIF4的构建
PCR扩增产生的KIF4编码区域通过BglⅡ-SalⅠ双酶切位点克隆至真核表达载体pEGFP-C1。
(2)转染pEGFP-KIF4质粒或pEGFP质粒至BGC细胞
取生长良好的BGC细胞,按3×104／孔接种到24孔板,培养过夜。用脂质体2000将0.5μg pEGFP-KIF4或pEGFP质粒转染入细胞。2天后,细胞转移入10cm平皿,在含1mg／mlG418的完全培养基中培养,每隔3天换一次液。2到3周后,在倒置荧光显微镜下挑选出单个阳性克隆,继续在1mg／mlG418的完全培养基中培养。最后,稳定表达GFP-KIF4或GFP的BGC细胞被筛选出来,通过Western blot进行检测。
(3) Western blot检测GFP-KIF4在稳筛细胞株中的表达
离心收集细胞,用裂解液(50mmol/L Tris-HCl,150mmol/L NaCl,0.5% NP-40, 0.5%dexycholate-Na,0.1% SDS, protease inhibitor cocktail)裂解细胞。40μg总蛋白通过12%SDS—聚丙烯酰胺凝胶电泳分离,并转移至PVDF膜。兔抗KIF4的抗体1：1000稀释来检测内源性KIF4和表达的GFP-KIF4融合蛋白。鼠抗Tubulin的单克隆抗体1：1000倍稀释,来显示微管蛋白作为内参。随后加入HRP标记的羊抗兔／鼠IgG的二抗,最后用ECL发光系统显色。
2、免疫荧光染色显示过表达KIF4引起的细胞多核性
生长在盖玻片上的稳筛细胞株用冷甲醇室温固定5min,然后用含5%羊血清、0.3%TritonX-100的PBS封闭。一抗用鼠抗Tubulin的抗体(1：10000稀释),二抗用TexasRed交联的羊抗鼠的抗体(1：10000稀释),分别染色2h。DNA用DAPI(1μg/ml)染色5min。PBS洗后,用FluoroGuard抗淬灭剂将盖玻片固定在干净的载玻片上,指甲油封边。
3、细胞增殖计数及MTT检测过表达KIF4对BGC增殖的影响
将两种稳筛细胞株接种于24孔板中,每孔500μl细胞悬液中含5000个细胞,置培养箱中培养。在接下来的9天内,每隔一天计数细胞数量。根据数据绘制细胞生长曲线。
将两种稳筛细胞株接种于96孔板中,每孔200μl细胞悬液中含5000个细胞,置培养箱中培养。在接下来的9天内,每隔一天采用MTT法检测细胞增殖情况。每孔加入20μl 5mg／mlMTT,继续培养4个小时。然后去除细胞培养基,加入200μlDMSO,20min后,在酶标仪上570nm测量吸光度值。根据数据绘制曲线。
4、克隆形成实验检测过表达KIF4对BGC增殖的影响
在35mm平皿中铺入2ml含0.6%琼脂的培养基,室温下凝固作为下层胶。1×104个细胞加入1ml含0.3%琼脂的培养基中,作为上层胶。培养3周后,用0.05%结晶紫染色细胞30min。拍摄照片并用Image-Pro Plus6.0进行克隆计数。
5、裸鼠体内成瘤检测过表达KIF4对BGC成瘤性的影响
实验用的雌性BABL／c小鼠(6-8周大)从中国科学院上海实验动物中心购进。取4×106个BGC-GFP-KIF4或BGC-GFP细胞和100 gMatrigel接种到无胸腺小鼠(nu／nu)的左腋下。每组5只。肿瘤的大小每周用游标卡尺进行测量。肿瘤的体积通过下列公式计算：体积=(长×宽2)／2。7周后,脱颈法处死小鼠,从小鼠体内取出肿瘤组织测量体积和称重并照相。
6、统计分析
使用SPSS16.0软件进行统计学分析。对正态分布的配对数据进行t检验。P<0.05为差异有统计学意义。
三、驱动蛋白KIF4对卵巢癌SK-OV-3迁移能力的影响
1.筛选稳定低表达KIF4的SK-OV-3细胞株
(1) pGPU6/GFP/Neo-KIF4质粒及pGPU6/GFP/Neo-NC质粒的构建
于文献资料中检索到KIF4的siRNA序列,它是针对KIF4的19nt的RNA片段。序列为5'GCAGATTGAAAGCCTAGAG3'.将此片段补充设计为shDNA,并克隆入pGPU6/GFP/Neo载体,命名为pGPU6/GFP/Neo-KIF4。阴性对照干扰序列为5'GTTCTCCGAACGTGTCACGT 3'。将此片段补充设计为shDNA,并克隆入pGPU6／GFP／Neo载体,命名为pGPU6／GFP／Neo-NC。
(2)转染pGPU6/GFP/Neo-KIF4质粒及pGPU6/GFP/Neo-NC质粒至SK-OV-3细胞
人卵巢癌细胞SK-OV-3用含10%胎牛血清的RPMI-1640,于37℃、5%C02的条件下培养,每周传代2-3次。当SK-OV-3培养至对数生长期,用0.25%胰酶消化,按每孔3×104／400μL接种于24孔板,次日当细胞融合率达70%左右时,用Lipofectamine2000分别转染pGPU6/GFP/Neo-KIF4及pGPU6/GFP/Neo-NC。每孔质粒：脂质体=0.5μg：0.5μL。用无血清RPMI-1640培养基50μL稀释脂质体0.5μL,室温静置5min。无血清RPMI-1640培养基50μL稀释质粒0.5gg,室温静置5min。混合上述液体,室温静置20min。将混合液100μL加入24孔板细胞中,轻摇混匀。转染4-6h后更换成含10%胎牛血清的新鲜培养基,继续培养。48h后将孔板中的细胞消化,转入10cm平皿中,用含1mg／mLG418的培养基进行筛选,隔3d换液,待形成阳性单克隆后,用枪头吸取细胞培养。转染pGPU6/GFP/Neo-NC的阳性克隆命名为NC对照组,转染pGPU6/GFP/Neo-KIF4的阳性克隆分别命名为KIF4敲除组1、KIF4敲除组2。
(3) Western blot检测KIF4的敲除情况
每组收集1×106个细胞,用RIPA裂解液冰上裂解40min,4℃最高速离心后取上清。使用BCA蛋白浓度检测试剂盒进行蛋白浓度测定,取50μg总蛋白上样。10%SDS-聚丙烯酰胺凝胶、稳流30mA、1.5h,电泳分离蛋白质。400mA,2h,转移至PVDF膜。5%脱脂奶粉封闭1h,以兔抗KIF4抗体(1：1000)孵育2h,辣根过氧化物酶偶联的IgG二抗(1：10000)1h,用ECL发光系统检测,暗室内X胶片显影。
2、低表达KIF4对卵巢癌细胞SK-OV-3迁移能力的影响
(1)细胞迁移实验
将细胞分为4组：SK-OV-3组、NC对照组、KIF4敲除组1、KIF4敲除组2。每组设3个复孔,在8μm孔径的Transwell小室中进行实验。取对数生长期的细胞,用0.25%胰酶消化,无血清RPMI-1640洗涤细胞两次,计数1×105／100μL个细胞,种于上室。下室加600μL含10%胎牛血清的RPMI-1640培养基,37℃、5%CO2培养22h。取出上室,PBS洗涤小室上下部2次,甲醇固定10min。1%伊红染色5min。PBS洗涤后,用棉签擦去上室面的细胞。小心用刀片将膜从小室上切下,膜下室面朝上置于载玻片上,干燥后,中性树胶封固。显微镜下观察。每组3个复孔中的每个孔于显微镜下随机选取5个视野拍照。用Image-Pro Plus 6.0进行计数分析。
(2)统计学处理
使用SPSS16.0软件进行统计学分析。对正态分布的配对数据进行t检验。P<0.05为差异有统计学意义。
研究结果
一、驱动蛋白KIF4在胃癌组织中的表达情况
1、驱动蛋白KIF4在胃癌中表达降低
免疫组织化学检测了23例胃癌样本及相应的癌旁组织。KIF4的表达水平根据免疫组化染色实验分成8级。结果显示,23例中有13(56.5%)例呈现癌组织比癌旁组织低表达。应用Image-Pro Plus6.0软件,测量样本KIF4染色的累积光密度,发现所有癌旁组织的累积光密度比癌组织要高4倍。
2、KIF4在胃癌组织中表达降低与胃癌分化有相关性
我们接下来研究KIF4的表达和临床病理特征,临床档案资料之间的联系。结果显示KIF4的低表达和胃癌的低分化之间有显著相关性。在19例低分化的胃癌中,13例呈现出KIF4在癌组织中较癌旁组织中低表达。但是,4例中分化的肿瘤样本中,没有一例癌组织比癌旁组织KIF4低表达。所有结果表明,驱动蛋白KIF4在胃癌组织中低表达,且表达程度与肿瘤分化级别密切相关。
二、驱动蛋白KIF4对胃癌细胞BGC增殖的影响
1、成功筛选稳定表达GFP-KIF4的BGC细胞株(BGC-GFP-KIF4)及稳定表达GFP的BGC细胞株(BGC-GFP)
(1)成功构建融合表达载体pEGFP-KIF4
重组质粒双酶切后电泳,获得的目的基因片段与预期大小一致。DNA测序证明重组载体pEGFP-KIF4构建成功。
(2)转染pEGFP-KIF4质粒或pEGFP质粒至BGC细胞
转染质粒至细胞中,2天后,荧光比率可达约80%。转入10cm平皿培养2-3周后,挑取荧光较强的单克隆进行培养。
(3) Western blot检测GFP-KIF4或GFP在稳筛细胞中的表达
Western blot结果显示pEGFP-KIF4转染的BGC细胞中有明显的特异性条带,而转染空质粒和空细胞的对照组均未见条带,表明重组质粒可在BGC细胞中表达外源KIF4即GFP-KIF4。
2、免疫荧光染色显示过表达KIF4引起细胞多核性
在BGC-GFP-KIF4细胞中存在比BGC-GFP细胞中更多的多核细胞。在BGC-GFP-KIF4细胞中有14.5±0.82%的多核细胞,而在BGC-GFP中只有4.4±0.46%,与BGC细胞中相似。
3、细胞增殖计数及MTT表明过表达KIF4对BGC增殖有抑制作用
细胞增殖计数及MTT每隔一天检测一次,一共持续9天。结果显示,前3天内,两株细胞间没有显著的差别。但是从第5天开始,BGC-GFP-KIF4相对于BGC-GFP和BGC细胞来说,增殖显著抑制。而在BGC-GFP和BGC之间没有增殖效率的不同,说明在BGC细胞表达GFP不会影响细胞的增殖。
4、克隆形成实验表明过表达KIF4对BGC增殖有抑制作用
为了检验细胞在不贴壁情况下的生存能力,利用软琼脂克隆形成实验来检测两种细胞系。1×104个细胞种植在35mm的软琼脂培养基中。3周后,细胞用结晶紫染色后拍照。计数细胞克隆。在BGC-GFP中有平均143±9个细胞克隆,而在BGC-GFP-KIF4中只有30±9个细胞克隆。这些结果说明在细胞不贴壁的情况下,当在BGC中过表达KIF4,细胞增殖被显著抑制。
5、裸鼠体内成瘤表明过表达KIF4对BGC成瘤性的抑制作用
4×106个BGC-GFP-KIF4或BGC-GFP细胞接种到无胸腺裸鼠的左腋下(每组5只),每周测量记录肿瘤大小,测量7周。由BGC-GFP-KIF4衍生出的肿瘤比BGC-GFP衍生出的生长缓慢很多。7周后,所有小鼠处死,分离肿瘤。然后将肿瘤样本称重并拍照。BG-GFP-KIF4生长出的肿瘤的平均重量(0.9±0.24g)显著低于BGC-GFP生长出的肿瘤(3.0±1.17g),大约要轻3倍。这些结果都表明,BGC-GFP-KIF4在裸鼠形成肿瘤的能力相对于BGC-GFP来说要弱很多。表明在BGC细胞过表达KIF4可抑制体内肿瘤的形成。
三、驱动蛋白KIF4对卵巢癌SK-OV-3迁移能力的影响
1、筛选稳定低表达KIF4的SK-OV-3细胞株
(1) pGPU6/GFP/Neo-KIF4质粒及pGPU6/GFP/Neo-NC质粒的构建
重组质粒双酶切后电泳,获得的目的基因片段与预期大小一致。DNA测序证明重组载体pGPU6/GFP/Neo-KIF4及pGPU6/GFP/Neo-NC构建成功。
(2)细胞培养及转染
pGPU6/GFP/Neo-NC及pGPU6/GFP/Neo-KIF4中含有编码绿色荧光蛋白GFP的基因,经细胞瞬时转染和G418稳定筛选后,稳筛成功的细胞在荧光显微镜下观察可发出绿色荧光,绿色荧光比率均可达到92%以上。
(3)免疫印迹法检测KIF4的表达
免疫印迹结果显示,与转染pGPU6/GFP/Neo-NC的NC对照组相比,转染pGPU6/GFP/Neo-KIF4质粒的阳性稳筛细胞株能显著抑制KIF4蛋白表达。
2、细胞迁移实验
SK-OV-3组、NC对照组、KIF4敲除组1、KIF4敲除组2下室面细胞平均数分别为122、112、298、312。KIF4敲除组迁移到下室面的细胞数明显多于SK-OV-3组和NC对照组,可达大约3倍。
结论及意义：
1.本研究成功构建了一个过表达质粒pEGFP-KIF4和两个敲除质粒pGPU6/GFP/Neo-KIF4及pGPU6/GFP/Neo-NC,三种载体的构建为进一步研究KIF4的功能提供了必要的条件。
2.本研究表明驱动蛋白KIF4在胃癌中表达降低,且与胃癌分化有相关性。为胃癌发病机制的研究奠定了基础,也为下一步的细胞水平实验指明了方向。
3.本研究证明在胃癌细胞BGC中过表达KIF4可抑制BGC的增殖和体内成瘤,为进一步深入研究KIF4功能奠定了基础,同时也为发现胃癌治疗新靶点提供实验依据。
4.本研究证明在SK-OV-3中低表达KIF4可使细胞迁移能力增强,为进一步研究KIF4在细胞迁移中的作用奠定了基础。
Objective:
To identify the expression of kinesin KIF4 in gastric caner and explore its effects on cell proliferation of gastric cancer cells BGC. Furthermoer, to investigate its effect on migration of ovarian cancer cells SK-OV-3.
Method:
一、The research of KIF4 expression in gastric cancer tissue
1、The expression condition of kinesin KIF4 in human gastric carcinoma
23 patients with the primary gastric cancer included in this study were performed surgery in Qilu Hospital, Shandong University between 2006 and 2008. After surgery, tumor samples and their adjacent tissues from the patients were paraffin-embedded and stored at room temperature. These samples were approved by the local ethics committee to be used in the study. Their clinicalpathological characteristics were obtained from the Qilu Hospital.
For immunohistochemical staining assay, paraffin-embedded tumor samples were sectioned at a thickness of 4-5μm and placed on clean slides. After blocking with goat sera, polyclonal anti-KIF4 antibodies were added onto the slides by 1:500 dilution. The immunostaining was carried out using Histostain-Plus Kit following the manufacturer's instructions. Negative control was performed using PBS solution instead of primary antibodies. Each slide was photographed with a digital camera on an invert microscope of OlympusⅨ81. Five pictures on each slide were randomly taken. The integrated optical density (IOD) of pictures was measured using Image-Pro Plus 6.0 software.
2、The correlation between KIF4 expression and clinical data
Statistical analysis was done using SPSS 11.0 software:The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples. Contingency table was used to analyze the relationship between KIF4 expression and clinicopathologic variables in gastric cancer patients.
二、The influence of KIF4 on proliferation of gastric cancer cell BGC
1. Select the stable cell line which expressed GFP-KIF4 or GFP
(1) Construction of pEGFP-KIF4 expression plasmids
The cDNAs of full-length coding region of KIF4 was generated by PCR and subcloned into BglⅡ-Sal I sites of the mammalian expression vector pEGFP-C1.
(2) Transfect pEGFP-KIF4 or pEGFP to BGC cell
Cell transfection was performed using Lipofectamine 2000 following the manufacturer's instruction. BGC cells stably expressing GFP-KIF4 or GFP were generated using G418-selection methods. In brief,3×10~4 of BGC cells were plated into each well of 24-well plate and cultured overnight. Then 0.5μg of pEGFP-KIF4 or pEGFP plasmids was transfected into the cells using Lipofectamne 2000. Two days later, cells were split into a 10cm dish and cultured in complete medium contained 1 mg/ml of G418. After two to three weeks, positive cell colonies were picked up under an invert fluorescent microscope and then continued to culture with 1mg/ml of G418 selection medium. Finally, the BGC cells stably expressing GFP-KIF4 (BGC-GFP-KIF4 cells) or GFP (BGC-GFP cells) were grown up and lysed for Western Blot assays or fixed for immunofluorescent staining assays.
(3) Western blot analysis for KIF4 protein expression
Cells were collected by centrifugation and lysed in lysis buffer.40μg of cell extract was resolved on 12% polyacrylamide gels using minigel apparatus and transferred to PVDF membrane. Affinity-purified rabbit antibodies against KIF4 were diluted 1:1000 to detect endogenous KIF4 and over expressed GFP-KIF4 fusion protein. The anti-a-tubulin monoclonal antibody was used with 1:1000 dilution. Blots were then exposed to HRP-conjugated goat anti-rabbit lgG(1:10000 dilution) followed by development using ECL reagent.
2. Immunofluorescence staining show the polynucleation of BGC-GFP-KIF4
Cells grown on coverslips were fixed with cold methanol for 5 minutes at room temperature and then blocked with PBS contained 5% goat serum and 0.3% Triton X-100. The staining was performed using mouse anti-a-tubulin (1:10000 dilution) followed by Texas Red conjugated goat anti-mouse antibodies (1:10000 dilution) for 2 hours at room temperature respectively. Finally, DNA was stained with DAPI (1μg/ml) for 5 minutes. After washing with PBS, coverslips were amounted on clean slides with FluoroGuard Antifade Reagent and sealed with Cytoseal 60.
3. Cell proliferate counting and MTT assay investigate the influence on proliferation of BGC when overexpressing KIF4
5000 cells were seeded into each well of 24-well plates and cultured in complete RPMI-1640 medium. In the following 9 days, cells were counting every two day as routine. Then cell growth curves were drew according to the data. Each independent experiment was performed three times.
5000 cells were seeded into each well of 96-well plates and cultured in complete RPMI-1640 medium. In the following 9 days, cells were assayed every two day by MTT method as routine. Briefly,20μl of 5mg/ml MTT was added into each well and continued to culture for four hours. Then the cell culture medium was removed followed by adding another 200μl of DMSO.30 minutes later, the plates were placed on a micro-plate auto-reader. Optical density (OD) was read at 570 nm wavelength, then cell growth curves were drew according to the data. Each independent experiment was performed three times.
4. Soft-agar colony-formation assay investigate the influence on proliferation of BGC when overexpressing KIF4
For cell colony formation assay, 1×104 cells were added to 1 ml of growth medium with 0.3%agar and layered onto 2 ml of 0.6%agar beds in 35mm dishes. After three weeks culture in soft agar, cells were stained with 0.05% crystal violet for half an hour and photographed. Cell colonies were counted using Image-Pro Plus 6.0 software. Assays were conducted in triplicate in three independent experiments.
5. Xenograft assays in nude mice investigate the potentiality of tumor formation when overexpressing KIF4 in BGC
Female BABL/c mice (6-8 weeks old) were purchased from SLAC laboratory animal Co. Ltd. A total of 4×10~6 cells of BGC-GFP-KIF4 or BGC-GFP cells and 100μg of Matrigel per animal were inoculated into the left flank of athymic nude (nu/nu) mice (n=5 for each group). All procedures complied with the protocols approved by the Institutional Animal Care Committee. The situation of tumor growth was recorded every week with a caliper-like instrument. Tumor volume was calculated according to the formula:volume=(lengthxwidth2)/2. At the end of 7 weeks, mice were sacrificed by cervical dislocation. Tumor tissues were removed from mice and the volume and weight of tumor tissues were measured and photographed.
6. Statistical analysis
Statistical analysis was done using SPSS 11.0 software. The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples.
三、The research of KIF4 on ovarian cancer cells SK-OV-3 migration.
1. Select the stable cell line which low expressing KIF4
(1)Construction of pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC expression plasmids
The shRNA toward KIF4 was generated by synthesis and subcloned into the mammalian knockdown vector pGPU6/GFP/Neo.
(2) Transfect pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC to SK-OV-3 cell
Cell transfection was performed using Lipofectamine 2000 following the manufacturer's instruction. SK-OV-3 cells stably expressing pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC were generated using G418-selection methods. In brief,3×104 of SK-OV-3 cells were plated into each well of 24-well plate and cultured overnight. Then 0.5μg of pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC plasmids was transfected into the cells using Lipofectamne 2000. Two days later, cells were split into a 10cm dish and cultured in complete medium contained 1 mg/ml of G418. After two to three weeks, positive cell colonies were picked up under an invert fluorescent microscope and then continued to culture with 1 mg/ml of G418 selection medium. Finally, the SK-OV-3 cells stably expressing pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC were grown up and lysed for Western Blot assays or fixed for immunofluorescent staining assays.
(3) Western blot analysis for KIF4 protein expression
Cells were collected by centrifugation and lysed in lysis buffer.40μg of cell extract was resolved on 12%polyacrylamide gels using minigel apparatus and transferred to PVDF membrane. Affinity-purified rabbit antibodies against KIF4 were diluted 1:1000 to detect endogenous KIF4. The anti-α-tubulin monoclonal antibody was used with 1:1000 dilution. Blots were then exposed to HRP-conjugated goat anti-rabbit lgG(1:10000 dilution) followed by development using ECL reagent.
2、Transwell assay investigate the influence on migration of SK-OV-3 when knockdown KIF4
(1) Transwell assay
Migration of SK-OV-3 cells were measured by Transwell chambers with 8.0μm pore membranes.The cells were seeded into the top chamber of the Transwell with a concentration of 1×10~5/100μL in FBS free medium.The bottom chamber of the Transwell contained 600μL 10%FBS medium.Cells were allowed to migration for 22 h at 37℃,5% CO_2.Then the Transwell top surface of the membrance was cleared of adherent cells with a cotton swab.Cells that had migrated to the bottom side of the membrance were fixed in methanol for 10min,stained with eosin.The number of migrated cells,counted under a microscope at 200×magnification,is the sum of cells found in the bottom side of the membrance and the lower chamber.
(2) Statistical analysis
Statistical analysis was done using SPSS 11.0 software. The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples.
Result:
一、The research of KIF4 on expression in gastric cancer tissue
1、Kinesin KIF4 appears reduced expression in human gastric carcinoma
Kinesin KIF4 is previously report to play an intriguing role in tumorigenesis. To investigate the critical role of kinesin KIF4 in human gastric cancer, we examined 23 gastric carcinoma samples along with corresponding adjacent tissue samples using immunohistochemical analysis. Immunohistochemical staining on the paraffin-embedded samples was performed using anti-KIF4 polyclonal antibodies as previously published. The expression level of kinesin KIF4 in each specimen is classified into eight grades according to immunohistochemical staining assay following the instruction of previous described methods. The result come out that 13 of 23 (56.5%) samples appeared reduced expression of KIF4 in carcinoma compare with their corresponding adjacent tissues. Using Image-Pro Plus 6.0 software, we measured the integrated optical density (IOD) of KIF4 staining in all above samples and found 4-fold lower level of KIF4 expression in total carcinoma than that of adjacent tissues. Immunohistochemical analysis clearly showed KIF4 localized in cytoplasm and nucleus in gastric tissues which is consistent with previous reports in other human tissues or in vitro cultured cells.
2、KIF4 appears reduced expression in human gastric carcinoma and associates with the differentiation of gastric carcinoma.
We next studied the associativity between expression of KIF4 and clinicopathologic features as well as clinical profiles of these samples. Our results indicated a significant association of low expression of KIF4 with poor differentiation of clinical gastric carcinoma. Among nineteen poorly differentiated carcinoma samples, thirteen samples showed lower expression of KIF4 in carcinoma compare with corresponding adjacent tissues. However none of four moderately differentiated carcinoma samples took place lower level of KIF4 than their adjacent tissues. All these results suggest that kinesin KIF4 is lowly expressed in human gastric cancer and its expression level is closely correlated with the tumor differentiation grade.
二、The influence of KIF4 on proliferation of gastric cancer cell BGC
1. Successfully select the stable cell line which expressed GFP-KIF4 or GFP
(1) Construction of pEGFP-KIF4 expression plasmids
Enzymes digestion, PCR and DNA sequencing confirmed that the combinant vector, pEGFP-KIF4 successfully constructed.
(2) Transfect pEGFP-KIF4 or pEGFP to BGC cell
The stable cell lines which expressing GFP-KIF4 or GFP were selected. The ratio which cell expressing green fluorescence protein is about 93%.
(3) Western blot analysis for KIF4 protein expression
Western blot results showed that GFP-KIF4 protein could express in BGC stable cell line. GFP-KIF4 fusion protein was highly expressed in BGC-GFP-KIF4 cells and even higher than endogenous KIF4 protein in the cells as well as in BGC and BGC-GFP cells.
2. Immunofluorescence staining show that over expression of KIF4 in gastric cancer cells resulted in multinucleation.
Given that low expression of KIF4 in clinical gastric carcinoma specimens, the effect of over expression of KIF4 in human gastric cancer cells such as BGC cells would be interestingly to survey. To study the critical role of KIF4 in the growth of BGC cells, we stably expressed GFP-KIF4 fusion protein in BGC cells (designated as BGC-GFP-KIF4 cells) as well as GFP in BGC cells (designated as BGC-GFP cells). The Western Blotting assay showed that GFP-KIF4 fusion protein was highly expressed in BGC-GFP-KIF4 cells and even higher than endogenous KIF4 protein in the cells as well as in BGC and BGC-GFP cells. We then performed immunofluorescent staining on the cells to examine morphological differences between these cells. Consistent with previous reports in in vitro cultured cells, GFP-KIF4 predominantly localized to the nucleus in BGC-GFP-KIF4 cells. Strikingly, we found more multinucleation exist in BGC-GFP-KIF4 cells than in BGC-GFP cells. There were 14.5±0.82%of BGC-GFP-KIF4 cells that come out multinucleation. In contrast, only 4.4±0.46% of BGC-GFP cells become multinucleated similar to maternal BGC cells.
3. Cell proliferate counting and MTT assay investigate the influence on proliferation of BGC when overexpressing KIF4
Over expression of KIF4 in BGC cells resulted in multinucleation and then potentially affected cell proliferation in vitro. To assess inhibition effect of cell proliferation by over expression of KIF4 in gastric cancer cells,we carried out cell proliferate counting and MTT assay in BGC-GFP-KIF4 cells as well as BGC-GFP cells. Assay was performed every two day for 9 days and three independent experiments have been achieved. The result showed that no significant difference occurred in above cells within three days. However, the proliferation of BGC-GFP-KIF4 cells were gradually inhibited from the fifth day when compare with BGC-GFP as well as BGC cells. There was no difference of cell proliferation rate between BGC-GFP and BGC cells indicating that expression of GFP protein in BGC cells did not affect the ability of cell proliferation.
4. Soft-agar colony-formation assay investigate the influence on proliferation of BGC when overexpressing KIF4
To examine cell viability in anchorage-independent condition, we performed soft-agar colony-formation assay with BGC-GFP-KIF4 and BGC-GFP cells.104 cells were plated on the soft-agar medium in a 35 mm dish. Three weeks later, the cells were stained with crystal violet and photographed. The cell colonies were scored and results from three independent experiments were analyzed together. Average 143±9 cell colonies were formed in BGC-GFP cells but only 30±9 colonies in BGC-GFP-KIF4 cells. These results suggested that cell growth were significantly inhibited in anchorage-independent condition in BGC cells because of over expression of KIF4.
5. Xenograft assays in nude mice investigate the potentiality of tumor formation when overexpressing KIF4 in BGC
As described above, the growth rate of BGC-GFP-KIF4 cells was noticeably lower than that of BGC-GFP cells in vitro. We next determined the ability of these cells giving rise to tumor in nude mice.4×10~6 of BGC-GFP-KIF4 or BGC-GFP cells were inoculated into the left flank of athymic nude mice (n=5 for each group) and tumor size was recorded weekly for 7 weeks. The tumor derived from BGC-GFP-KIF4 cells grew much slowly compare with BGC-GFP cells during this period. Seven weeks later, all mice were killed and dissected to isolate the final xenografts. Then the tumor samples were weighed and photographed. The weight of tumors derived from BGC-GFP-KIF4 cells (0.9±0.24g) was significantly less than that from BGC-GFP cells (3.0±1.17g). It was about 3-fold lighter of tumors from BGC-GFP-KIF4 cells than that from BGC-GFP cells. These results indicated that the ability of BGC-GFP-KIF4 cells to form tumor in nude mice was much weaker than that of BGC-GFP cells suggesting that over expression of ectopic KIF4 protein in BGC cells can inhibit tumor formation in vivo.
三、The research of KIF4 on ovarian cancer cells SK-OV-3 migration.
1. Select the stable cell line which low expressing KIF4
(1)Construction of pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC expression plasmids
Enzymes digestion, PCR and DNA sequencing confirmed that the combinant vector, pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC successfully constructed.
(2) Transfect pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC to BGC cell
The stable cell lines which expressing GFP-KIF4 or GFP were selected. The ratio which cell expressing green fluorescence protein is about 92%.
(3) Western blot analysis for KIF4 protein expression
Western blot results showed that KIF4 protein was lowly expressed in SK-OV-3 cells which was transfected pGPU6/GFP/Neo-KIF4.
2、Transwell assay investigate the influence on migration of SK-OV-3 when knockdown KIF4
The migration of SK-OV-3 cells was determined by transwell chambers assay.Migratory ability of pGPU6/GFP/Neo-KIF4 transfected SK-OV-3 cells was increased significantly compared to that pGPU6/GFP/Neo-NC transfected and Mock SK-OV-3 cells(P<0.05).
Conclusions:
1. We successfully constructed one fusion expression vectors pEGFP-KIF4, two knockdown vectors pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC,which could produce a marked effect in vitro efficiently. This can provide requirement for further study of KIF4 function.
2. This study confirmed kinesin KIF4 appears reduced expression in human gastric carcinoma and associates with the differentiation of gastric carcinoma.This study provided necessary experimental data for further research on the function of KIF4.
3. In this study, both of our in vitro and in vivo studies showed an apparent inhibition of proliferation in BGC cells by way of over expression of KIF4. These results strongly imply that chromokinesin KIF4 functions as an inhibitor for the proliferation of human gastric cancer cells.
4. This study confirmed silencing KIF4 in human ovarian cancer cell SK-OV-3 increases its migration capability.
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18. Mazumdar M, Lee JH, Sengupta K, Ried T, Rane S, Misteli T: Tumor formation via loss of a molecular motor protein. Curr Biol 2006,16(15):1559-1564.
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20. Kawai H, Ishii A, Washiya K, Konno T, Kon H, Yamaya C, Ono I, Minamiya Y, Ogawa J: Estrogen receptor alpha and beta are prognostic factors in non-small cell lung cancer. Clin Cancer Res 2005, 11(14):5084-5089.
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17. Midorikawa R, Takei Y, Hirokawa N:KIF4 motor regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity. Cell 2006,125(2):371-383.
18. Mazumdar M, Lee JH, Sengupta K, Ried T, Rane S, Misteli T: Tumor formation via loss of a molecular motor protein. Curr Biol 2006,16(15):1559-1564.
19. Mazumdar M, Sundareshan S, Misteli T: Human chromokinesin KIF4A functions in chromosome condensation and segregation. J Cell Biol 2004,166(5):613-620.
20. Kawai H, Ishii A, Washiya K, Konno T, Kon H, Yamaya C, Ono I, Minamiya Y, Ogawa J: Estrogen receptor alpha and beta are prognostic factors in non-small cell lung cancer. Clin Cancer Res 2005, 11(14):5084-5089.
21. Lee YM, Lee S, Lee E, Shin H, Hahn H, Choi W, Kim W: Human kinesin superfamily member 4 is dominantly localized in the nuclear matrix and is associated with chromosomes during mitosis. Biochem J 2001,360(Pt 3):549-556.
22. Kaplan DR, Miller FD:When a motor goes bad:a kinesin regulates neuronal survival. Cell 2006,125(2):224-226.
23. Verhey K J, Hammond J W. Traffic control:regulation of kinesin motors. Nat Rev Mol Cell Biol,2009,10(11):765-777.
24. Ozlii N, Monigatti F, Renard B Y, et al. Binding partner switching on microtubules and aurora-B in the mitosis to cytokinesis transition. Mol Cell Proteomics,2010,9(2):336-350.
25. Mazumdar M, Lee J H, Sengupta K, et al. Tumor formation via loss of a molecular motor protein. Curr Biol,2006,16:1559-1564.
26.王玉珍,姜慧卿,扈彩霞.Rho信号转导通路与细胞迁移.医学综述杂志,2006,12(11)：651-653.
27. Watanabe T, Noritake J, Kaibuchi K. Regulation of microtubules in cell migration.Trends Cell Biol,2005,15(2):76-83.
28. Mary S, Charrasse S, Meriane M, et al. Biogenesis of N-Cadherin-dependent cell-cell contacts in living fibroblasts is a microtubule-dependent kinesin-driven mechanism.Mol Biol Cell,2002,13(1):285-301.
29. Jimbo T, Kawasaki Y, Koyama R,et al. Identification of a link between the tumour suppressor APC and the kinesin superfamily.Nat Cell Biol,2002,4:323-327.