PLA2G2A基因过表达对卵巢癌细胞生物学行为影响的研究
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摘要
背景与目的
上皮性卵巢癌是世界范围内女性死亡的主要原因之一。由于卵巢癌发病隐匿,缺乏早期症状,大多数患者就诊时多已经是晚期,且预后极差。尽管新型化疗药物的应用极大提高了患者的生存率,但是卵巢癌的总体死亡率仍居高不。卵巢癌的发生是一个多基因参与、多阶段发展的复杂过程,其中涉及到原癌基因的激活及抑癌基因的失活等多个过程。且大多数恶性肿瘤患者的死亡往往不是由于原发肿瘤而是由于远处的转移病灶。尽管目前发现了一些与肿瘤转移相关的基因,但是它们并不能完全阐释肿瘤侵袭及转移的机制。因此,发现与肿瘤发生发展及转移相关的新基因对改善卵巢癌的早期诊断、治疗,预后有着极其重要的意义,而PLA2G2A就是我们发现的其中之一。
磷脂酶A2是一种能催化甘油磷脂生成脂肪酸和溶血磷脂的酯酶。PLA2G2A属是磷脂酶家族中的一员,是一种分子量仅有16kDa的分泌型的磷脂酶。PLA2G2A目前已知的功能大多与炎症反应、免疫反应、抗血栓形成、细胞增殖、缺血性损伤及变态反应相关。
PLA2G2A在前列腺癌及结直肠癌等多种恶性肿瘤中表达,且在其中发挥各自不同的功能。尽管PLA2G2A一直被认为是一个潜在的抑癌基因,但是又并不像经典的原癌基因及抑癌基因一样发挥作用,使其一直备受争议。例如,研究表明,PLA2G2A在前列腺癌、胃癌及肠癌中表达上调,PLA2G2A促进前列腺癌向高级别转化,这一证据支持PLA2G2A的原癌基因的作用。相反,高表达PLA2G2A的胃癌患者的5年生存率却明显提高。且近期有研究表明,PLA2G2A可能通过上游的β-catenin依赖的Wnt信号通路抑制胃癌的侵袭。
到目前为止,PLA2G2A在卵巢癌中的生物学功能尚未有研究,故我们通过在卵巢癌细胞系H08910pm过表达PLA2G2A研究其在卵巢癌发生发展中的作用,以期能为卵巢癌的基因靶向治疗提供可能的新靶点。
方法
为了明确PLA2G2A在卵巢癌不同细胞系中的表达量差异,首先分析PLA2G2A在卵巢癌细胞系A2780, H08910, H08910pm, SKOV3及OVCAR3中PLA2G2A的mRNA水平的表达情况。通过特异性引物利用PCR技术从人新鲜胎盘组织中扩增出PLA2G2A完整开放阅读框架的cDNA。然后将cDNA片段克隆入经EcoRI-XhoI酶切处理的pcDNA3.1载体构建过表达载体pcDNA3.1-PLA2G2A(3.1-PLA2G2A)。随后按照脂质体2000说明书转染H08910pm细胞系。24小时后以1：10传代并换用新鲜含10%血清的1640培养基。48小时后换用含750ug／mL G418的培养基筛选稳定转染的细胞,2周后即可观察到单个克隆,随之在375ug/mL G418的1640培养基里扩增培养。通过RT-PCR和western blot验证过表达细胞系在mRNA及蛋白水平上的表达情况。此外,我们用同样的方法构建了空载体pcDNA3.1-vector(3.1-vector)。
对于过表达前后的H08910pm细胞系采用MTT实验分析检测细胞的增值能力的差异；对于过表达前后的H08910pm细胞系采用克隆形成实验检测单个细胞独立生存的能力的变化；对于过表达前后的H08910pm细胞系采用Transwell小室实验及细胞划痕实验分析侵袭及迁移能力的差异；对于过表达前后的H08910pm细胞系采用流式细胞仪PI单染法检测细胞周期,分析PLA2G2A过表达前后细胞G0/G1期周期阻滞情况的变化,并进行统计学分析；制备过表达前后细胞的肿瘤条件培养基,采用Transwell小室的迁移实验分析其对HUVEC迁移的影响；提取转染前后细胞的蛋白,采用western blot实验分析与侵袭及转移可能的相关通路蛋白的差异。此外我们从甲基化角度通过甲基化特异性PCR分析了PLA2G2A低表达的原因。
结果
实时定量PCR检测PLA2G2A在卵巢癌细胞系A2780, H08910, H08910pm, SKOV3及OVCAR3中均表达较低。
过表达载体经过双向测序及双酶切验证成功,经实时定量PCR及Western blot检测证实转染成功并筛选出稳定过表达的细胞系,且过表达效果良好。
MTT法及克隆形成实验检测了细胞系3.1-PLA2G2A及3.1-vector对卵巢癌细胞生长的影响。结果显示3.1-PLA2G2A较3.1-vector明显抑制卵巢癌细胞的增殖(P<0.01)。克隆形成实验显示3.1-PLA2G2A形成的克隆数明显少于3.1-vector形成的克隆数(168±60vs824±97,P<0.01)。由此可知,PLA2G2A过表达后明显抑制卵巢癌细胞的生长。
Transwell侵袭、迁移实验及划痕实验评价PLA2G2A对卵巢癌侵袭及转移的影响。tanswell侵袭实验,3.1-PLA2G2A穿出的细胞数明显少于3.1-vector穿出的细胞数(184±22.86vs312±23.55, P<0.01); transwell迁移实验,3.1-PLA2G2A穿出的细胞数明显少于3.1-vector穿出的细胞数(126±17.74vs281±38.97,P<0.01)；细胞划痕实验,3.1-PLA2G2A迁移距离明显少于3.1-vector迁移距离(0.24±0.03vs0.48±0.016,P<0.01)。由此可见,PLA2G2A过表达后明显抑制卵巢癌细胞的运动。
流式细胞仪检测3.1-PLA2G2A及3.1-vector的GO/G1期阻滞情况差异。结果显示PLA2G2A过表达后G1期细胞比例为少于空载体(66.9±3.6%vs79.1±1.0%,P<0.05)。
Transwell小室实验评价3.1-PLA2G2A条件培养基对HUVEC的迁移能力的影响。3.1-PLA2G2A条件培养基处理后的HUVEC穿出的细胞数明显少于3.1-vector处理的HUVEC穿出的细胞数(165±17.40vs393±51.99,P<0.01)。Western blot分析了PLA2G2A可能影响卵巢癌侵袭及转移的可能的相关通路蛋白。结果显示PLA2G2A过表达后会降低磷酸化β-catenin的表达,结果导致β-catenin的累积活化Wnt信号通路,反过来调节下游基因的表达。
甲基化特异性PCR分析了PLA2G2A启动子区域的-186、-111及-82区域甲基化位点的情况,结果显示,上皮性卵巢癌各细胞系中均存在3个位点甲基化状态,这可能是其低表达的机制之一
结论
PLA2G2A在上皮性卵巢癌细胞系中均低表达,其启动子区域特异性位点的甲基化是导致其低表达的机制之一；PLA2G2A过表达后可以明显抑制卵巢癌细胞的增殖、侵袭、迁移、诱导细胞周期G1期阻滞及抑制HUVEC的迁移,而这些过程可能是通过降解β-catenin的磷酸化来实现的。
BACKGROUND AND OBJECT
Epithelial ovarian cancer is a leading cause of death in women around the world. Due to the lack of obvious symptoms, the majority of patients is only identified in the advanced stages of the disease. The prognosis for ovarian cancer patients is very poor. Although new chemotherapeutic agents have significantly improved five-year survival rate, the overall mortality of ovarian cancer has remained unchanged. The tumorigenesis of ovarian cancer is a polygenic disease and a multistep complicated process involved, which includes the activation of pro-oncogenes and inactivation of suppressor genes. However, most cancer related deaths are not due to the growth of the primary tumor but result from its invasive spread to secondary sites. Though some candidate genes were found to be related to metastasis, neither of them could be interpreted the mechanism of invasion and metastasis completely. Consequently, it is significantly necessary to identify the novel genes related to the tumorigenesis, development and progress to improve the early diagnosis and treatment of ovarian cancer, and PLA2G2A is one of them we found.
Phospholipase A2(PLA2) is a esterase that cleave glycerophospholipids at the sn-2ester bond to release a fatty acid andlysophospholipid. PLA2G2A belongs to a family of phospholipases and is a low molecular weight (16kDa) secreted phospholipases. The major known functions of PLA2G2A are mostly related to inflammatory responses, antimicrobial defense, anticoagulation, cell proliferation, ischemic injury and allergic diseases.
PLA2G2A is expressed in various types of cancers, including prostate cancer, colorectal cancer and so on, which plays different roles in these cancers. Although PLA2G2A has been proposed as a potential tumor suppressor, evidence supporting this model is conflicting and it does not behave like a classical oncogene or tumor suppressor gene. For example, PLA2G2A is upregulated in prostate, gastric, intestinal human tumors. Several studies of human prostate cancer support an oncogenic role for sPLA2-IIA, particularly in its progression to advanced cancer. In contrast, in human gastric cancer, patients expressing high levels of sPLA2-IIA showed a highly significant survival advantage. A recent study reported that PLA2G2A may function as an inhibitor of gastric cancer invasion in vitro and β-catenin-dependent Wnt-signaling may serve as its up-stream regulator.
Till now, the biological functions in tumor development of PLA2G2A in ovarian cancer have not been discussed. Consequently, PLA2G2A was overexpressed in ovarian cancer cells to identify how PLA2G2A might contribute to ovarian cancer development and progression and provide the potential therapeutic target for ovarian cancer.
METHOD
PLA2G2A mRNA expression level was examined in ovarian serous cancer cell lines A2780, HO8910, HO8910pm, SKOV3and OVCAR3.
The cDNA representing the complete open reading frame of PLA2G2A was amplified from the cDNA of fresh human placenta tissue using the specific primers as follows:forward,5'-CCGGAATTCGCCACCATGAAGACCCTCCTACTGT-3';revers-e,5'-CCGCTCGAGTCAGCAACGAGGGGTGCT-3',and then was cloned into the EcoRI-XhoI vector fragment derived from the pcDNA3.1vector (Invitrogen) to generate pcDNA3.1-PLA2G2A(3.1-PLA2G2A) cells. The expression plasmid was verified by sequencing of both strands and was used to transfect the HO8910pm cells using lipofectamine2000transfection reagent (Invitrogen) according to the manufacturer's protocol. After24h, the cells for stable transfection cells were passaged at a1:10dilution into the fresh growth medium. After24h,750ug/mL G418(Invitrogen) was added in order to select the stable transfected cell lines for2weeks and individual colonies were isolated, expanded and maintained in375ug/mL G418. The overexpression of PLA2G2A in these clones was confirmed by RT-PCR and western blot analysis. The empty pcDNA3.1plasmid was used similarly to establish pcDNA3.1-vector (3.1-vector).
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to assess cellular proliferative ability between3.1-Vector and3.1-PLA2G2A. The viability of cells was assessed by clonogenic assays between3.1-Vector and3.1-PLA2G2A. To assess whether PLA2G2A overexpression affects migration and invasion of HO8910pm cells, both cell migration and invasion ability were evaluated by a two-chamber assay. To assess the effect of PLA2G2A overexpression on cell cycle, a cell cycle assay was performed using a FACSCalibur flow cytometer (BD Biosciences). In order to evaluate the ability of conditional medium to regulate the migration of HUVEC, the HUVEC migration ability was evaluated by a two-chamber assay. At last, we evaluated correlations among overexpression of PLA2G2A β-Catenin, Bcl-2, p65and AKT, western blot was performed to assess their relationship in ovarian cancer to discuss the possible related pathway proteins.
Methylation-specific PCR (MS-PCR)
Genomic DNA was extracted from epithelial ovarian cancer cells according to the manufacturer's instructions of DNA extraction kit. Sodium bisulfite treatment of the genomic DNA was performed the CpGenome DNA modification kit according to the manufacturer's instructions. The locations of CpG sites within the PLA2G2A gene promoter have been described previously. Primers and PCR conditions for MSP are available on request. M standard, U standard and3DW were used as positive and negative control.
RESULTS
The mRNA expression levels of all the five types of ovarian cancer cell lines A2780, HO8910, HO8910pm, SKOV3and OVCAR3were very low and almost cannot be detected by RT-PCR.
The overexpression of PLA2G2A in transfected HO8910pm cells was confirmed by RT-PCR and western blot analysis. Cells transfected with3.1-PLA2G2A showed significant increased PLA2G2A in both mRNA and protein levels compared with the control empty vector cell line
MTT assay showed that3.1-PLA2G2A inhibited celluar proliferation compared to the3.1-vector cells (P<0.01). Overexpression of PLA2G2A in HO8910pm cells dramatically reduced colony formation efficiency (168±60vs824±97, P<0.01).
3.1-PLA2G2A cells showed significant decreased invasive ability in comparison with the control cells (184±22.86vs312±23.55, P<0.01).Similarly, migration of3.1-PLA2G2A cells was greatly decreased compared with the empty vector control cells (126±17.74vs281±38.97,P<0.01).
In the cell cycle assay performed using a FACSCalibur flow cytometer, overexpression of PLA2G2A caused a moderately cell cycle arrest in the G1phase in3.1-PLA2G2A cells (66.9±3.6%vs79.1±1.0%, P<0.05).
Migration ability of HUVECs accompanied with overexpression of PLA2G2A was obviously decreased than that of empty vector control (165±17.40vs393±51.99, P<0.01).
Western blot was performed to assess the relationship between overexpression of PLA2G2A and β-Catenin. It showed that overexpression of PLA2G2A would decrease the expression of phosphorylation of β-Catenin, leading to the accumulation of β-Catenin and consequent Wnt pathway hyperactivation which promoted the expression of downstream PLA2G2A gene. Additional, Bcl-2,p65and AKT were also evaluated the different expression in transfected ovarian cancer cells, but the results didn't appear obvious differences.
A number of potential binding sites were found in sPLA2-IIA promoter region from-260to+20, which included those for signal transducer and activator of transcription. methylation-specific PCR (MSP) was used to verified whether the three CpG sites(-186,-111,-82) were methylated as shown in Jurkat Leukemia Cells, of which site-186is a potential binding site for the y-IRE binding domain, site-111is part of the DNA binding region for NF-Y/CCAAT box-binding factor-82is within the potential Spl and site-82is within the potential Sp1.As shown in figure(9), three CpG sites were all in presence of methylation,but site-82was less methylation to some extent.
CONCLUSION
PLA2G2A mRNA expression was decreased in ovarian serous cancer cells, and the methylation in the specific binding site of promoter might be one of the reasons of low expression of PLA2G2A in ovarian cancer. Overexpression of PLA2G2A inhibited proliferation, invasion, migration and induced G1arrest of ovarian cancer cells. It also could decrease migration of HUVEC. All the above processes might be mediated by degrading expression of phosphalation β-Catenin.
上皮性卵巢癌是世界范围内女性死亡的主要原因之一。由于卵巢癌发病隐匿,缺乏早期症状,大多数患者就诊时多已经是晚期,且预后极差。尽管新型化疗药物的应用极大提高了患者的生存率,但是卵巢癌的总体死亡率仍居高不。卵巢癌的发生是一个多基因参与、多阶段发展的复杂过程,其中涉及到原癌基因的激活及抑癌基因的失活等多个过程。且大多数恶性肿瘤患者的死亡往往不是由于原发肿瘤而是由于远处的转移病灶。尽管目前发现了一些与肿瘤转移相关的基因,但是它们并不能完全阐释肿瘤侵袭及转移的机制。因此,发现与肿瘤发生发展及转移相关的新基因对改善卵巢癌的早期诊断、治疗,预后有着极其重要的意义,而PLA2G2A就是我们发现的其中之一。
磷脂酶A2是一种能催化甘油磷脂生成脂肪酸和溶血磷脂的酯酶。PLA2G2A属是磷脂酶家族中的一员,是一种分子量仅有16kDa的分泌型的磷脂酶。PLA2G2A目前已知的功能大多与炎症反应、免疫反应、抗血栓形成、细胞增殖、缺血性损伤及变态反应相关。
PLA2G2A在前列腺癌及结直肠癌等多种恶性肿瘤中表达,且在其中发挥各自不同的功能。尽管PLA2G2A一直被认为是一个潜在的抑癌基因,但是又并不像经典的原癌基因及抑癌基因一样发挥作用,使其一直备受争议。例如,研究表明,PLA2G2A在前列腺癌、胃癌及肠癌中表达上调,PLA2G2A促进前列腺癌向高级别转化,这一证据支持PLA2G2A的原癌基因的作用。相反,高表达PLA2G2A的胃癌患者的5年生存率却明显提高。且近期有研究表明,PLA2G2A可能通过上游的β-catenin依赖的Wnt信号通路抑制胃癌的侵袭。
到目前为止,PLA2G2A在卵巢癌中的生物学功能尚未有研究,故我们通过在卵巢癌细胞系H08910pm过表达PLA2G2A研究其在卵巢癌发生发展中的作用,以期能为卵巢癌的基因靶向治疗提供可能的新靶点。
方法
为了明确PLA2G2A在卵巢癌不同细胞系中的表达量差异,首先分析PLA2G2A在卵巢癌细胞系A2780, H08910, H08910pm, SKOV3及OVCAR3中PLA2G2A的mRNA水平的表达情况。通过特异性引物利用PCR技术从人新鲜胎盘组织中扩增出PLA2G2A完整开放阅读框架的cDNA。然后将cDNA片段克隆入经EcoRI-XhoI酶切处理的pcDNA3.1载体构建过表达载体pcDNA3.1-PLA2G2A(3.1-PLA2G2A)。随后按照脂质体2000说明书转染H08910pm细胞系。24小时后以1：10传代并换用新鲜含10%血清的1640培养基。48小时后换用含750ug／mL G418的培养基筛选稳定转染的细胞,2周后即可观察到单个克隆,随之在375ug/mL G418的1640培养基里扩增培养。通过RT-PCR和western blot验证过表达细胞系在mRNA及蛋白水平上的表达情况。此外,我们用同样的方法构建了空载体pcDNA3.1-vector(3.1-vector)。
对于过表达前后的H08910pm细胞系采用MTT实验分析检测细胞的增值能力的差异；对于过表达前后的H08910pm细胞系采用克隆形成实验检测单个细胞独立生存的能力的变化；对于过表达前后的H08910pm细胞系采用Transwell小室实验及细胞划痕实验分析侵袭及迁移能力的差异；对于过表达前后的H08910pm细胞系采用流式细胞仪PI单染法检测细胞周期,分析PLA2G2A过表达前后细胞G0/G1期周期阻滞情况的变化,并进行统计学分析；制备过表达前后细胞的肿瘤条件培养基,采用Transwell小室的迁移实验分析其对HUVEC迁移的影响；提取转染前后细胞的蛋白,采用western blot实验分析与侵袭及转移可能的相关通路蛋白的差异。此外我们从甲基化角度通过甲基化特异性PCR分析了PLA2G2A低表达的原因。
结果
实时定量PCR检测PLA2G2A在卵巢癌细胞系A2780, H08910, H08910pm, SKOV3及OVCAR3中均表达较低。
过表达载体经过双向测序及双酶切验证成功,经实时定量PCR及Western blot检测证实转染成功并筛选出稳定过表达的细胞系,且过表达效果良好。
MTT法及克隆形成实验检测了细胞系3.1-PLA2G2A及3.1-vector对卵巢癌细胞生长的影响。结果显示3.1-PLA2G2A较3.1-vector明显抑制卵巢癌细胞的增殖(P<0.01)。克隆形成实验显示3.1-PLA2G2A形成的克隆数明显少于3.1-vector形成的克隆数(168±60vs824±97,P<0.01)。由此可知,PLA2G2A过表达后明显抑制卵巢癌细胞的生长。
Transwell侵袭、迁移实验及划痕实验评价PLA2G2A对卵巢癌侵袭及转移的影响。tanswell侵袭实验,3.1-PLA2G2A穿出的细胞数明显少于3.1-vector穿出的细胞数(184±22.86vs312±23.55, P<0.01); transwell迁移实验,3.1-PLA2G2A穿出的细胞数明显少于3.1-vector穿出的细胞数(126±17.74vs281±38.97,P<0.01)；细胞划痕实验,3.1-PLA2G2A迁移距离明显少于3.1-vector迁移距离(0.24±0.03vs0.48±0.016,P<0.01)。由此可见,PLA2G2A过表达后明显抑制卵巢癌细胞的运动。
流式细胞仪检测3.1-PLA2G2A及3.1-vector的GO/G1期阻滞情况差异。结果显示PLA2G2A过表达后G1期细胞比例为少于空载体(66.9±3.6%vs79.1±1.0%,P<0.05)。
Transwell小室实验评价3.1-PLA2G2A条件培养基对HUVEC的迁移能力的影响。3.1-PLA2G2A条件培养基处理后的HUVEC穿出的细胞数明显少于3.1-vector处理的HUVEC穿出的细胞数(165±17.40vs393±51.99,P<0.01)。Western blot分析了PLA2G2A可能影响卵巢癌侵袭及转移的可能的相关通路蛋白。结果显示PLA2G2A过表达后会降低磷酸化β-catenin的表达,结果导致β-catenin的累积活化Wnt信号通路,反过来调节下游基因的表达。
甲基化特异性PCR分析了PLA2G2A启动子区域的-186、-111及-82区域甲基化位点的情况,结果显示,上皮性卵巢癌各细胞系中均存在3个位点甲基化状态,这可能是其低表达的机制之一
结论
PLA2G2A在上皮性卵巢癌细胞系中均低表达,其启动子区域特异性位点的甲基化是导致其低表达的机制之一；PLA2G2A过表达后可以明显抑制卵巢癌细胞的增殖、侵袭、迁移、诱导细胞周期G1期阻滞及抑制HUVEC的迁移,而这些过程可能是通过降解β-catenin的磷酸化来实现的。
BACKGROUND AND OBJECT
Epithelial ovarian cancer is a leading cause of death in women around the world. Due to the lack of obvious symptoms, the majority of patients is only identified in the advanced stages of the disease. The prognosis for ovarian cancer patients is very poor. Although new chemotherapeutic agents have significantly improved five-year survival rate, the overall mortality of ovarian cancer has remained unchanged. The tumorigenesis of ovarian cancer is a polygenic disease and a multistep complicated process involved, which includes the activation of pro-oncogenes and inactivation of suppressor genes. However, most cancer related deaths are not due to the growth of the primary tumor but result from its invasive spread to secondary sites. Though some candidate genes were found to be related to metastasis, neither of them could be interpreted the mechanism of invasion and metastasis completely. Consequently, it is significantly necessary to identify the novel genes related to the tumorigenesis, development and progress to improve the early diagnosis and treatment of ovarian cancer, and PLA2G2A is one of them we found.
Phospholipase A2(PLA2) is a esterase that cleave glycerophospholipids at the sn-2ester bond to release a fatty acid andlysophospholipid. PLA2G2A belongs to a family of phospholipases and is a low molecular weight (16kDa) secreted phospholipases. The major known functions of PLA2G2A are mostly related to inflammatory responses, antimicrobial defense, anticoagulation, cell proliferation, ischemic injury and allergic diseases.
PLA2G2A is expressed in various types of cancers, including prostate cancer, colorectal cancer and so on, which plays different roles in these cancers. Although PLA2G2A has been proposed as a potential tumor suppressor, evidence supporting this model is conflicting and it does not behave like a classical oncogene or tumor suppressor gene. For example, PLA2G2A is upregulated in prostate, gastric, intestinal human tumors. Several studies of human prostate cancer support an oncogenic role for sPLA2-IIA, particularly in its progression to advanced cancer. In contrast, in human gastric cancer, patients expressing high levels of sPLA2-IIA showed a highly significant survival advantage. A recent study reported that PLA2G2A may function as an inhibitor of gastric cancer invasion in vitro and β-catenin-dependent Wnt-signaling may serve as its up-stream regulator.
Till now, the biological functions in tumor development of PLA2G2A in ovarian cancer have not been discussed. Consequently, PLA2G2A was overexpressed in ovarian cancer cells to identify how PLA2G2A might contribute to ovarian cancer development and progression and provide the potential therapeutic target for ovarian cancer.
METHOD
PLA2G2A mRNA expression level was examined in ovarian serous cancer cell lines A2780, HO8910, HO8910pm, SKOV3and OVCAR3.
The cDNA representing the complete open reading frame of PLA2G2A was amplified from the cDNA of fresh human placenta tissue using the specific primers as follows:forward,5'-CCGGAATTCGCCACCATGAAGACCCTCCTACTGT-3';revers-e,5'-CCGCTCGAGTCAGCAACGAGGGGTGCT-3',and then was cloned into the EcoRI-XhoI vector fragment derived from the pcDNA3.1vector (Invitrogen) to generate pcDNA3.1-PLA2G2A(3.1-PLA2G2A) cells. The expression plasmid was verified by sequencing of both strands and was used to transfect the HO8910pm cells using lipofectamine2000transfection reagent (Invitrogen) according to the manufacturer's protocol. After24h, the cells for stable transfection cells were passaged at a1:10dilution into the fresh growth medium. After24h,750ug/mL G418(Invitrogen) was added in order to select the stable transfected cell lines for2weeks and individual colonies were isolated, expanded and maintained in375ug/mL G418. The overexpression of PLA2G2A in these clones was confirmed by RT-PCR and western blot analysis. The empty pcDNA3.1plasmid was used similarly to establish pcDNA3.1-vector (3.1-vector).
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to assess cellular proliferative ability between3.1-Vector and3.1-PLA2G2A. The viability of cells was assessed by clonogenic assays between3.1-Vector and3.1-PLA2G2A. To assess whether PLA2G2A overexpression affects migration and invasion of HO8910pm cells, both cell migration and invasion ability were evaluated by a two-chamber assay. To assess the effect of PLA2G2A overexpression on cell cycle, a cell cycle assay was performed using a FACSCalibur flow cytometer (BD Biosciences). In order to evaluate the ability of conditional medium to regulate the migration of HUVEC, the HUVEC migration ability was evaluated by a two-chamber assay. At last, we evaluated correlations among overexpression of PLA2G2A β-Catenin, Bcl-2, p65and AKT, western blot was performed to assess their relationship in ovarian cancer to discuss the possible related pathway proteins.
Methylation-specific PCR (MS-PCR)
Genomic DNA was extracted from epithelial ovarian cancer cells according to the manufacturer's instructions of DNA extraction kit. Sodium bisulfite treatment of the genomic DNA was performed the CpGenome DNA modification kit according to the manufacturer's instructions. The locations of CpG sites within the PLA2G2A gene promoter have been described previously. Primers and PCR conditions for MSP are available on request. M standard, U standard and3DW were used as positive and negative control.
RESULTS
The mRNA expression levels of all the five types of ovarian cancer cell lines A2780, HO8910, HO8910pm, SKOV3and OVCAR3were very low and almost cannot be detected by RT-PCR.
The overexpression of PLA2G2A in transfected HO8910pm cells was confirmed by RT-PCR and western blot analysis. Cells transfected with3.1-PLA2G2A showed significant increased PLA2G2A in both mRNA and protein levels compared with the control empty vector cell line
MTT assay showed that3.1-PLA2G2A inhibited celluar proliferation compared to the3.1-vector cells (P<0.01). Overexpression of PLA2G2A in HO8910pm cells dramatically reduced colony formation efficiency (168±60vs824±97, P<0.01).
3.1-PLA2G2A cells showed significant decreased invasive ability in comparison with the control cells (184±22.86vs312±23.55, P<0.01).Similarly, migration of3.1-PLA2G2A cells was greatly decreased compared with the empty vector control cells (126±17.74vs281±38.97,P<0.01).
In the cell cycle assay performed using a FACSCalibur flow cytometer, overexpression of PLA2G2A caused a moderately cell cycle arrest in the G1phase in3.1-PLA2G2A cells (66.9±3.6%vs79.1±1.0%, P<0.05).
Migration ability of HUVECs accompanied with overexpression of PLA2G2A was obviously decreased than that of empty vector control (165±17.40vs393±51.99, P<0.01).
Western blot was performed to assess the relationship between overexpression of PLA2G2A and β-Catenin. It showed that overexpression of PLA2G2A would decrease the expression of phosphorylation of β-Catenin, leading to the accumulation of β-Catenin and consequent Wnt pathway hyperactivation which promoted the expression of downstream PLA2G2A gene. Additional, Bcl-2,p65and AKT were also evaluated the different expression in transfected ovarian cancer cells, but the results didn't appear obvious differences.
A number of potential binding sites were found in sPLA2-IIA promoter region from-260to+20, which included those for signal transducer and activator of transcription. methylation-specific PCR (MSP) was used to verified whether the three CpG sites(-186,-111,-82) were methylated as shown in Jurkat Leukemia Cells, of which site-186is a potential binding site for the y-IRE binding domain, site-111is part of the DNA binding region for NF-Y/CCAAT box-binding factor-82is within the potential Spl and site-82is within the potential Sp1.As shown in figure(9), three CpG sites were all in presence of methylation,but site-82was less methylation to some extent.
CONCLUSION
PLA2G2A mRNA expression was decreased in ovarian serous cancer cells, and the methylation in the specific binding site of promoter might be one of the reasons of low expression of PLA2G2A in ovarian cancer. Overexpression of PLA2G2A inhibited proliferation, invasion, migration and induced G1arrest of ovarian cancer cells. It also could decrease migration of HUVEC. All the above processes might be mediated by degrading expression of phosphalation β-Catenin.
引文
1. Shield, K., et al., Multicellular spheroids in ovarian cancer metastases: Biology and pathology. Gynecol Oncol,2009.113(1):p.143-8.
2. Haruta, S., et al., Molecular genetics and epidemiology of epithelial ovarian cancer (Review). Oncol Rep,2011.26(6):p.1347-56.
3. Praml, C., et al., Secretory type Ⅱ phospholipase A2 (PLA2G2A) expression status in colorectal carcinoma derived cell lines and in normal colonic mucosa. Oncogene,1998.17(15):p.2009-12.
4. Kudo, I. and M. Murakami, Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat,2002.68-69:p.3-58.
5. Leung, S.Y., et al., Phospholipase A2 group ⅡA expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis. Proc Natl Acad Sci U S A,2002.99(25):p.16203-8.
6. Belinsky, G.S., et al., Expression of secretory phospholipase A2 in colon tumor cells potentiates tumor growth. Mol Carcinog,2007.46(2):p.106-16.
7. Praml, C., et al., Human homologue of a candidate for the Moml locus, the secretory type Ⅱ phospholipase A2 (PLA2S-II), maps to 1p35-36.1/D1S199. Cancer Res,1995.55(23):p.5504-6.
8. Laye, J.P. and J.H. Gill, Phospholipase A2 expression in tumours:a target for therapeutic intervention? Drug Discov Today,2003.8(15):p.710-6.
9. Dong, Z., et al., Secretory phospholipase A2-Ila is involved in prostate cancer progression and may potentially serve as a biomarker for prostate cancer. Carcinogenesis,2010.31(11):p.1948-55.
10. Ganesan, K., et al., Inhibition of gastric cancer invasion and metastasis by PLA2G2A, a novel beta-catenin/TCF target gene. Cancer Res,2008.68(11):p. 4277-86.
11. Rajoria, S., et al., Molecular target based combinational therapeutic approaches in thyroid cancer. J Transl Med,2012.10:p.81.
12. Menschikowski, M., et al., Involvement of epigenetic mechanisms in the regulation of secreted phospholipase A2 expressions in Jurkat leukemia cells. Neoplasia,2008.10(11):p.1195-203.
13. Bast, R.C., Jr., B. Hennessy, and G.B. Mills, The biology of ovarian cancer: new opportunities for translation. Nat Rev.Cancer,2009.9(6):p.415-28.
14. Itamochi, H., Targeted therapies in epithelial ovarian cancer:Molecular mechanisms of action. World J Biol Chem,2010.1(7):p.209-20.
15. Zerbini, G., M. Lorenzi, and A. Palini, Tumor angiogenesis. N Engl J Med, 2008.359(7):p.763; author reply 764.
16. Liekens, S., E. De Clercq, and J. Neyts, Angiogenesis:regulators and clinical applications. Biochem Pharmacol,2001.61(3):p.253-70.
2. Haruta, S., et al., Molecular genetics and epidemiology of epithelial ovarian cancer (Review). Oncol Rep,2011.26(6):p.1347-56.
3. Praml, C., et al., Secretory type Ⅱ phospholipase A2 (PLA2G2A) expression status in colorectal carcinoma derived cell lines and in normal colonic mucosa. Oncogene,1998.17(15):p.2009-12.
4. Kudo, I. and M. Murakami, Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat,2002.68-69:p.3-58.
5. Leung, S.Y., et al., Phospholipase A2 group ⅡA expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis. Proc Natl Acad Sci U S A,2002.99(25):p.16203-8.
6. Belinsky, G.S., et al., Expression of secretory phospholipase A2 in colon tumor cells potentiates tumor growth. Mol Carcinog,2007.46(2):p.106-16.
7. Praml, C., et al., Human homologue of a candidate for the Moml locus, the secretory type Ⅱ phospholipase A2 (PLA2S-II), maps to 1p35-36.1/D1S199. Cancer Res,1995.55(23):p.5504-6.
8. Laye, J.P. and J.H. Gill, Phospholipase A2 expression in tumours:a target for therapeutic intervention? Drug Discov Today,2003.8(15):p.710-6.
9. Dong, Z., et al., Secretory phospholipase A2-Ila is involved in prostate cancer progression and may potentially serve as a biomarker for prostate cancer. Carcinogenesis,2010.31(11):p.1948-55.
10. Ganesan, K., et al., Inhibition of gastric cancer invasion and metastasis by PLA2G2A, a novel beta-catenin/TCF target gene. Cancer Res,2008.68(11):p. 4277-86.
11. Rajoria, S., et al., Molecular target based combinational therapeutic approaches in thyroid cancer. J Transl Med,2012.10:p.81.
12. Menschikowski, M., et al., Involvement of epigenetic mechanisms in the regulation of secreted phospholipase A2 expressions in Jurkat leukemia cells. Neoplasia,2008.10(11):p.1195-203.
13. Bast, R.C., Jr., B. Hennessy, and G.B. Mills, The biology of ovarian cancer: new opportunities for translation. Nat Rev.Cancer,2009.9(6):p.415-28.
14. Itamochi, H., Targeted therapies in epithelial ovarian cancer:Molecular mechanisms of action. World J Biol Chem,2010.1(7):p.209-20.
15. Zerbini, G., M. Lorenzi, and A. Palini, Tumor angiogenesis. N Engl J Med, 2008.359(7):p.763; author reply 764.
16. Liekens, S., E. De Clercq, and J. Neyts, Angiogenesis:regulators and clinical applications. Biochem Pharmacol,2001.61(3):p.253-70.