【作者】 张军；

【导师】 程建新；

【作者基本信息】 河北医科大学 ， 外科学， 2009， 博士

【摘要】 背景:卵巢上皮性癌起源于卵巢表面上皮,是最致命的女性生殖系统恶性肿瘤,其预后差,死亡率居妇科恶性肿瘤之首。卵巢上皮性癌有多种不同的组织学类型:包括浆液性癌、粘液性癌、子宫内膜样癌、透明细胞癌等。其中浆液性癌是最常见的组织学类型,占全部上皮性卵巢癌的一半以上。目前,有关卵巢上皮性癌的发生机制、分子遗传学和流行病学资料仍未完全阐明,而且不同组织学类型的卵巢上皮性癌在发生机制和预后等方面都存在许多差异。近年来,随着对卵巢浆液性癌的临床病理形态学特征的的深入了解和分子遗传学的发展,有关卵巢浆液性癌的发生机制和临床病理形态特征有了新的认识,并初步建立了卵巢低级别浆液性癌与高级别浆液性癌存在不同的发病机制的二元论模型:低分级浆液性癌通过良性-交界性-恶性过程逐步发展形成,而高分级浆液性癌是从正常卵巢表面上皮、包涵囊肿或输卵管上皮直接发展形成。但有关浆液性卵巢癌的发生机制尚未完全阐明,低级别的浆液性卵巢癌的发生是否经过良性-交界性-恶性过程逐步演变形成还需要分子遗传学方面进一步证实,与高级别浆液性癌之间是否存在某些共同的分子病理基础仍然是关注的焦点问题。肿瘤的发生是一个多因素、多步骤复杂的生物学行为,其中抑癌基因在肿瘤的形成过程中发挥着重要作用,其表达产物能直接或间接地负性调节细胞的生长或能促进细胞的分化、衰老和死亡,从而抑制细胞增生或癌变。Ras同源基因家族成员Ⅰ(ras homologue memberⅠ,ARHI)作为ras超家族中第一个抑癌印迹基因,其通过多种途径参与信号转导和细胞周期的调控从而抑制细胞生长,促进细胞的凋亡。氧化还原酶的双色氨酸域基因(WW domain-containing oxi- doreductase,WWOX)基因就是跨越了染色体脆性部位FRA16D的一个新的抑癌基因。WWOX可以增强多种抑癌基因的功能,抑制原癌基因的表达,从而发挥抑制肿瘤作用。PTEN基因即第10号染色体缺失的磷酸酶和张力蛋白同源物基因(phosphataseand tension homologdeleted on chromosome ten, PTEN),是一种具有磷酸酶活性的抑癌基因, PTEN作为PIP3的磷酸酶,抑制PI3K的磷酸化,阻断AKT及其下游激酶的活性,使细胞周期阻滞在G1期或促使细胞凋亡,对细胞的生长起负调节作用。研究证实上述三个重要的抑癌基因在人类多种恶性肿瘤中都存在不同程度的表达降低或缺失,但有关卵巢浆液性癌发生发展过程中ARHI、WWOX和PTEN表达情况和各自所起的作用以及相互关系,同时三者在癌变过程中的可能调控机制尚无系统阐述。卵巢上皮性癌死亡率高,预后差。有关其预后因素的研究始终是妇科肿瘤工作者关注的焦点。既往针对卵巢上皮性癌的预后相关因素进行了很多研究工作,但多数都集中在一些临床分期、病理分级、病理类型、淋巴结转移、手术后化疗和残留灶的大小等传统的预后因素方面,而且由于涉及不同的组织学类型,往往不能对某一特定的类型的预后做出更加准确的判断。目前,探索除传统的预后因素之外的一些对预测某种特定类型的卵巢上皮性癌的预后有价值的分子指标是有关卵巢癌预后研究的另一热点,尚无一个因素能够完全准确的预测其预后。抑癌基因ARHI、WWOX和PTEN与卵巢浆液性癌预后关系尚未见报道。目的:本研究运用免疫组织化学、RT-PCR、FCM和蛋白印迹方法检测卵巢浆液性良性肿瘤、交界性肿瘤和浆液性癌中,ARHI、WWOX和PTEN表达的变化规律,分析其与重要的信号转导转录因子STAT3和E2F1的相关性,探讨上述三种抑癌基因的表达变化规律及其可能的调控机制在卵巢浆液性癌发生中的意义。同时运用Kaplan-Meier单因素生存分析,筛选影响卵巢浆液性癌预后的相关因素,判断各因素是否为独立的预测因素及各因素的预测价值,Cox多因素生存分析比较各因素对预后影响的程度,确定各因素的风险系数,评估分析抑癌基因ARHI、WWOX和PTEN蛋白表达情况对判断卵巢浆液性癌预后的价值。方法:1研究对象:选取2000年1月至2003年6月河北医科大学第二医院临床资料完整、经手术切除并经病理证实并有随访记录的卵巢浆液性癌标本56例,卵巢浆液性良性肿瘤35例,卵巢浆液性交界性肿瘤18例,另取因其他疾病手术切除的正常卵巢组织25例。用于各指标蛋白表达的免疫组织化学检测及预后多因素分析。留取河北医科大学第四医院2006～2008年因卵巢肿瘤收住院接受手术治疗患者的新鲜肿瘤组织93例,其中卵巢浆液性癌45例,卵巢浆液性良性肿瘤32例,卵巢浆液性交界性肿瘤16例,另取正常卵巢组织15例。用于mRNA的检测、FCM和蛋白印迹。2免疫组织化学采用免疫组织化学二步法检测各指标蛋白的表达。步骤按二步法免疫组化检测试剂盒进行。苏木素轻度复染,乙醇梯度脱水,二甲苯透明,中性树胶封片,光镜观察。阴性对照以磷酸盐缓冲液(PBS)代替一抗而其他条件均相同。3流式细胞定量检测(FCM)分别取冻存的各肿瘤组织,4%甲醛固定,流式细胞定量检测各指标蛋白表达情况。4细胞总蛋白的提取和定量胞浆裂解液分别加入,以提取细胞的总蛋白。将提取的蛋白应用紫外分光光度计进行定量。5蛋白印迹经SDS-PAGE电泳、转膜后,ECL发光法显示各蛋白阳性荧光条带,由X线胶片感光将结果记录下来,扫描后再经Bio1D图像分析系统对杂交条带进行定量分析。以β-肌动蛋白(β-actin)表达作为内参照,以各蛋白与β-肌动蛋白的比值分别表示其相对表达量。6细胞总RNA的提取、鉴定及定量采用异硫氰酸胍一步法提取细胞总RNA。1%琼脂糖电泳鉴定其完整性。紫外分光光度计进行定量。7 RT-PCR反转录合成cDNA,建立PCR反应体系,94℃变性5min,94℃50s, 56℃30s, 72℃30s,30次循环扩增,PCR产物1.5%琼脂糖凝胶电泳90V 40min。采用凝胶分析软件(BIO-LD)进行定量,以目的基因与内参照基因基因GAPDH的比值表示目的基因相对表达量。8卵巢浆液性癌预后Kaplan-Meier单因素及COX多因素分析对56例有随访的卵巢浆液性癌患者进行总结分析,分别以36个月和60个月为研究截止点,计算3年和5年生存率。分析抑癌基因与患者预后的关系,并筛选确定影响预后的相关因素,COX多因素生存分析确定独立的预后因素及影响程度。9统计学处理与分析采用SPSS 13.0软件进行统计学处理,计数资料比较采用行×列表资料的x2检验,多个样本率的两两比较采用分割法;计量资料采用单因素方差分析,用SNK法进行两两比较;Spearman等级相关分析各指标间的相关关系;Kaplan-Meier单因素生存分析,计算患者生存率,Log-Rank检验比较生存率间差异有无显著性,筛选预后相关因素,判断各因素是否为独立的预测因素及各因素的预测价值,Cox多因素生存分析比较各因素对预后影响的程度,确定各因素的风险系数。检验水准α=0.05。结果第一部分卵巢浆液性肿瘤中抑癌基因ARHI、WWOX和PTEN表达1.卵巢浆液性肿瘤中抑癌基因ARHI表达1.1卵巢浆液性肿瘤中抑癌基因ARHI mRNA表达RT-PCR结果, 45例卵巢浆液性癌、16例卵巢浆液性交界性肿瘤和32例卵巢浆液性良性肿瘤组织中分别有18例、7例和28例检测到了ARHI mRNA的表达,阳性表达率分别为40%、43.8%和87.5%。统计分析表明,卵巢浆液性癌和交界性肿瘤中ARHI mRNA阳性表达率明显低于正常卵巢组织和浆液性良性肿瘤,差异有统计学意义(P<0.05)。但浆液性癌与交界性肿瘤之间,正常卵巢与浆液性良性肿瘤之间ARHI mRNA阳性表达率的差异无统计学意义。凝胶分析软件(BIO-LD)定量分析结果,卵巢浆液性癌组织的ARHI mRNA相对表达量为0.12±0.06,卵巢浆液性交界性肿瘤相对表达量为0.15±0.04,明显低于正常卵巢组织和浆液性良性肿瘤,差异有统计学意义(P<0.05)。1.2卵巢浆液性肿瘤中抑癌基因ARHI蛋白表达1.2.1 ARHI蛋白印迹结果Western Blotting检测结果,26例卵巢浆液性良性肿瘤、8例卵巢浆液性交界性囊腺瘤和19例卵巢浆液性癌在对应的26kd位置出现了阳性荧光条带,ARHI蛋白表达阳性率分别为81.3%、50%和42.2%。卵巢浆液性癌和交界性囊腺瘤ARHI蛋白表达阳性率明显低于正常卵巢和浆液性良性肿瘤(P<0.05)。阳性条带密度扫描定量结果,卵巢浆液性癌中ARHI蛋白表达相对量为0.17±0.12,与正常卵巢和浆液性囊腺瘤和交界性囊腺瘤比明显降低,差异有统计学意义(P<0.05),其余各组间无统计学差异。1.2.2 ARHI蛋白FCM检测结果FCM结果,卵巢浆液性癌和卵巢浆液性交界性肿瘤ARHI蛋白表达的FI值(0.894±0.008和0.914±0.06)明显低于正常卵巢和良性肿瘤,差异有统计学意义(P<0.05)。1.2.3 ARHI蛋白免疫组织化学结果35例浆液性良性肿瘤、18例交界性囊腺瘤和56例浆液性癌中,ARHI蛋白表达分别为27例、10例和22例。卵巢浆液性癌和浆液性交界性肿瘤ARHI蛋白的表达阳性率分别为39.3%和55.6%,明显低于正常卵巢组织和卵巢浆液性良性肿瘤(P<0.05)。但浆液性癌与交界性囊腺瘤之间,正常卵巢与浆液性囊腺瘤之间ARHI阳性表达率的差异无统计学意义。2卵巢浆液性肿瘤中抑癌基因WWOX表达2.1卵巢浆液性肿瘤中抑癌基因WWOX mRNA表达RT-PCR结果,45例卵巢浆液性癌、16例卵巢浆液性交界性肿瘤和32例卵巢浆液性良性肿瘤中分别有21例、12例和29例检测到WWOX mRNA表达,阳性表达率分别为46.7%、75.0%和90.6%。卵巢浆液性癌中WWOX mRNA阳性表达率明显低于其余各组,差异有统计学意义(P<0.05)。其余各组之间差异无统计学意义。凝胶分析软件(BIO-LD)定量分析结果,卵巢浆液性癌的WWOX mRNA相对表达量为0.25±0.08显著低于正常卵巢组织、卵巢浆液性良性肿瘤和卵巢浆液性交界性肿瘤(P<0.05)。2.2卵巢浆液性肿瘤中抑癌基因WWOX蛋白表达2.2.1 WWOX蛋白印迹结果Western Blotting检测结果,正常卵巢组织、卵巢浆液性囊腺瘤、卵巢浆液性交界性囊腺瘤和卵巢浆液性癌WWOX蛋白表达阳性率分别为86.7%、78.1%、68.8%和44.4%。卵巢浆液性癌WWOX蛋白表达阳性率明显低于正常卵巢、浆液性囊腺瘤和交界性囊腺瘤(P<0.05)。阳性条带密度扫描定量结果,卵巢浆液性癌中WWOX蛋白表达相对量为0.11±0.04,与正常卵巢和浆液性囊腺瘤和交界性囊腺瘤比明显降低(p<0.05),其余各组间无统计学差异。2.2.2 WWOX蛋白FCM检测结果FCM结果,卵巢浆液性癌WWOX蛋白表达的FI值(0.930±0.02)明显低于正常卵巢、卵巢囊腺瘤和交界性囊腺瘤的FI值,差异有统计学意义(P<0.05)。2.2.3 WWOX蛋白免疫组织化学结果35例浆液性囊腺瘤、18例交界性囊腺瘤和56例浆液性癌中,分别有30例、16例和27例呈阳性表达,WWOX蛋白在卵巢浆液性癌中阳性率为48.2%,明显低于正常卵巢组织(92.0%)、卵巢浆液性囊腺瘤(85.7%)和交界性囊腺瘤(88.9%) (P<0.05),但其余各组之间无统计学差异。3卵巢浆液性肿瘤中抑癌基因PTEN表达3.1卵巢浆液性肿瘤中抑癌基因PTEN mRNA表达RT-PCR结果,45例卵巢浆液性癌、16例卵巢浆液性交界性囊腺瘤和32例卵巢浆液性囊腺瘤组织中PTEN mRNA阳性表达率分别为51.1%、81.3%和90.6%。卵巢浆液性癌中PTEN mRNA阳性表达率明显低于其余各组,差异有统计学意义(P<0.05)。其余各组之间差异无统计学意义。凝胶分析软件(BIO-LD)定量分析结果,卵巢浆液性癌的PTEN mRNA相对表达量为0.31±0.08显著低于正常卵巢组织、卵巢浆液性囊腺瘤和卵巢浆液性交界性囊腺瘤(P<0.05)。3.2卵巢浆液性肿瘤中抑癌基因PTEN蛋白表达3.2.1 PTEN蛋白印迹结果Western Blotting检测结果,正常卵巢组织、卵巢浆液性囊腺瘤、卵巢浆液性交界性囊腺瘤和卵巢浆液性癌PTEN蛋白表达阳性率分别为93.3%、84.4%、75.0%和53.3%。卵巢浆液性癌PTEN蛋白表达阳性率明显低于正常卵巢、浆液性囊腺瘤和交界性囊腺瘤(P<0.05)。阳性条带密度扫描定量结果,卵巢浆液性癌中PTEN蛋白表达相对量为0.34±0.12比正常卵巢和浆液性囊腺瘤和交界性囊腺瘤明显降低(p<0.05),其余各组间无统计学差异。3.2.2 PTEN蛋白FCM检测结果FCM结果,卵巢浆液性癌PTEN蛋白表达的FI值(0.908±0.023)明显低于正常卵巢、卵巢囊腺瘤和交界性囊腺瘤的FI值,差异有统计学意义(P<0.05)。3.2.3 PTEN蛋白免疫组织化学结果35例浆液性囊腺瘤、18例交界性囊腺瘤和56例浆液性癌中,分别有31例、15例和29例呈阳性表达。卵巢浆液性癌中PTEN蛋白表达阳性率为51.8%,明显低于正常卵巢组织(100%)、卵巢浆液性囊腺瘤(88.6%)和交界性囊腺瘤(83.3%) (P<0.05),但其余各组之间无统计学差异。4 ARHI、WWOX和PTEN蛋白在低级别和高级别浆液性癌中的表达比较ARHI在低级别和高级别卵巢浆液癌中的阳性表达率无明显差异;WWOX和PTEN蛋白在高级别中的阳性表达率明显低于其在低级别中的阳性表达率(P<0.05)。第二部分Stat3和E2F1在卵巢浆液性肿瘤中的表达1 STAT3蛋白在卵巢浆液性肿瘤组织中的表达1.1 STAT3蛋白免疫组织化学染色结果STAT3在卵巢浆液性癌和浆液性交界性囊腺瘤中的阳性表达率为87.5%(49/56)和77.8%(14/18),强阳性表达率为71.4%(40/56)和50.0%(9/18),均显著高于其在正常卵巢组织和卵巢浆液性囊腺瘤中的阳性表达率(P<0.05)。浆液性癌中的阳性表达和强阳性表达率也高于交界性囊腺瘤,但差异无统计学意义( P>0.05)。正常卵巢与浆液性囊腺瘤之间,STAT3的阳性表达率无统计学意义。1.2 STAT3蛋白流式细胞检测结果FCM结果,卵巢浆液性癌STAT3蛋白表达的FI值(1.192±0.031)明显高于正常卵巢、卵巢囊腺瘤和交界性囊腺瘤的FI值,差异有统计学意义(P<0.05)。2 E2F1蛋白在卵巢浆液性肿瘤组织中的表达2.1 E2F1蛋白免疫组织化学染色结果35例浆液性囊腺瘤、18例交界性囊腺瘤和56例浆液性癌中,分别有7、12、10和46例E2F1蛋白表达呈阳性。卵巢浆液性癌组织中,E2F1的阳性表达率为82.1%,明显高于正常卵巢组织、卵巢浆液性囊腺瘤和交界性囊腺瘤(P<0.05)。交界性囊腺瘤中,E2F1的阳性表达率虽也高于正常卵巢和浆液性囊腺瘤,但差异无统计学意义(P>0.05)。2.2 E2F1蛋白流式细胞检测结果FCM结果,卵巢浆液性癌E2F1蛋白表达的FI值(1.139±0.023)明显高于正常卵巢、卵巢囊腺瘤和交界性囊腺瘤的FI值,差异有统计学意义(P<0.05)。3 STAT3和E2F1蛋白表达与卵巢浆液性癌临床病理特征的关系统计分析显示,STAT3和E2F1蛋白的表达在卵巢浆液性癌的病理分级、有无淋巴结转移、有无腹膜转移和临床分期中,差异均无统计学意义(P>0.05)4卵巢浆液性癌ARHI与STAT3和E2F1蛋白表达的相关性研究结果显示,ARHI蛋白表达缺失的34例患者中,33例STAT3蛋白表达增强、31例E2F1蛋白表达增强。卵巢浆液性癌中ARHI蛋白表达降低或缺失,而STAT3和E2F1蛋白的表达明显增强。提示卵巢浆液性癌中,ARHI蛋白表达强度与STAT3和E2F1蛋白表达强度分别呈负相关关系(P<0.05)。5卵巢浆液性癌WWOX与STAT3和E2F1蛋白表达的相关性研究结果显示, WWOX蛋白表达缺失的29例患者中,28例STAT3蛋白表达增强、27例E2F1蛋白表达增强。提示卵巢浆液性癌中,WWOX蛋白表达强度与STAT3和E2F1蛋白表达强度呈负相关关系(P<0.05)。6卵巢浆液性癌PTEN与STAT3和E2F1蛋白表达的相关性研究结果显示, PTEN蛋白表达缺失的27例患者中,26例STAT3蛋白表达增强、24例E2F1蛋白表达增强。卵巢浆液性癌中PTEN蛋白的表达与STAT3和E2F1蛋白表达呈相反的趋势,但未见明显的呈负相关关系(P>0.05)。第三部分ARHI、WWOX和PTEN与卵巢浆液性癌预后的关系1 ARHI蛋白表达与卵巢浆液性癌预后的关系结合ARHI蛋白表达结果,对56例有随访的卵巢浆液性癌患者进行分析,ARHI蛋白阳性患者和ARHI蛋白阴性患者的3年和5年生存率分别为(68.2%/54.5%)和(32.4%/17.6%),ARHI蛋白阳性的卵巢浆液性腺癌患者的3年和5年生存率均高于ARHI蛋白阴性的患者(P=0.008,P=0.003)。统计分析显示,ARHI蛋白表达在卵巢浆液性癌的病理分级、有无淋巴结转移、有无腹膜转移和临床分期中,差异均无统计学意义。2 WWOX蛋白表达与卵巢浆液性癌预后及临床病理特征的关系结合WWOX蛋白表达结果,对56例有随访的卵巢浆液性癌患者进行分析。WWOX蛋白阳性和阴性患者的3年和5年生存率分别为(63.0%/44.4%)和(31.0%/20.7%)WWOX蛋白阳性的卵巢浆液性癌患者的3年和5年生存率均高于WWOX蛋白阴性的患者(P=0.023,P=0.031)。统计分析显示,WWOX蛋白表达在卵巢浆液性癌的病理分级中差异有统计学意义(P<0.05);在有无淋巴结转移、有无腹膜转移和临床分期中,差异均无统计学意义。3 PTEN蛋白表达与卵巢浆液性癌预后及临床病理特征的关系结合PTEN蛋白表达结果,对56例有随访的卵巢浆液性癌患者进行分析, PTEN蛋白阳性和阴性患者的3年和5年生存率分别为(62.1%/44.8%)和(29.6%/18.5%)。PTEN蛋白阳性的卵巢浆液性癌患者的3年和5年生存率均高于PTEN蛋白阴性的患者(P=0.028,P=0.025)。统计分析显示,PTEN蛋白表达在卵巢浆液性癌的病理分级中差异有统计学意义(P<0.05)。在有无淋巴结转移、有无腹膜转移和临床分期中差异无统计学意义。4卵巢浆液性癌的预后多因素分析结果4.1卵巢浆液性癌的预后因素的筛选经Kaplan-Meier单因素生存分析和Log-Rank检验表明,临床分期、病理分级、术后是否正规化疗和术后有无肉眼可见的残留灶是影响卵巢浆液性癌患者预后的相关因素。4.2卵巢浆液性癌患者COX多因素生存分析结果结果显示临床分期、手术后化疗、ARHI表达、病理分级进入了COX比例风险回归模型中是影响卵巢浆液性癌的独立预后因素,而手术后残留灶、WWOX及PTEN蛋白表达未能进入COX模型不是影响预后的独立因素。多因素分析确定的独立预后因素对卵巢浆液性癌的影响程度依次是临床分期、病理分级、手术后化疗和ARHI蛋白的表达。结论:1抑癌基因ARHI、WWOX和PTEN的mRNA和蛋白表达在卵巢浆液性癌中都存在不同程度的降低和缺失,提示卵巢浆液性癌发生过程中三种抑癌基因均丧失了抑癌基因的功能,从而在卵巢浆液性癌的发生中发挥了一定的作用。2抑癌基因ARHI mRNA和蛋白表达降低或缺失程度高于WWOX和PTEN基因,提示三个抑癌基因中ARHI可能在卵巢浆液性癌的发生中起关键作用。3不同级别的卵巢浆液性癌中抑癌基因表达存在差异,进一步证实低级别和高级别浆液性癌存在不同的发生机制。4 STAT3和E2F1在卵巢浆液性癌中表达增强,提示E2F1蛋白过表达抑制了ARHI的表达,从而失去了对STAT3的负性调节作用,进一步激活下游一系列促增殖抑凋亡的基因,可能是卵巢浆液性癌的调控机制之一。5除传统的预后因素外,抑癌基因ARHI、WWOX和PTEN蛋白表达也是影响卵巢浆液性癌的预后相关因素,而且ARHI是独立的预后因素,可能成为卵巢浆液性癌基因治疗的新靶点。6综合分析包括抑癌基因ARHI、WWOX和PTEN蛋白表达在内的各种预后因素,有助于指导临床更加准确的评价患者的预后和合理治疗。更多还原

【Abstract】 BackgroudOvarian cancer is composed of a heterogeneous group of tumors that are derived from the surface epithelium of the ovary or from surface inclusions. They are the most lethal malignant tumors of female reproductive system and the mortality rate is first of all gynecological tumors. They are classified into serous, mucinous, endometrioid, clear cell, and Brenner (transitional) types corresponding to the different types of epithelia in the organs of the female reproductive tract. Among, serous carcinoma accounts for about 50% of all ovarian epithelial carcinomas. At present, the pathogenesis, molecular genetics, and epidemiology of ovarian epithelial carcinomas are still unclear. Moreover, a lot of differences of pathogenesis and prognosis are present among different ovarian epithelial carcinoma types.Recently, following the further understanding of clinical pathomorphology characteristic about ovarian serous carcinoma and the development of molecular genetics, we have a new recognition about the pathogenesis and pathomorphology characteristic of ovarian serous carcinoma, and propose a dualistic theory model for ovarian serous carcinogenesis. One pathway involves a stepwise progression from serours borderline tumor (SBT) to noninvasive and then invasive micropapillary serous carcinomas (MPSC). The other pathway is characterized by rapid progression from the ovarian surface epithelium or inclusion cysts to a conventional (high-grade) serous carcinoma. Despite considerable efforts aimed at elucidating the molecular mechanisms of ovarian serous carcinoma, its pathogenesis is still poorly understood and the dualistic theory model needs to be confirmed by moleclular genetics. In addition,we have focus on whether low and high grade serous carcinoma have a common moleclular base or not. Oncogenesis is a multistepwise process during which oncogenes are activated and the function of tumor suppressor genes is lost. Tumor suppressor genes play a important role in the process by down-regulation cell growth or promoting cell differentiation and death.ARHI is a maternally imprinted putative human tumor suppressor gene that maps to chromosome 1p31 and that encodes a 26-kDa small G protein with 60% homology to rap and ras. It inhibits cell growth, slows motility, prevents invasion, and induces apoptosis by signal transduction and cell cycle regulation. WWOX (WW domain-contaig oxidoreductase) gene, located on chromosome 16q 23.3-24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene. It can enhance many tumor suppressor genes and inhibit oncogenes.Many studies confirmed that loss expression of three tumor suppressor genes be very common in human carcinoma including ovarian cancer and breast caner. But the expression of three tumor suppressor genes in ovarian serous carcinoma, contribution to oncogenesis of it, and regulation mechanism were not explained.Ovarian epithelial carcinomas have a poor prognosis with high mortality. All the while, the studies on ovarian epithelial carcinomas prognosis were paied close attention by gynecology worker. Before, many studies about ovarian epithelial carcinomas prognosis were undertaked, which invoving in clinical stage, histological gade, pathological category, lymphatic metastasis, chemotherapy, and residual focal size. Owing to different histological types, it is difficult to more exactly evaluate ovarian epithelial carcinomas prognosis. At present, it has becamed a hot spot that searching for molecular targets which predicting prognosis besides traditional prognosis factors. The relationship between ARHI, WWOX, PTEN and prognosis of ovarian serous carcinoma was not reported.Objective The variance in ARHI, WWOX and PTEN expression were detected in ovarian serous benign tumor, borderline tumor and carcinoma with immunohistochemistry staining, RT-PCR, flow cytometry, and western blotting. The correlation between ARHI, WWOX, PTEN and STAT3, E2F1 were analysed. On the base, we explore that the rule of variance in ARHI, WWOX, PTEN expression and regulation mechanism in oncogenesis of ovarian serous carcinoma. Meanwhile, the prognosis factors were screen with Kaplan-Meier univariate survival analysis and COX multivariate survival analysis was used to determine the risk coefficient of each prognosis factor and different layers in each factor and analyze the relationship of ARHI expression and prognosis.Method1 Research object The 109 epithelial ovarian tumors investigated were collected from patients diagnosed between 2001 and 2003 at the Second Hospital of HeBei Medical University. 35 of the tumors were serous benign tumors, 18 of the tumors were serous borderline tumors, and 56 of the tumors were ovarian serous carcinomas with complete clinical information and follow-up data. 25 normal ovarian tissues were selected as control. Formalin-fixed and paraffin-embedded tissue blocks from all samples were used in this study with immunohistochemistry staining and survival analysis. 93 fresh ovarian tumor tissues were abtained from patients at The Fouth Hospital of HeBei Medical University. 32 of the tumors were serous benign tumors, 16 of the tumors were serous borderline tumors, and 45 of the tumors were ovarian serous carcinomas. 15 normal ovarian tissues were selected as control. All tissues were fresh-frozen and stored at -80℃for RT-PCR, FCM, and Western Blotting.2 ImmunohistochemistryThe protein expressin of ARHI, WWOX, PTEN, STAT3 and E2F1 were detected in 25 normal ovarian tissues and 109 ovarian serous tumors with immnohistochemisty ElivisionTM plus two-step. The immunocytochemistry procession was also carried out according to manufacturer’s instructions. Hematoxylin stsining, grad alcohol dehydration, dimethyl benzene transparence, neutral gum mounting, observed under light microscope. Negative control was carried out in identical condition excluding PBS insteading antibody.3 Flow cytometryThe fresh frozen tissues were fixed with 4% formaldehyde and The protein expressin of ARHI, WWOX, PTEN, STAT3 and E2F1 were detected with Flow cytometry according to manufacturer’s instructions.4 Protein extraction and quantitationThe fresh frozen tissues were splited in total cell lysates. The protein concentration was determined using Coomassie brilliant blue (CBB) reagent in spectrophotometer.5 Western BlottingFifty micrograms of protein were electrophoretically separated using a 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with primery monoclone antibodies and secondary monoclone antibodies. Immunoreactive bands were visualized using a ECL system. Band density was quantified using Snygene-Image Systems and normalized toβ-actin.6 RNA extraction, identify and quantitationTotal RNA from fresh ovarian serous tumors was isolated with guanidinium isothiocyanate (GITC). The integrity of total RNA was identfied at 90V on 1% agarose gels containing EB (0.5μg/ml). The UV Spectro- photometer was used for the quantitation of the total RNA.7 RT-PCRTotal RNA was reverse transcribed using reverse transcriptase with oligo dT at 43°C for 1.5 h to synthesize cDNA.Then the PCR was carried out in a complex mixture such as cDNA,dNTP,Taq DNA polymerase,primers and so on, denaturation for 5 mins, 94℃50s, 56℃30s, 72℃30s, 30 circulations . After amplification, each sample was analyzed by 1.5% agarose gel electrophoresis for 35 mins and visualized by ethidium bromide staining. The fragment for glyceraldehydes -3-phosphate dehydrogenase(GAPDH)cDNA gene was used as an internal control and the relative expression was quantitated as standardization on GAPDH mRNA content by BIO-LD densitometric image analyzer.8 Survival analysis of 56 ovarian serous carcinoma patiens by Kaplan-Meier univariate survival analysis and COX multivariate survival analysis56 ovarian serous carcinoma patiens with follow-up were analysed by Kaplan-Meier univariate survival analysis, the sum survival rates in 3 and 5 years were calculated by means of 36 months and 60 months as cut-off point. The correlation between tumor suppressor genes and prognosis was analysed, prognosic factors were screened, and the risk coefficient of independent factors were determined by COX multivariate survival analysis.9 Statistics analysisThe statistics analysises in the study were carried out by SPSS13.0 statistical package. The measurement datas were expressed as means±SD. x2 test, one-way ANOVA, SNK, and Spearman rank correlation were used for numeration and measurement datas respectively. Kaplan-Meier univariate survival analysis was used to calculate the survival rate; the difference was test by Log-Rank test. COX multivariate survival analysis was used to determine the risk coefficient of each prognosis factor and different layers in each factor.α=0.05.ReultsPart one Tumor suppressor gene ARHI,WWOX,and PTEN expression in ovarin seruous tumors1 ARHI mRNA and protein expression in ovarin seruous tumors1.1 ARHI mRNA expression in ovarin seruous tumorsThe result of RT-PCR showed that ARHI mRNA expression were detected in 28 of 32 ovarian serous benign tumors, 7 of 16 ovarian serous borderline tumors, and 18 of 45 ovarian serous carcinomas respectively. The positive expression rates of ARHI mRNA in ovarian serous carcinomas and ovarian serous borderline tumors were 40% and 43.8% respectively, significantly lower than that in ovarian serous benign tumors and normal ovaries (P<0.05), but the statistical differences were not found between borderline tumors and serous carcinomas, benign tumors and normal ovaries. Quantitative analysis of ARHI mRNA demonstrated that the relative quantities of ARHI mRNA were 0.12±0.06 in serous carcinomas, 0.15±0.04 in serous borderline tumors, statistically decreased than that in benign tumors and normal ovaries (P<0.05).1.2 ARHI protein expression in ovarin seruous tumors1.2.1 Western BlottingWestern Blotting showed that positive bands appeared in 26kd location for 26 of 32 benign tumors, 8 of 16 borderline tumors, 19 of 45 serous carcinomas. The positive expression rates of ARHI protein were 50%, 42.2% in borderline tumors and serous carcinomas respectively, obviously lower that in benign tumors and normal ovaries (P<0.05). Quantitative analysis of positive band density scanning diplay that the relative quantitiy of ARHI protein in serous carcinomas was 0.17±0.12, significantly decreased than that in borderline tumors, benign tumors, and normal ovaris (P<0.05).1.2.2 FCMThe FCM revealed that the FI in serous carcinomas and borderline tumors were 0.894±0.008 and 0.914±0.06 respectively, obviously decreased compare to benign tumors and normal ovaries.1.2.3 ImmunohistochemistryThe result of ICH showed that ARHI protein expression was detected in 22 serous carcinomas, 10 borderline tumors, and 27 benign tumors samples. The positve rates in serous carcinomas and borderline tumors were 39.3% and 55.6%, significantly lower than that in benign tumors and normal ovaries (P<0.05), but the statistical differences were not found between borderline tumors and serous carcinomas, benign tumors and normal ovaries.2 WWOX mRNA and protein expression in ovarin seruous tumors2.1 WWOX mRNA expression in ovarin seruous tumors The result of RT-PCR showed that WWOX mRNA expression were detected in 29 of 32 ovarian serous benign tumors, 12 of 16 ovarian serous borderline tumors, and 21 of 45 ovarian serous carcinomas respectively. The positive expression rates of WWOX mRNA in ovarian serous carcinomas was 46.7%, significantly lower than that in ovarian serous borderline tumors, benign tumors and normal ovaries (P<0.05). But the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries. Quantitative analysis of WWOX mRNA demonstrated that the relative quantities were 0.25±0.08 in serous carcinomas, statistically decreased compare to borderline tumors, benign tumors and normal ovaries (P<0.05).2.2 WWOX protein expression in ovarin seruous tumors2.2.1 Western BlottingWestern Blotting showed that the positive expression rates of WWOX protein in serous carcinomas was 44.4%, significantly lower than 68.8% in borderline tumors, 78.1% in benign tumors, and 86.7% in normal ovaries (P<0.05). Quantitative analysis of positive band density scanning diplaied that the relative quantitiy in serous carcinomas was 0.11±0.04, significantly decreased compare to borderline tumors, benign tumors, and normal ovaris (P<0.05).2.2.2 FCMThe FCM revealed that the FI in serous carcinomas was 0.930±0.02, obviously decreased compare to borderline tumors, benign tumors, and normal ovaries.2.2.3 ImmunohistochemistryThe result of IHC showed that WWOX protein expression was detected in 27 serous carcinomas, 16 borderline tumors, and 30 benign tumors samples. The positve rates in serous carcinomas was 48.2%, significantly lower than that in borderline tumors, benign tumors, and normal ovaries (P<0.05), but the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries.3 PTEN mRNA and protein expression in ovarin seruous tumors 3.1 PTEN mRNA expression in ovarin seruous tumorsThe positive expression rates of PTEN mRNA in ovarian serous carcinomas was 51.1%, significantly lower than 81.3% in borderline tumors, 90.6% in benign tumors, and 100% in normal ovaries (P<0.05). But the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries. Quantitative analysis of PTEN mRNA demonstrated that the relative quantities were 0.31±0.08 in serous carcinomas, statistically decreased compare to borderline tumors, benign tumors and normal ovaries (P<0.05).3.2 PTEN protein expression in ovarin seruous tumors3.2.1 Western BlottingWestern Blotting showed that the positive expression rates of PTEN protein in serous carcinomas was 53.3%, significantly lower than 75.0% in borderline tumors, 84.4% in benign tumors, and 93.3% in normal ovaries (P<0.05). Quantitative analysis of positive band density scanning diplaied that the relative quantitiy in serous carcinomas was 0.34±0.12, significantly decreased compare to borderline tumors, benign tumors, and normal ovaris (P<0.05).3.2.2 FCMThe FCM revealed that the FI in serous carcinomas was 0.908±0.023, obviously decreased compare to borderline tumors, benign tumors, and normal ovaries.3.2.3 ImmunohistochemistryThe result of IHC showed that PTEN protein expression was detected in 29 serous carcinomas, 15 borderline tumors, and 31 benign tumors samples. The positve rates in serous carcinomas was 51.8%, significantly lower than that 83.3% in borderline tumors, 88.6% in benign tumors, and 100% in normal ovaries (P<0.05), but the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries.4 Protein expressions of ARHI, WWOX, and PTEN in low grade and high grade ovarian serous carcinoma The results showed that the differnce of protein expressions of ARHI was not found between low and high grade. But the positive rates of WWOX and PTEN protein expressions in high grade were significantly lower than that in low grade (P<0.05).Part two Expression of STAT3 and E2F1 protein in ovarian serous tumors1 Expression of STAT3 protein in ovarian serous tumors1.1 ImmunohistochemistryThe result of ICH showed that the positve rates in serous carcinomas and borderline tumors were 87.5% (49/56) and 77.8% (14/18), significantly higher than that in benign tumors and normal ovaries (P<0.05), but the statistical differences were not found between borderline tumors and serous carcinomas, benign tumors and normal ovaries.1.2 FCMThe FCM revealed that the FI in serous carcinomas was 1.192±0.031, obviously increased compare to borderline tumors, benign tumors, and normal ovaries.2 Expression of E2F1 protein in ovarian serous tumors2.1 ImmunohistochemistryThe result of ICH showed that the positve rates in serous carcinomas was 82.1%, significantly higher than that in borderline tumors, benign tumors, and normal ovaries (P<0.05), but the statistical differences were not found among borderline tumors, benign tumors, and normal ovaries.2.2 FCMThe FCM revealed that the FI in serous carcinomas was 1.139±0.023, obviously increased compare to borderline tumors, benign tumors, and normal ovaries.3 The relationship between STAT3, E2F1 and clinicpathological characteristics in serous carcinomasThe statistical differences of STAT3, E2F1 protein expression were not found in different grades, stages, lymph node metastasis, and peritoneum metastasis.4 The correlation between ARHI protein and STAT3, E2F1 protein in in serous carcinomasStatistically, negtive correlations were found between ARHI protein expression and STAT3, E2F1 protein expression in serous carcinomas.5 The correlation between WWOX protein and STAT3, E2F1 protein in in serous carcinomasStatistically, negtive correlations were found between WWOX protein expression and STAT3, E2F1 protein expression in serous carcinomas.6 The correlation between PTEN protein and STAT3, E2F1 protein in in serous carcinomasStatistically, negtive correlations were not found between PTEN protein expression and STAT3, E2F1 protein expression in serous carcinomas.Part three The effect of ARHI, WWOX and PTEN protein expression on prognosis in ovarain serous carcinomas1 Correlation between ARHI protein expression and prognosis, clinicpathological characteristics in ovarain serous carcinomasThe result of Kaplan-Meier univariate survival analysis showed that the expression of ARHI was a related factor of prognosis in ovarian serous carcinoma patients. The survival rates of 3 and 5 years were 68.2% and 54.5% in ovarian serous carcinoma patients with ARHI positive expression, significantly higher than 32.4% and 17.6% in patients with ARHI negitive expression.The statistical differences of ARHI protein expression were not found in different grades, stages, lymph node metastasis, and peritoneum metastasis.2 Correlation between WWOX protein expression and prognosis, clinicpathological characteristics in ovarain serous carcinomasThe result of Kaplan-Meier univariate survival analysis showed that the expression of WWOX was a related factor of prognosis in ovarian serous carcinoma patients. The survival rates of 3 and 5 years were 63.0% and 44.4% in ovarian serous carcinoma patients with WWOX positive expression, significantly higher than 31.0% and 20.7% in patients with WWOX negitive expression.The statistical differences of WWOX protein expression were found in different grades, but not in lymph node metastasis, peritoneum metastasis, and stages.3 Correlation between PTEN protein expression and prognosis, clinicpathological characteristics in ovarain serous carcinomasThe result of Kaplan-Meier univariate survival analysis showed that the expression of PTEN was a related factor of prognosis in ovarian serous carcinoma patients. The survival rates of 3 and 5 years were 62.1% and 44.8% in ovarian serous carcinoma patients with PTEN positive expression, significantly higher than 29.6% and 18.5% in patients with PTEN negitive expression.The statistical differences of PTEN protein expression were found in different grades, but not in lymph node metastasis, peritoneum metastasis, and stages.4 Survival analyses of ovarain serous carcinoma patients4.1 Screening relative prognosis factors of ovarain serous carcinomaKaplan-Meier univariate survival analysis showed clinical stage, pathology grades, residual disease and chemotherapy had effects on survival, were prognosis factors of patients with serous ovarian carcinoma.4.2 COX multivariate survival analysis of ovarain serous carcinoma patientsCOX multivariate survival analysis showed that clinical stage was the important independent prognosis factor, followed by pathology grades, chemotherapy, and ARHI expression (the independent risk coefficients were 2.810, 1.966, 0.547 and 0.428 respectively), but residual disease, WWOX, and PTEN expression were not.Conclusion1 Protein and mRNA expression of ARHI, WWOX and PTEN were decreased in ovarain serous carcinoma, suggesting that loss of function of tumor suppressor gene may contribute to oncogenesis of ovarian serous carcinoma.2 Proteins expression of STAT3 and E2F1 in ovarain serous carcinoma were increased, suggesting that E2F1 overexpression inhibited ARHI expression and down-regulation of STAT3 by ARHI were losed and many down stream genes promoting proliferation and inducing apoptosis were actived.3 The differnces of tumor suppressor genes exppression between low and high grade confirmed a dualistic theory model for ovarian serous carcinogenesis.4 Protein and mRNA expression of ARHI was significantly decreased compared to WWOX and PTEN, suggestting that ARHI may play a main role in oncogenesis of ovarian serous carcinoma.5 Beside conventional prognosis factors, expression of ARHI, WWOX and PTEN had effects on survival of ovarain serous carcinoma patients, moreover ARHI protein expression was an independant prognosis factor.6 It was very useful for more exactly evaluating prognosis and guiding efficient therapy that analyzing independent prognosis factors including ARHI, WWOX, and PTEN expression.更多还原