PRMT5对上皮性卵巢癌生物学行为影响的实验研究
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摘要
卵巢癌是女性常见生殖系统肿瘤,病死率居妇科恶性肿瘤首位。超过80%的卵巢癌为上皮性来源(Epithelial Ovarian Cancer, EOC),其侵袭性高,对化疗反应差,常导致患者不良预后。近30年来,虽然手术技巧不断提高,新的有效化疗药物也不断更新,但上皮性卵巢癌的5年生存率也只有从36%提高到44%。因此研究卵巢癌特别是上皮性卵巢癌的发生、发展机制,从分子水平上寻找合理有效的治疗靶点,是提高卵巢癌生存率、延长生存期的关键。
蛋白质的精氨酸甲基化广泛存在于生物体内,发挥许多重要的作用,如参与转录后调节、RNA加工、细胞增殖、分化、凋亡和肿瘤形成等,而催化此反应的酶称做蛋白质精氨酸甲基转移酶(Protein Arginine Methyltransferase, PRMTs)。在人类中,到目前为止已经鉴定出11种该家族的成员(PRMT1-11),除了PRMT2, PRMT10和PRMT11,该家族的其他成员均具有酶和催化精氨酸甲基化的活性。根据其催化精氨酸甲基化方式的不同,PRMTs家族主要分为2型,I型：包括PRMT1、PRMT3、PRMT4、PRMT6和PRMT8,催化形成单甲基(MMA)和不对称双甲基(aDMA); Ⅱ型：催化形成单甲基(MMA)和对称双甲基(sDMA),包括PRMT5、PRMT7和PRMT9。
精氨酸甲基转移酶5(PRMT5)是Ⅱ型中最早鉴定出的成员,位于人类染色体14q11.2,有2个亚型,分别长620个和637个氨基酸。PRMT5(?)具有重要的生物学作用,包括核糖体的生物合成,高尔基体的组装,生殖细胞特化,细胞分化,增殖和凋亡等。另外(?)PRMT5也能与其他基因相互作用,如周期蛋白cyclin E1、肿瘤抑制基因ST7和NM23、P53、程序性细胞死亡蛋白PDCD4、转录因子E2F-1和E-cadherin(?)等,参与了多种细胞内的生物过程,调控细胞的生长增殖,凋亡及侵袭转移过程。在人类的多种疾病中存在PRMT5的异常表达,特别是在一些肿瘤中,包括白血病和淋巴瘤,胃癌,乳腺癌、结直肠癌和肺癌等,而且研究也发现PRMT5与乳腺癌和结直肠癌患者的预后密切相关。总之越来越多的研究表明PRMT5参与了人类癌症的发生。
然而,关于PRMT5在卵巢肿瘤发生发展过程中的作用还未知,因此本研究拟对PRMT5在卵巢癌中的作用进行探讨研究。
第一部分：PRMT5在卵巢癌组织中的表达及其意义
研究目的：
1、检测并分析PRMT5在正常卵巢上皮、卵巢上皮性良性、交界性及恶性肿瘤中的表达是否存在差异。
2、分析PRMT5表达与卵巢癌不同临床病理特征如病理类型、组织分化、临床分期、淋巴结转移、术后病灶残留等之间的关系。
3、分别检测卵巢癌同一手术标本中PRMT5和Ki-67的表达,分析两者之间是否有相关性,以评价PRMT5在卵巢癌细胞增殖中的作用。
4、分析PRMT5(?)的表达与卵巢癌患者预后的关系。
研究方法：
1.收集2005.1-2008.12山东大学齐鲁医院妇科住院治疗的正常卵巢组织12例、卵巢上皮性良性肿瘤20例,交界性肿瘤14例及卵巢癌118例的手术标本,并收集患者的完整临床及术后随访资料,本研究获山东大学齐鲁医院伦理委员会批准并得患者知情同意。
2.应用免疫组化SP法检测不同标本组织中的PRMT5和Ki-67的表达。
3.应用SPSS13.0软件对数据进行分析,卡方检验或者Fisher's精确检验分析PRMT5在不同卵巢组织、不同临床病理指标中的表达差异；Spearman检验法分析PRMT5和Ki-67表达相关性：单因素和多因素Cox回归分析卵巢癌患者的预后影响因素；Kaplan-Meier法绘制患者的生存曲线,Log-rank检验比较患者的生存率差别。
结果：
1. PRMT5的阳性表达主要位于细胞核和细胞浆中,在卵巢癌中的高表达率为83.1%(98/118),显著高于正常卵巢组织的高表达率33．3%(4/12,P<0.05)及卵巢良性肿瘤中的高表达率30%(6/20,P<0.05)；与交界性肿瘤中的高表达率64.3%(9/14)相比,无统计学差异。
2. PRMT5在卵巢癌浆液性病理类型、临床晚期(Ⅲ/Ⅳ)、低分化(G3)、有淋巴结转移和术后有病灶残留的组织切片中高表达(P均<0.05)。
3.在Ki-67高表达的55例中,有54例高表达PRMT5(98.2%),在Ki-67低表达的63例中,有44例高表达PRMT5(69.2%), PRMT5在两组中的比较具有显著统计学差异。PRMT5与Ki-67的表达呈显著正相关性(r=0.377,P<0.001)。
4.118例卵巢癌患者截止到随访结束,中位总生存时间为40.0个月,95%CI：19.1-60.1个月,中位无进展生存时间为20.0个月,95%CI：16.8-23.2个月。Kaplan-Meier (?)生存曲线显示PRMT5高表达组与低表达相比,其总生存率(Overall Survival, OS)和无进展生存率(Progression-free Survival, PFS)均明显降低(Log-rank检验,P均<0.001)。单因素Cox生存分析结果显示：在OS分析中,卵巢癌的浆液性病理类型、年龄≥60岁、临床分期Ⅲ/Ⅳ、肿瘤低分化(G3)和PRMT5的高表达等因素对患者的OS是不利因素(P均<0.05)。而在PFS中,除了淋巴结转移外其余的临床病理特征及Ki.67高表达均为患者PFS的不利因素(P均<0.05)。
5.Cox多因素回归分析中,PRMT5高表达、年龄≥60岁,临床Ⅲ/Ⅳ期对OS是独立的不良预后指标(P均<0.05)；而PRMT5高表达、残留病灶存在、临床Ⅲ/Ⅳ期为PFS独立的不良预后因素,能显著降低患者的PFS(P均<0.05)。
结论：
1. PRMT5在上皮性卵巢良性、交界性及恶性肿瘤中的表达逐渐升高,且PRMT5的高表达与卵巢癌的不良临床病理特征相关,说明PRMT5参与了卵巢肿瘤的发生发展过程。
2. PRMT5与Ki-67的表达呈显著正相关,说明PRMT5可能参与了卵巢癌细胞的生长和增殖过程。
3.单因素和多因素的生存分析结果显示PRMT5对于卵巢癌的预后是一个独立的因素,高表达PRMT5能显著降低患者的OS和PFS。
第二部分：靶向沉默PRMT5的表达对卵巢癌细胞系生物学行为的影响及机制研究
研究目的：
通过转染靶向PRMT5的siRNA来抑制卵巢癌细胞系A2780和SKOV3I中PRMT5f的表达,观察靶向降调其表达对卵巢癌细胞系生物学行为如生长增殖、凋亡和侵袭转移等能力的影响,探讨PRMT5在卵巢癌分子发病中的作用机制,为卵巢癌的基因治疗提供理论依据。
研究方法：
1.设计并体外化学合成靶向PRMT5(?)的2条siRNA序列(siP1和siP2)及阴性对照siRNA序列(siC),并在脂质体LipofectamineTM2000(?)的介异下转染进入卵巢癌细胞系A2780和SKOV3。
2.应用Real-time PCR和Western blot技术分别检测卵巢癌细胞系转染48小时后的PRMT5mRNA和转染72小时后的PRMT5蛋白的变化,筛选出具有最佳干扰效果的siRN八序列,作为后续研究的序列。
3.应用CCK8、BrdU(?)参入和Ki-67标记实验分别检测转染PRMT5-siRNA后的卵巢癌细胞系的生长和增殖能力的变化。
4. Annexin V-FITC/PI双染法流式细胞仪检测转染PRMT5-siRNA后卵巢癌细胞系凋亡的变化。
5.应用Transwell小室技术,检测转染PRMT5-siRNA后卵巢癌细胞系迁移和侵袭能力的变化。
6. Western blot技术检测PRMT5表达抑制后E2F-1、cIF4E和E-cadherin蛋白的
变化。
7. SPSS13.0统计软件对数据进行分析。计量资料用平均值±标准差表示,组间差异应用独立样本的t检验分析。
结果：
1.靶向PRMT5的两条siRNA序列和阴性对照序列均成功转染卵巢癌细胞A2780和SKOV3,转染效率90%以上。
2. Real-time PCR和Western blot实验结果均显示：siP1(?)序列能够显著减少A2780和SKOV3中PRMT5的mRNA和蛋白的表达；而siP2(?)序列只能减少SKOV3细胞中的PRMT5的表达,而对A2780的作用微弱。综合实验结果,siP1序列能够显著抑制A2780和SKOV3细胞中PRMT5的表达,筛选作为后续实验的干扰序列。
3.在CCK8实验中,转染后72小时始,siP1组与siC组和正常未转染组(Normal)相比,细胞生存率开始明显降低,且随着时间延长,生存率降低更明显,两种细胞系的结果类似。观察至120小时,A2780-siP1组生存率约24.4±0.9%,SKOV3-siP1组生存率约36.9±1.7%,均明显低于siC组(P均<0.001)。
4.A2780细胞中,siP1组的BrdU阳性率明显低于siC组(11.7±1.5%vs.33.3±1.5%,P<0.001)：SKOV3细胞中,siP1组的BrdU阳性率亦明显低于siC组(18.0±2.0%vs.35.3±1.5%,P<0.001)。
5.在A2780细胞中,siP1组与siC组相比,Ki.67的标记指数(LI)明显下降(12.4±3_3%vs.50.2±8.0%,P<0.001)；在SKOV3细胞中,在siPl组和siC组中的LI分别为2.3±1.0%和23.6±6.6%,siP1组有明显降低(P<0.001)。
6.A2780细胞中,siP1组明显高于siC组的凋亡率(29.4±0.9%vs.16.3±0.8%,P<0.001)：SKOV3中,siP1组亦明显高于siC组的凋亡率(28.9±0.6%vs.14.1±1.4%,P<0.001)。
7. Transwell体外迁移和侵袭实验均表明：在A2780和SKOV3细胞中,siP1组的穿膜细胞数均明显少于siC组和Normal组,差异有统计学意义(P分别<0.001)。
8.在转染后72小时,Western blot实验显示：A2780和SKOV3细胞中的E2F-1蛋白和E-cadherin蛋白表达明显上调。
结论：
1. PRMT5参与了卵巢癌细胞的增殖、凋亡、迁移和侵袭的过程,干扰其表达能够显著减慢细胞的增殖,促进细胞的凋亡和减弱其迁移和侵袭的能力。说明PRMT5在卵巢癌的发生发展中有着重要的作用,同时也说明PRMT5有可能成为卵巢癌基因治疗的候选基因。
2.PRMT5通过调控E2F-1的表达参与卵巢癌细胞增殖凋亡过程,调控E-cadherin(?)内表达参与卵巢癌细胞的迁移和侵袭过程。
Ovarian cancer is one of the leading causes of death from gynecological malignancies. Over80%of ovarian cancers are of epithelial origin (EOC), which show highly invasive, respond poorly to chemotherapies and are usually detected at advanced stages, resulting in poor prognosis. Despite the improvement of surgical techniques and the advent of more effective therapeutics, the overall5-year survival of epithelial ovarian cancer has increased only from36%to44%during the last three decades. To improve better prognostic and predictive assays and develop new therapeutic strategies for HOC, understanding the molecular mechanisms underlying HOC is critical.
Arginine methylation is becoming an important type of posttranslational modification and is widely appreciated, playing a vital role in the regulation of diverse cellular procession that range from transcription and RNA procession to signaling, differentiation, apoptosis and tumorigenesis. The methylation of arginine residues is catalyzed by the protein arginine methyltransferase(PRMTs) family of enzymes. To date, PRMTs have been found11members in human, most of PRMTs except for PRMT2, PRMT10, and PRMT11have been shown to have enzymatic activity and can catalyze arginine methylation. Based on the type of modification they catalyze, PRMTs are mainly classified as type1and type11enzymes. All these two enzymes catalyze the formation of a mono-methylated (MMA) intermediate, subsequently, type I PRMTs (PRMT1,3,4,6and8) further catalyze the production of asymmetric dimethylation of arginine residues (aDMA), and type II PRMTs (PRMT5,7and PRMT9) catalyze the formation of symmetric dimethylation of arginine residues (sDMA).
Protein arginine methyltransferase5(PRMT5) the first identified type II enzyme, is in human chromosome14q11.2and has two isoforms with637and620amino acids in length. It is involved in a variety of biological processes including ribosome biogenesis, assembly of the Golgi apparatus, cellular differentiation, germ cell specification, cellular proliferation, and apoptosis. In addition, it has been shown to interact with many other genes, such as cyclin E1, ST7and NM23, p53, E2F-1, PDCD4and E-cadherin to participate many cell processes. In human, aberrant expression of PRMT5are found in a wide variety of human diseases, especially in many cancers, including leukemia and lymphoma, breast cancer, colorectal cancer and lung cancer. Moreover, it is relevant to poor outcome in breast cancer and colorectal cancer patients.
However, little is known about the role of PRMT5in ovarian cancer. In this study, we investigated the role of PRMT5in epithelial ovarian cancer.
Part I:PRMT5is Overexpression in Epithelial Ovarian Cancers and is Associated with Poor Disease Prognosis
Objective:
1. To detect the difference of PRMT5expression in normal epithelium cells, epithelial ovarian benign tumors, borderline tumors and ovarian cancers.
2. To valuate the relationship between PRMT5overexpression and clinicopathologic features of the patients.
3. To examine the same set of EOC specimens with Ki-67, and investigate the relationship between PRMT5and Ki-67.
4. Evaluate the relevance of PRMT5expression to the prognosis of ovarian cancer patients.
Methods:
1. EOCs (n=118), epithelial ovarian benign tumors (n=20), borderline tumors (n=14) and normal ovaries (n=12) samples were collected from the Department of Gynecology, Qilu Hospital of Shandong University between January2005and December2008. The clinical and follow-up data of ovarian cancer patients wre collected. The study was approved by the Institutional Medical Ethics Committee of Qilu Hospital of Shandong University and informed consent were obtained by patients.
2. PRMT5and Ki-67expression were examined by immunohistochemistry (IHC) of standard streptavidin-peroxidase complex method.
3. All statistical analyses were performed with SPSS13.0statistical software. IHC data were analyzed using Chi-square test or Fisher's exact test. The Spearman test was used to analyze the relationship between PRMT5and Ki-67scores. Univariate and multivariate Cox regression analyses were used to evaluate the prognostic significance of PRMT5and other variables of ROC. Kaplan-Meier method was used to calculate the survival curves, and Log-rank test was used to compare the overall survival and progression-free survival between patient subgroups.
Results:
1. The positive staining of PRMT5was observed both in nuclear and cytoplasm of cells. High expression of PRMT5was observed in83.1%(98/118) of EOCs, in33.3%(4/12)of normal ovaries, in30%(6/20) of benign tumors, in64.3%(9/14) of borderline tumors. The level of PRMT5in EOCs was higher than normal ovaries and benign tumors (P<0.05, respectively), and there was no significant difference between borderline tumors and ovarian cancers.
2. High expression of PRMT5was significantly associated with serous subtype, advanced FIGO stage(Ⅲ/Ⅳ), poor differentiation(G3), lymph node invasion, and presence of residual tumor (P<0.05, respectively).
3. There was a significant positive correlation between PRMT5and Ki-67expression (r=0.377, P<0.001) in ovarian cancers, in which tumors with high Ki-67expression had a significantly higher expression of PRMT5(69.2%vs.98.2%, P<0.001).
4. At the end of the follow-up period, of the118ovarian cancer patients, the median OS time was40months (95%CI:19.1-60.1), and median PFS time was20months (95%CI:16.8-23.2).
5. In Univariate Cox analyses, serous subtype, high PRMT5expression, older age (>60years), advanced FIGO stage (Ⅲ/Ⅳ), and poor tumor differentiation (G3) were unfavorable predictors of OS (P<0.05, respectively), and all clinico-pathological variables, except lymph node invasion, were unfavorable predictors of PFS (P<0.05, respectively). Kaplan-Meier survival curves indicated that patients with high PRMT5expression had a significantly poor OS and PFS than those with low PRMT5expression (Log-rank test, P<0.001, respectively).
6. In multivariate Cox analysis, the high expression of PRMT5, older age and advanced FIGO stage retained their significance as independent prognostic factors of poorer OS and the high expression of PRMT5, advanced FIGO stage and presence of residual tumor were found to be independent prognostic factors for shorter PFS.
Conclusions:
1. The expression of PRMT5was increased gradually between ovarian benign, borderline and malignant tumors and overexpression of PRMT5was associated with poor clinicopathologic features of the patients. These results demonstrate that PRMT5may play a crucial role as an oncogene in the development of EOC.
2. The expression of PRMT5was positively correlated with the expression of Ki-67, which indicated that PRMT5may be involved in the regulation of ovarian cancer growth and proliferation.
3. In univariate and multivariate survival analyses, high level of PRMT5was identified as an independent factor for poor prognosis in patients with EOC.
Part II:Effects of PRMT5-silencing on Proliferation, Apoptosis, Migration and Invasion of Ovarian Cancer Cells
Objective:
To investigate the effects of PRMT5-siRNA-mediated gene silencing on proliferation, apoptosis, migration and invasion of ovarian cancer cell lines A2780and SKOV3. To explore the role of PRMT5in ovarian cancer cells in vitro, and to develop therapeutic potential of PRMT5in ovarian cancer.
Methods:
1. The two human PRMT5siRNA sequences siP1and siP2, control-siRNA(siC) were designed and chemically synthesize. They were transfected into ovarian cancer cell lines A2780and SKOV3with LipofectamineTM2000.
2. Silencing of PRMT5was assayed for mRNA and protein expression level at48hr and72hr after transfection, respectively. The most efficient and specific sequence to silence the expression of PRMT5in two cells was screened.
3. CCK8, BrdU incorporation and Ki-67labelling assays were used to detect the proliferation activity of ovarian cancer cells after PRMT5-siRNA was transfection.
4. Annexin V-FITC/PI staining by How cytometry analysis was used to detect the apoptosis of ovarian cancer cells after PRM15-siRNA was transfection.
5. Transwell assay was employed to test ovarian cancer cell migration and invasion after PRMT5-siRNA was transfection.
6. E2F-1,eIF4E and E-cadherin protein were detected by Western blot assay after PRMT5-siRNA was transfection.
7. Quantitative data were expressed as means±SD, the significance of the difference between groups was evaluated with independent-samples t test. All calculations were performed using SPSS13.0statistical software.
Results:
1. Two PRMT5siRNA (siP1and siP2) and scrambled control (siC) were respectively and successfully transfected into ovarian cancer cell line, and the efficiency of transfection was up to90%.
2. Compared with normal control or cells transfected with siC, siP1could reduce PRMT5mRNA and protein in A2780and SKOV3cells significantly; siP2could reduce PRMT5expression in SKOV3, but the silence effect of siP2in A2780was weak. According to the results, siPl was the most efficient and specific sequence to silence the expression of PRMT5in two cell lines.
3.72hr after transfection, the cell viability started to show lower in the cells transfected with siPl as compared with the cells transfected with siC and normal control (P<0.05). Similar results were observed in both A2780and SKOV3cells lines. The inhibition of growth was in a time-dependent manner.120hr after transfection, the cell viability of siPl group was24.4±0.9%in A2780, and was36.9±1.7%in SKOV3, both of them were significantly lower than the cell viability in siC group (P<0.001, respectively)
4.48hr after transfection, compared to control sample that transfected with siC, cells treated with siPl showed significantly decreased BrdU incorporation,11.7±1.5%vs.33.3±1.5%in A2780, and18.0±2.0%vs.35.3±1.5%in SKOV3(P<0.05, respectively).
5.72hr after transfection, compared to siC, cells transfected with siPl showed significantly decreased positive percentage of Ki-67,12.4±3.3%vs.50.2±8.0%in A2780,2.3±1.0%vs.23.6±6.6%in SKOV3(P<0.001, respectively).
6.72hr after transfection, the percentage of apoptosis cells in siPl cells was higher than that in siC cells (29.4±0.9%vs.16.3±0.8%, P<0.001) in A2780; the percentage of apoptosis was28.9±0.6%vs.14.1±1.4%(P<0.001) in SKOV3.
7. Both tumor cell migration and invasion assays indicated that transfection of PRMT5-siRNA could reduce the migration and invasion power of ovarian cancer cells. The number of migration/invasion cells with siPl group was lower than that with siC and normal control (P<0.001, respectively).
8. E2F-1and E-cadherin protein were increased after PRMT5-siRNA transfection in ovarian cancer cells.
Conclusions:
1. PRMT5could significantly promote the proliferation, migration and invasion, inhibit the apoptosis of ovarian cancer cell line, suggesting that PRMT5could play an important role in the tumorigenesis and development of ovarian cancer. PRMT5may be a novel therapeutic target for epithelial ovarian cancer.
2. PRMT-siRNA could inhibit the proliferation, induce apoptosis via up-regulation E2F-1and reduce the migration/invasion ability via up-regulation E-cadherin in ovarian cancer.
蛋白质的精氨酸甲基化广泛存在于生物体内,发挥许多重要的作用,如参与转录后调节、RNA加工、细胞增殖、分化、凋亡和肿瘤形成等,而催化此反应的酶称做蛋白质精氨酸甲基转移酶(Protein Arginine Methyltransferase, PRMTs)。在人类中,到目前为止已经鉴定出11种该家族的成员(PRMT1-11),除了PRMT2, PRMT10和PRMT11,该家族的其他成员均具有酶和催化精氨酸甲基化的活性。根据其催化精氨酸甲基化方式的不同,PRMTs家族主要分为2型,I型：包括PRMT1、PRMT3、PRMT4、PRMT6和PRMT8,催化形成单甲基(MMA)和不对称双甲基(aDMA); Ⅱ型：催化形成单甲基(MMA)和对称双甲基(sDMA),包括PRMT5、PRMT7和PRMT9。
精氨酸甲基转移酶5(PRMT5)是Ⅱ型中最早鉴定出的成员,位于人类染色体14q11.2,有2个亚型,分别长620个和637个氨基酸。PRMT5(?)具有重要的生物学作用,包括核糖体的生物合成,高尔基体的组装,生殖细胞特化,细胞分化,增殖和凋亡等。另外(?)PRMT5也能与其他基因相互作用,如周期蛋白cyclin E1、肿瘤抑制基因ST7和NM23、P53、程序性细胞死亡蛋白PDCD4、转录因子E2F-1和E-cadherin(?)等,参与了多种细胞内的生物过程,调控细胞的生长增殖,凋亡及侵袭转移过程。在人类的多种疾病中存在PRMT5的异常表达,特别是在一些肿瘤中,包括白血病和淋巴瘤,胃癌,乳腺癌、结直肠癌和肺癌等,而且研究也发现PRMT5与乳腺癌和结直肠癌患者的预后密切相关。总之越来越多的研究表明PRMT5参与了人类癌症的发生。
然而,关于PRMT5在卵巢肿瘤发生发展过程中的作用还未知,因此本研究拟对PRMT5在卵巢癌中的作用进行探讨研究。
第一部分：PRMT5在卵巢癌组织中的表达及其意义
研究目的：
1、检测并分析PRMT5在正常卵巢上皮、卵巢上皮性良性、交界性及恶性肿瘤中的表达是否存在差异。
2、分析PRMT5表达与卵巢癌不同临床病理特征如病理类型、组织分化、临床分期、淋巴结转移、术后病灶残留等之间的关系。
3、分别检测卵巢癌同一手术标本中PRMT5和Ki-67的表达,分析两者之间是否有相关性,以评价PRMT5在卵巢癌细胞增殖中的作用。
4、分析PRMT5(?)的表达与卵巢癌患者预后的关系。
研究方法：
1.收集2005.1-2008.12山东大学齐鲁医院妇科住院治疗的正常卵巢组织12例、卵巢上皮性良性肿瘤20例,交界性肿瘤14例及卵巢癌118例的手术标本,并收集患者的完整临床及术后随访资料,本研究获山东大学齐鲁医院伦理委员会批准并得患者知情同意。
2.应用免疫组化SP法检测不同标本组织中的PRMT5和Ki-67的表达。
3.应用SPSS13.0软件对数据进行分析,卡方检验或者Fisher's精确检验分析PRMT5在不同卵巢组织、不同临床病理指标中的表达差异；Spearman检验法分析PRMT5和Ki-67表达相关性：单因素和多因素Cox回归分析卵巢癌患者的预后影响因素；Kaplan-Meier法绘制患者的生存曲线,Log-rank检验比较患者的生存率差别。
结果：
1. PRMT5的阳性表达主要位于细胞核和细胞浆中,在卵巢癌中的高表达率为83.1%(98/118),显著高于正常卵巢组织的高表达率33．3%(4/12,P<0.05)及卵巢良性肿瘤中的高表达率30%(6/20,P<0.05)；与交界性肿瘤中的高表达率64.3%(9/14)相比,无统计学差异。
2. PRMT5在卵巢癌浆液性病理类型、临床晚期(Ⅲ/Ⅳ)、低分化(G3)、有淋巴结转移和术后有病灶残留的组织切片中高表达(P均<0.05)。
3.在Ki-67高表达的55例中,有54例高表达PRMT5(98.2%),在Ki-67低表达的63例中,有44例高表达PRMT5(69.2%), PRMT5在两组中的比较具有显著统计学差异。PRMT5与Ki-67的表达呈显著正相关性(r=0.377,P<0.001)。
4.118例卵巢癌患者截止到随访结束,中位总生存时间为40.0个月,95%CI：19.1-60.1个月,中位无进展生存时间为20.0个月,95%CI：16.8-23.2个月。Kaplan-Meier (?)生存曲线显示PRMT5高表达组与低表达相比,其总生存率(Overall Survival, OS)和无进展生存率(Progression-free Survival, PFS)均明显降低(Log-rank检验,P均<0.001)。单因素Cox生存分析结果显示：在OS分析中,卵巢癌的浆液性病理类型、年龄≥60岁、临床分期Ⅲ/Ⅳ、肿瘤低分化(G3)和PRMT5的高表达等因素对患者的OS是不利因素(P均<0.05)。而在PFS中,除了淋巴结转移外其余的临床病理特征及Ki.67高表达均为患者PFS的不利因素(P均<0.05)。
5.Cox多因素回归分析中,PRMT5高表达、年龄≥60岁,临床Ⅲ/Ⅳ期对OS是独立的不良预后指标(P均<0.05)；而PRMT5高表达、残留病灶存在、临床Ⅲ/Ⅳ期为PFS独立的不良预后因素,能显著降低患者的PFS(P均<0.05)。
结论：
1. PRMT5在上皮性卵巢良性、交界性及恶性肿瘤中的表达逐渐升高,且PRMT5的高表达与卵巢癌的不良临床病理特征相关,说明PRMT5参与了卵巢肿瘤的发生发展过程。
2. PRMT5与Ki-67的表达呈显著正相关,说明PRMT5可能参与了卵巢癌细胞的生长和增殖过程。
3.单因素和多因素的生存分析结果显示PRMT5对于卵巢癌的预后是一个独立的因素,高表达PRMT5能显著降低患者的OS和PFS。
第二部分：靶向沉默PRMT5的表达对卵巢癌细胞系生物学行为的影响及机制研究
研究目的：
通过转染靶向PRMT5的siRNA来抑制卵巢癌细胞系A2780和SKOV3I中PRMT5f的表达,观察靶向降调其表达对卵巢癌细胞系生物学行为如生长增殖、凋亡和侵袭转移等能力的影响,探讨PRMT5在卵巢癌分子发病中的作用机制,为卵巢癌的基因治疗提供理论依据。
研究方法：
1.设计并体外化学合成靶向PRMT5(?)的2条siRNA序列(siP1和siP2)及阴性对照siRNA序列(siC),并在脂质体LipofectamineTM2000(?)的介异下转染进入卵巢癌细胞系A2780和SKOV3。
2.应用Real-time PCR和Western blot技术分别检测卵巢癌细胞系转染48小时后的PRMT5mRNA和转染72小时后的PRMT5蛋白的变化,筛选出具有最佳干扰效果的siRN八序列,作为后续研究的序列。
3.应用CCK8、BrdU(?)参入和Ki-67标记实验分别检测转染PRMT5-siRNA后的卵巢癌细胞系的生长和增殖能力的变化。
4. Annexin V-FITC/PI双染法流式细胞仪检测转染PRMT5-siRNA后卵巢癌细胞系凋亡的变化。
5.应用Transwell小室技术,检测转染PRMT5-siRNA后卵巢癌细胞系迁移和侵袭能力的变化。
6. Western blot技术检测PRMT5表达抑制后E2F-1、cIF4E和E-cadherin蛋白的
变化。
7. SPSS13.0统计软件对数据进行分析。计量资料用平均值±标准差表示,组间差异应用独立样本的t检验分析。
结果：
1.靶向PRMT5的两条siRNA序列和阴性对照序列均成功转染卵巢癌细胞A2780和SKOV3,转染效率90%以上。
2. Real-time PCR和Western blot实验结果均显示：siP1(?)序列能够显著减少A2780和SKOV3中PRMT5的mRNA和蛋白的表达；而siP2(?)序列只能减少SKOV3细胞中的PRMT5的表达,而对A2780的作用微弱。综合实验结果,siP1序列能够显著抑制A2780和SKOV3细胞中PRMT5的表达,筛选作为后续实验的干扰序列。
3.在CCK8实验中,转染后72小时始,siP1组与siC组和正常未转染组(Normal)相比,细胞生存率开始明显降低,且随着时间延长,生存率降低更明显,两种细胞系的结果类似。观察至120小时,A2780-siP1组生存率约24.4±0.9%,SKOV3-siP1组生存率约36.9±1.7%,均明显低于siC组(P均<0.001)。
4.A2780细胞中,siP1组的BrdU阳性率明显低于siC组(11.7±1.5%vs.33.3±1.5%,P<0.001)：SKOV3细胞中,siP1组的BrdU阳性率亦明显低于siC组(18.0±2.0%vs.35.3±1.5%,P<0.001)。
5.在A2780细胞中,siP1组与siC组相比,Ki.67的标记指数(LI)明显下降(12.4±3_3%vs.50.2±8.0%,P<0.001)；在SKOV3细胞中,在siPl组和siC组中的LI分别为2.3±1.0%和23.6±6.6%,siP1组有明显降低(P<0.001)。
6.A2780细胞中,siP1组明显高于siC组的凋亡率(29.4±0.9%vs.16.3±0.8%,P<0.001)：SKOV3中,siP1组亦明显高于siC组的凋亡率(28.9±0.6%vs.14.1±1.4%,P<0.001)。
7. Transwell体外迁移和侵袭实验均表明：在A2780和SKOV3细胞中,siP1组的穿膜细胞数均明显少于siC组和Normal组,差异有统计学意义(P分别<0.001)。
8.在转染后72小时,Western blot实验显示：A2780和SKOV3细胞中的E2F-1蛋白和E-cadherin蛋白表达明显上调。
结论：
1. PRMT5参与了卵巢癌细胞的增殖、凋亡、迁移和侵袭的过程,干扰其表达能够显著减慢细胞的增殖,促进细胞的凋亡和减弱其迁移和侵袭的能力。说明PRMT5在卵巢癌的发生发展中有着重要的作用,同时也说明PRMT5有可能成为卵巢癌基因治疗的候选基因。
2.PRMT5通过调控E2F-1的表达参与卵巢癌细胞增殖凋亡过程,调控E-cadherin(?)内表达参与卵巢癌细胞的迁移和侵袭过程。
Ovarian cancer is one of the leading causes of death from gynecological malignancies. Over80%of ovarian cancers are of epithelial origin (EOC), which show highly invasive, respond poorly to chemotherapies and are usually detected at advanced stages, resulting in poor prognosis. Despite the improvement of surgical techniques and the advent of more effective therapeutics, the overall5-year survival of epithelial ovarian cancer has increased only from36%to44%during the last three decades. To improve better prognostic and predictive assays and develop new therapeutic strategies for HOC, understanding the molecular mechanisms underlying HOC is critical.
Arginine methylation is becoming an important type of posttranslational modification and is widely appreciated, playing a vital role in the regulation of diverse cellular procession that range from transcription and RNA procession to signaling, differentiation, apoptosis and tumorigenesis. The methylation of arginine residues is catalyzed by the protein arginine methyltransferase(PRMTs) family of enzymes. To date, PRMTs have been found11members in human, most of PRMTs except for PRMT2, PRMT10, and PRMT11have been shown to have enzymatic activity and can catalyze arginine methylation. Based on the type of modification they catalyze, PRMTs are mainly classified as type1and type11enzymes. All these two enzymes catalyze the formation of a mono-methylated (MMA) intermediate, subsequently, type I PRMTs (PRMT1,3,4,6and8) further catalyze the production of asymmetric dimethylation of arginine residues (aDMA), and type II PRMTs (PRMT5,7and PRMT9) catalyze the formation of symmetric dimethylation of arginine residues (sDMA).
Protein arginine methyltransferase5(PRMT5) the first identified type II enzyme, is in human chromosome14q11.2and has two isoforms with637and620amino acids in length. It is involved in a variety of biological processes including ribosome biogenesis, assembly of the Golgi apparatus, cellular differentiation, germ cell specification, cellular proliferation, and apoptosis. In addition, it has been shown to interact with many other genes, such as cyclin E1, ST7and NM23, p53, E2F-1, PDCD4and E-cadherin to participate many cell processes. In human, aberrant expression of PRMT5are found in a wide variety of human diseases, especially in many cancers, including leukemia and lymphoma, breast cancer, colorectal cancer and lung cancer. Moreover, it is relevant to poor outcome in breast cancer and colorectal cancer patients.
However, little is known about the role of PRMT5in ovarian cancer. In this study, we investigated the role of PRMT5in epithelial ovarian cancer.
Part I:PRMT5is Overexpression in Epithelial Ovarian Cancers and is Associated with Poor Disease Prognosis
Objective:
1. To detect the difference of PRMT5expression in normal epithelium cells, epithelial ovarian benign tumors, borderline tumors and ovarian cancers.
2. To valuate the relationship between PRMT5overexpression and clinicopathologic features of the patients.
3. To examine the same set of EOC specimens with Ki-67, and investigate the relationship between PRMT5and Ki-67.
4. Evaluate the relevance of PRMT5expression to the prognosis of ovarian cancer patients.
Methods:
1. EOCs (n=118), epithelial ovarian benign tumors (n=20), borderline tumors (n=14) and normal ovaries (n=12) samples were collected from the Department of Gynecology, Qilu Hospital of Shandong University between January2005and December2008. The clinical and follow-up data of ovarian cancer patients wre collected. The study was approved by the Institutional Medical Ethics Committee of Qilu Hospital of Shandong University and informed consent were obtained by patients.
2. PRMT5and Ki-67expression were examined by immunohistochemistry (IHC) of standard streptavidin-peroxidase complex method.
3. All statistical analyses were performed with SPSS13.0statistical software. IHC data were analyzed using Chi-square test or Fisher's exact test. The Spearman test was used to analyze the relationship between PRMT5and Ki-67scores. Univariate and multivariate Cox regression analyses were used to evaluate the prognostic significance of PRMT5and other variables of ROC. Kaplan-Meier method was used to calculate the survival curves, and Log-rank test was used to compare the overall survival and progression-free survival between patient subgroups.
Results:
1. The positive staining of PRMT5was observed both in nuclear and cytoplasm of cells. High expression of PRMT5was observed in83.1%(98/118) of EOCs, in33.3%(4/12)of normal ovaries, in30%(6/20) of benign tumors, in64.3%(9/14) of borderline tumors. The level of PRMT5in EOCs was higher than normal ovaries and benign tumors (P<0.05, respectively), and there was no significant difference between borderline tumors and ovarian cancers.
2. High expression of PRMT5was significantly associated with serous subtype, advanced FIGO stage(Ⅲ/Ⅳ), poor differentiation(G3), lymph node invasion, and presence of residual tumor (P<0.05, respectively).
3. There was a significant positive correlation between PRMT5and Ki-67expression (r=0.377, P<0.001) in ovarian cancers, in which tumors with high Ki-67expression had a significantly higher expression of PRMT5(69.2%vs.98.2%, P<0.001).
4. At the end of the follow-up period, of the118ovarian cancer patients, the median OS time was40months (95%CI:19.1-60.1), and median PFS time was20months (95%CI:16.8-23.2).
5. In Univariate Cox analyses, serous subtype, high PRMT5expression, older age (>60years), advanced FIGO stage (Ⅲ/Ⅳ), and poor tumor differentiation (G3) were unfavorable predictors of OS (P<0.05, respectively), and all clinico-pathological variables, except lymph node invasion, were unfavorable predictors of PFS (P<0.05, respectively). Kaplan-Meier survival curves indicated that patients with high PRMT5expression had a significantly poor OS and PFS than those with low PRMT5expression (Log-rank test, P<0.001, respectively).
6. In multivariate Cox analysis, the high expression of PRMT5, older age and advanced FIGO stage retained their significance as independent prognostic factors of poorer OS and the high expression of PRMT5, advanced FIGO stage and presence of residual tumor were found to be independent prognostic factors for shorter PFS.
Conclusions:
1. The expression of PRMT5was increased gradually between ovarian benign, borderline and malignant tumors and overexpression of PRMT5was associated with poor clinicopathologic features of the patients. These results demonstrate that PRMT5may play a crucial role as an oncogene in the development of EOC.
2. The expression of PRMT5was positively correlated with the expression of Ki-67, which indicated that PRMT5may be involved in the regulation of ovarian cancer growth and proliferation.
3. In univariate and multivariate survival analyses, high level of PRMT5was identified as an independent factor for poor prognosis in patients with EOC.
Part II:Effects of PRMT5-silencing on Proliferation, Apoptosis, Migration and Invasion of Ovarian Cancer Cells
Objective:
To investigate the effects of PRMT5-siRNA-mediated gene silencing on proliferation, apoptosis, migration and invasion of ovarian cancer cell lines A2780and SKOV3. To explore the role of PRMT5in ovarian cancer cells in vitro, and to develop therapeutic potential of PRMT5in ovarian cancer.
Methods:
1. The two human PRMT5siRNA sequences siP1and siP2, control-siRNA(siC) were designed and chemically synthesize. They were transfected into ovarian cancer cell lines A2780and SKOV3with LipofectamineTM2000.
2. Silencing of PRMT5was assayed for mRNA and protein expression level at48hr and72hr after transfection, respectively. The most efficient and specific sequence to silence the expression of PRMT5in two cells was screened.
3. CCK8, BrdU incorporation and Ki-67labelling assays were used to detect the proliferation activity of ovarian cancer cells after PRMT5-siRNA was transfection.
4. Annexin V-FITC/PI staining by How cytometry analysis was used to detect the apoptosis of ovarian cancer cells after PRM15-siRNA was transfection.
5. Transwell assay was employed to test ovarian cancer cell migration and invasion after PRMT5-siRNA was transfection.
6. E2F-1,eIF4E and E-cadherin protein were detected by Western blot assay after PRMT5-siRNA was transfection.
7. Quantitative data were expressed as means±SD, the significance of the difference between groups was evaluated with independent-samples t test. All calculations were performed using SPSS13.0statistical software.
Results:
1. Two PRMT5siRNA (siP1and siP2) and scrambled control (siC) were respectively and successfully transfected into ovarian cancer cell line, and the efficiency of transfection was up to90%.
2. Compared with normal control or cells transfected with siC, siP1could reduce PRMT5mRNA and protein in A2780and SKOV3cells significantly; siP2could reduce PRMT5expression in SKOV3, but the silence effect of siP2in A2780was weak. According to the results, siPl was the most efficient and specific sequence to silence the expression of PRMT5in two cell lines.
3.72hr after transfection, the cell viability started to show lower in the cells transfected with siPl as compared with the cells transfected with siC and normal control (P<0.05). Similar results were observed in both A2780and SKOV3cells lines. The inhibition of growth was in a time-dependent manner.120hr after transfection, the cell viability of siPl group was24.4±0.9%in A2780, and was36.9±1.7%in SKOV3, both of them were significantly lower than the cell viability in siC group (P<0.001, respectively)
4.48hr after transfection, compared to control sample that transfected with siC, cells treated with siPl showed significantly decreased BrdU incorporation,11.7±1.5%vs.33.3±1.5%in A2780, and18.0±2.0%vs.35.3±1.5%in SKOV3(P<0.05, respectively).
5.72hr after transfection, compared to siC, cells transfected with siPl showed significantly decreased positive percentage of Ki-67,12.4±3.3%vs.50.2±8.0%in A2780,2.3±1.0%vs.23.6±6.6%in SKOV3(P<0.001, respectively).
6.72hr after transfection, the percentage of apoptosis cells in siPl cells was higher than that in siC cells (29.4±0.9%vs.16.3±0.8%, P<0.001) in A2780; the percentage of apoptosis was28.9±0.6%vs.14.1±1.4%(P<0.001) in SKOV3.
7. Both tumor cell migration and invasion assays indicated that transfection of PRMT5-siRNA could reduce the migration and invasion power of ovarian cancer cells. The number of migration/invasion cells with siPl group was lower than that with siC and normal control (P<0.001, respectively).
8. E2F-1and E-cadherin protein were increased after PRMT5-siRNA transfection in ovarian cancer cells.
Conclusions:
1. PRMT5could significantly promote the proliferation, migration and invasion, inhibit the apoptosis of ovarian cancer cell line, suggesting that PRMT5could play an important role in the tumorigenesis and development of ovarian cancer. PRMT5may be a novel therapeutic target for epithelial ovarian cancer.
2. PRMT-siRNA could inhibit the proliferation, induce apoptosis via up-regulation E2F-1and reduce the migration/invasion ability via up-regulation E-cadherin in ovarian cancer.
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