LPA_2调控LPA诱导的卵巢癌细胞uPA分泌和细胞侵袭的实验研究
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摘要
研究背景
卵巢癌是死亡率最高的妇科恶性肿瘤。由于缺乏有效的早期筛查手段,2/3以上的卵巢癌患者就诊时已届晚期,其5年生存率仅为20%～30%,而早期卵巢癌患者的5年生存率可达90%。卵巢癌发病隐匿,进展迅速,虽然近20年来在卵巢癌治疗方面取得了长足进展,卵巢癌手术技巧不断提高,新型化疗药物、化疗方案亦陆续投入临床使用,但这些传统的治疗手段均未能显著改善患者的长期生存率。局部浸润与转移是关系到卵巢癌临床治疗成败的关键,而侵袭转移又是一个多步骤、多因素参与的极其复杂的过程。因此研究卵巢癌侵袭转移机制及主要调控因素意义重大,可以为临床治疗提供新思路和分子治疗的靶点。
溶血磷脂酸(Lysophosphatidic acid,LPA)是新近发现的具有生长因子样作用的脂类小分子物质,主要通过与其特异性G蛋白偶联受体结合发挥多种生物学作用,诱导多种细胞发生增生性和形态学改变。许多晚期肿瘤患者的腹水中都可检测到高水平的LPA。在卵巢癌,研究显示LPA不仅存在于晚期患者的腹水,在早期患者的血浆中也可检测到LPA水平升高,且其特异性和敏感性明显高于CA125。近年发现LPA及其受体信号通路在许多恶性肿瘤的发生、发展、侵袭和转移过程中发挥重要作用,如甲状腺癌、胰腺癌、胃癌、结直肠癌、卵巢癌等。现已知的LPA受体有5种,LPA_1/EDG2、LPA_2/EDG4、LPA_3/EDG7,以及最近识别的LPA_4/GPR23/p2y9和LPA_5/PPARγ。LPA通过与这些受体结合刺激肿瘤细胞的增殖、黏附、侵袭、迁移及侵袭相关细胞因子的分泌。体外研究显示:LPA_1在正常和永生化的卵巢表面上皮细胞中高表达,而LPA_2/EDG4,LPA_3/EDG7在卵巢癌组织和癌细胞系中高表达,提示LPA受体有望成为卵巢癌治疗的新靶点。
尿激酶型纤溶酶原激活剂(urokinase plasminogen activator,uPA)是降解细胞外基质(extracellular matrix,ECM)最重要的酶类之一,它在肿瘤细胞侵袭和转移中的作用已被证实。uPA与卵巢、乳腺和结肠细胞的恶性转化有关。在卵巢癌,细胞内uPA的水平与卵巢癌的预后呈负相关。与其他癌症相比,uPA在卵巢癌组织和腹水中明显的高表达。
uPA既可通过催化纤溶酶原转化成纤溶酶导致基底膜的降解,从而加速癌细胞的侵袭和转移,也可通过与其特异性受体(urokinase plasminogen activatorreceptor,uPAR)结合刺激细胞的迁移和增殖。研究显示:LPA能够上调卵巢癌细胞uPA的分泌而不影响uPAR的表达,抑制uPA蛋白表达可以明显降低LPA诱导的卵巢癌细胞的侵袭,因此针对LPA及其与uPA关系的研究将有助于我们更好的了解LPA在卵巢癌侵袭、转移中的作用。
RNA干扰技术作为生物学领域的一种全新的技术,越来越受到广大研究者的重视并在肿瘤基因治疗中有着巨大的发展潜力。选择对肿瘤有治疗作用的靶基因是利用RNA干扰技术进行基因治疗的关键所在。在LPA诱导的肿瘤细胞侵袭转移的研究中发现,尽管其他受体也起作用,但LPA受体2(LPA_2)在LPA诱导的侵袭相关因子的分泌中起关键作用。故本研究选择LPA_2作为RNA干扰基因治疗的靶点。
基于以上报道,本研究首先观察LPA对人卵巢癌SKOV-3细胞uPA蛋白分泌和细胞侵袭能力的影响;其次,通过脂质体介导法将针对LPA_2的小干扰RNA转染人卵巢癌SKOV-3细胞,观察转染后卵巢癌SKOV-3细胞中LPA_2 mRNA和蛋白的表达情况;同时研究LPA_2受体抑制后对LPA诱导的uPA蛋白分泌、细胞侵袭和迁移能力的影响,探讨LPA_2受体在LPA诱导的卵巢癌侵袭转移中的作用。
第一部分溶血磷脂酸对卵巢癌SKOV-3细胞uPA分泌的影响
目的:研究发现,在卵巢癌患者的血浆和腹水中LPA的水平明显升高,且其水平与卵巢癌的生物学行为有一定的相关性。本研究旨在探讨溶血磷脂酸(lysophosphatidic acid,LPA)对人卵巢癌SKOV-3细胞尿激酶型纤溶酶原激活剂(uPA)分泌和细胞侵袭能力的影响。
方法:
1.常规培养的人卵巢癌SKOV-3细胞,加入不同浓度的LPA(0、2、20、40、80μmol/L)作用24小时后,收集细胞上清液,采用酶联免疫吸附试验(ELISA)检测溶血磷脂酸作用后,人卵巢癌SKOV-3细胞上清液中uPA蛋白的表达。
2.常规培养的人卵巢癌SKOV-3细胞,加入80μmol/L LPA,作用0、3、8、12、24小时后,收集细胞上清液,采用酶联免疫吸附试验(ELISA)检测溶血磷脂酸作用后,人卵巢癌SKOV-3细胞上清液中uPA蛋白的表达。
3.常规培养的人卵巢癌SKOV-3细胞,加入0或80μmol/L LPA后,利用Matrigel Transwell小室法检测LPA对SKOV-3细胞体外侵袭能力的影响。
结果:
1.加入不同浓度的LPA,作用24小时后,2μmol/L LPA即可引起uPA分泌增加,但与对照相比无明显的统计学差异(P＞0.05);而加入20、40、80μmol/LLPA后,uPA蛋白分泌量明显增加(P＜0.001)。
2.加入80μmol/L LPA,作用不同时间后,与对照相比,8小时后LPA诱导的uPA产量即明显增加(P＜0.01)。
3.Matrigel Transwell小室检测80μmol/L LPA作用后,穿透人工基底膜的SKOV-3细胞数较无LPA作用的对照组明显增多(219.4±23.6 vs.67±10.9)(P＜0.001)。
结论:
1.低浓度的LPA即可诱导uPA分泌增加,且随着LPA浓度的加大,uPA产量明显增多;二者间呈现明显的浓度依赖性。
2.相同浓度LPA作用于SKOV-3细胞,随着时间的延长uPA分泌量增多,二者间呈现明显的时间依赖性。
3.LPA可以明显增加SKOV-3细胞的体外侵袭能力。
第二部分siRNA沉默LPA_2基因对LPA诱导的卵巢癌SKOV-3细胞uPA产量和细胞侵袭的影响
目的:运用RNA干扰技术抑制卵巢癌SKOV-3细胞中LPA_2基因的表达,观察LPA_2基因沉默对LPA诱导的uPA产量和细胞侵袭、迁移能力的影响,探讨RNA干扰技术在抗肿瘤基因治疗方面的应用前景以及沉默LPA_2基因作为卵巢肿瘤治疗靶点的可能性。
方法:
1.复苏、培养卵巢癌细胞株SKOV-3至对数生长期,将实验分成三组,即正常对照组、阴性对照组和干扰组。
2.经脂质体介导将不同浓度梯度的siRNA转染至SKOV-3细胞中,观察转染效果,筛选出最佳转染浓度。
3.将特异性siRNA及阴性对照siRNA分别转染至干扰组和阴性对照组,正常对照组未作任何siRNA的转染,仅加转染液。
4.转染36小时后采用半定量RT-PCR和Western blot检测LPA_2 mRNA和蛋白的表达情况。
5.采用酶联免疫吸附实验(ELISA)检测干扰前、后LPA诱导的uPA产量的变化,采用Matrigel Transwell小室法和Transwell Chemotaxis实验检测干扰后LPA对卵巢癌细胞体外侵袭和迁移能力的影响。
结果:
1.半定量RT-PCR和Western blot结果显示siRNA转染后,SKOV-3细胞LPA_2 mRNA表达受抑制、蛋白表达水平降低。
2.ELISA结果显示干扰后80μmol/L LPA诱导的uPA产量下降,干扰组uPA蛋白表达(OD值:0.344±0.039)较阴性对照组uPA蛋白表达(OD值:0.746±0.031)明显降低(P＜0.001)。
3.Matrigel Transwell小室实验结果显示,80μmol/L LPA刺激后,干扰组穿过Matrigel膜的细胞数(36.2±3.3),明显低于阴性对照组(178±17.2)(P＜0.001)。
4.Transwell Chemotaxis实验结果显示,80μmol/L LPA刺激后,干扰组进入下室的细胞数(57±7.6)较阴性对照组(220.4±25.5)明显下降(P＜0.001)。
结论:
1.针对LPA_2基因的特异性siRNA成功转染SKOV-3细胞后,能够特异性抑制、降解同源mRNA,有效阻滞LPA_2 mRNA和蛋白的表达,可作为特异性阻断LPA_2基因表达、评价其功能的良好手段。
2.经RNA干扰技术特异性沉默LPA_2基因后,能够明显抑制LPA诱导的SKOV-3细胞uPA的分泌及细胞的侵袭和迁移能力,提示LPA_2可能成为卵巢癌治疗的靶基因。
Background:
Ovarian cancer accounts for more deaths than all other gynecologic malignancies combined.Because of the obscure early symptoms and the absence of effective measures of universal inspection and early diagnosis of ovarian cancer,more than 2/3 of the patients with ovarian cancer have reached an advanced stage when they were diagnosed,with little hope to get good curative effect,and the 5-year survival is only 20%-30%,while the survival rate of patients diagnosed in early stage could reach up to 90%.Although three therapeutic advances of malignant ovarian carcinoma in the late twenty years,which are comprehensive stage laparotomy, cytoreductive surgery and omentectomy,and paclitaxel-cisplatin combined chemotherapy,cannot improve the patients' survival rate in the long run.The invasion and metastasis of ovarian cancer are the most important influencing factors to success or failure of treatment.The metastasis of tumors is a complicated process including multiple steps and factors.So it has an important significance to investigate the metastasis and invasive mechanism of epithelial ovarian cancer,and can provide new methods and potential targets for molecule treatment.
LPA is a small molecular lipid functionally related with growth factors.Its signaling pathways play many biological functions mainly through G protein-coupled receptor.LPA induces proliferative and/or morphological effects.Recent years,LPA and its pathways were found to play important roles in the appearance,development, invasion and transfer of malignant tumors,such as thyroid cancer,pancreatic cancer, gastric cancer,colorectal cancer,ovarian caner and so on.To date,five LPA receptors have been found,namely LPA1/EDG-2,LPA2/EDG-4,LPA3/EDG-7, LPA4/GPR23/p2y9,and LPA5/PPARγ.LPA stimulates the proliferation,adhesion, invasion and metastasis,and the production of various invasion-associated factors through these receptors.In vitro studies have shown that LPA_1 is highly expressed in the normal and immortalized ovarian cells,while LPA_2/EDG4 and LPA_3/EDG7 mRNA are highly expressed in ovarian tissues and tumor cell lines.Thus LPA receptor is hopeful to become a new target for treatment of ovarian carcinoma.
Urokinase plasminogen activator(uPA)is one of the most important enzymes to degrade extracellular matrix(ECM).The function it performs in the invasion and metastasis of tumor cells is also testified,uPA has been linked to the malignant transformation in ovarian,breast and colon cells.In ovarian cancer,cellular uPA levels correlate inversely with prognosis.In comparison with other cancers,uPA is highly expressed in ovarian cancer cell lines and ascites.
uPA contributes to metastasis and migration because it catalyzes the conversion of plasminogen to plasmin,thus leading to degradation of the basement membrane.It also stimulates cellular migration and proliferation pltentially by binding to its specific cell surface receptor(uPAR).Recent research shows that LPA can induce uPA but not uPAR the secretion of uPA not uPAR and accelerate the uPA-induced invasion of tumor cells.Therefore,research on the relationship between LPA and uPA will help us understand the function of LPA in the invasion and metastasis of ovarian cancer.
As a totally new way in biological field RNA interference technology has very huge potential and is getting more and more importance from the investigators.To choose the target gene which has therapeutic action on tumor is the key of the RNA interference.The studies on LPA-induced metastasis of tumor cells show that LPA_2 receptor plays an important part role in the secretion of invasion-associated cell factors in response to LPA,although other LPA receptors could also mediate the response.So we choose LPA_2 receptor to be the target gene to RNA interference.
Based on the above report,this research will firstly observe the role of LPA on the production of uPA in human ovarian cancer cell line SKOV-3.Secondly,LPA_2 siRNA was transfected into SKOV-3 by using Lipofectamine~(TM)2000.The transfection effect was identified through detecting LPA_2 mRNA level by semiquantitative RT-PCR and protein level by Western blot,respectively. Furthermore,the role of inhibiting LPA_2 pathway in LPA-induced uPA regulation was observed,and the function of LPA2 in LPA-induced invasion and metastasis of ovarian cancer would be discussed.
PartⅠ:Effect of lysophosphatidic acid(LPA)on urokinase plasminogen activator(uPA)secretion of ovarian cancer SKOV-3 cells
Objective:To investigate the effect of LPA on the uPA secretion and cell invasion ability of ovarian cancer SKOV-3 cells.
Methods:Human ovarian cancer cell lines were cultured in various concentrations of LPA(0,2,20,40,80μmol/L)for 24 hours and cultured in 80μmol/L LPA for different time-points up to 24 hours(0,3,8,12,24h).Then cell culture supernatant was collected,and LPA-induced uPA expression was analyzed using uPA ELISA.After LPA stimulation with the concentrations of 0 or 80μmol/L, the invasiveness of SKOV-3 cells was analyzed by using Matrigel Transwell Assay.
Results:
1.After 24h of incubation with various concentrations of LPA,2μmol/L of LPA can cause the increase of uPA secretion in comparison with the control group(P＞0.05).While after adding LPA with concentration of 20,40,80μmol/L,the uPA secretion increased obviously(P＜0.001).
2.After incubation with 80μmol/L LPA in different time-points up to 24 hours, the secretion of uPA increased obviously in comparison with the control group after 8 hours(P＜0.01).
3.The number of SKOV-3 cells that penetrated the Matrigel after 80μmol/L LPA stimulation increased obviously in comparison with control,group(219.4±23.6 vs.67±10.9)(P＜0.001).
Conclusions:
1.The low concentration of LPA could induce uPA secretion.With the increase of LPA concentrations,the production of uPA significantly increased,there was obvious dose-dependent manner between LPA and uPA.
2.LPA treatment at 80μmol/L induced considerably uPA secretion in time-dependent manner.
3.LPA could significantly increase the invasion ability of SKOV-3 cells in vitro.
PartⅡ:Effect of inhibition of LPA_2 by siRNA on LPA-induced uPA production and cell invasion of ovarian cancer SKOV-3 cells
Objective:The purpose of this study is to investigat the inhibition effect of RNA interference(RNAi)on the expression of LPA_2 in ovarian cancer SKOV-3 cells,and to observe its impact on the output of LPA-induced uPA and on cell invasion. Meanwhile,we will explore the applying prospects of RNAi technique on anti-tumor genetic treatment and the possibility that knock-down of LPA_2 gene as a treatment target of ovarian cancer.
Methods:
1.Ovarian cancer cell strain SKOV-3 was resuscitated and cultivated until it came to logarithmic growing period,and then they were divided into three groups, which were normal control group,negative control group and siRNA group.
2.The siRNA of different concentrations of grads were transfected into SKOV-3 cells in order to observe the transfection effects,and then the optimal transfection concentration was selected.
3.The specific siRNA and negative control siRNA were transfected into siRNA group and negative control group respectively.The normal control group was not transfected by any siRNA,only transfection liquid.
4.The expression of LPA2 mRNA and protein were detected by using the semi-quantitative RT-PCR and Western blot after 36 hours.Meanwhile,the production of LPA-induced uPA was measured by ELISA analyse before and after the interference.The impacts of LPA on the ex-vivo invasion ability of ovarian cancer using Matrigel invasion assay.
Results:
1.The results of semi-quantitative RT-PCR and Western blot indicated that after transfection of LPA_2-specifical siRNA,the expression of LPA2 mRNA and protein in SKOV-3 cells were obviously reduced.
2.ELISA results indicated that the production of uPA induced by LPA of 80μmol/L after interference decreased,which of siRNA group(uPA OD 450nm: 0.344±0.039)decreases obviously in comparsion with that of negative control group (uPA OD 450nm:0.746±0.031)(P＜0.001).
3.The experiment of Matrigel invasion membrane indicated that the number (36.2±3.3)of cells that penetrated through Marigel chamber in siRNA group after the stimulation of 80μmol/L LPA were lower than that of negative control group (178±17.2)(P＜0.001).
4.The experiment of Transwell Chemotaxis chamber indicated that the number (57±7.6)of cells that entered the lower well in siRNA group after the LPA stimulation decreased obviously than that of negative control group(220.4±25.5)(P＜0.001).
Conclusions:
1.The specific siRNA aiming at LPA2 gene can be successfully transfected into SKOV-3 cells and then inhibites and degrades congenetic mRNA.It can also block the expression of LPA2 mRNA and protein effectively,and can be used as a new tool of blocking LPA2 gene expression and evaluating its biological function.
2.The inhibition of LPA_2 gene by using RNAi can reduce the LPA-induced uPA secretion and the ability cell invasion and migration in SKOV-3 cells,which indicates that LPA2 is probable to work as target gene in the therapy of ovarian cancer.
卵巢癌是死亡率最高的妇科恶性肿瘤。由于缺乏有效的早期筛查手段,2/3以上的卵巢癌患者就诊时已届晚期,其5年生存率仅为20%～30%,而早期卵巢癌患者的5年生存率可达90%。卵巢癌发病隐匿,进展迅速,虽然近20年来在卵巢癌治疗方面取得了长足进展,卵巢癌手术技巧不断提高,新型化疗药物、化疗方案亦陆续投入临床使用,但这些传统的治疗手段均未能显著改善患者的长期生存率。局部浸润与转移是关系到卵巢癌临床治疗成败的关键,而侵袭转移又是一个多步骤、多因素参与的极其复杂的过程。因此研究卵巢癌侵袭转移机制及主要调控因素意义重大,可以为临床治疗提供新思路和分子治疗的靶点。
溶血磷脂酸(Lysophosphatidic acid,LPA)是新近发现的具有生长因子样作用的脂类小分子物质,主要通过与其特异性G蛋白偶联受体结合发挥多种生物学作用,诱导多种细胞发生增生性和形态学改变。许多晚期肿瘤患者的腹水中都可检测到高水平的LPA。在卵巢癌,研究显示LPA不仅存在于晚期患者的腹水,在早期患者的血浆中也可检测到LPA水平升高,且其特异性和敏感性明显高于CA125。近年发现LPA及其受体信号通路在许多恶性肿瘤的发生、发展、侵袭和转移过程中发挥重要作用,如甲状腺癌、胰腺癌、胃癌、结直肠癌、卵巢癌等。现已知的LPA受体有5种,LPA_1/EDG2、LPA_2/EDG4、LPA_3/EDG7,以及最近识别的LPA_4/GPR23/p2y9和LPA_5/PPARγ。LPA通过与这些受体结合刺激肿瘤细胞的增殖、黏附、侵袭、迁移及侵袭相关细胞因子的分泌。体外研究显示:LPA_1在正常和永生化的卵巢表面上皮细胞中高表达,而LPA_2/EDG4,LPA_3/EDG7在卵巢癌组织和癌细胞系中高表达,提示LPA受体有望成为卵巢癌治疗的新靶点。
尿激酶型纤溶酶原激活剂(urokinase plasminogen activator,uPA)是降解细胞外基质(extracellular matrix,ECM)最重要的酶类之一,它在肿瘤细胞侵袭和转移中的作用已被证实。uPA与卵巢、乳腺和结肠细胞的恶性转化有关。在卵巢癌,细胞内uPA的水平与卵巢癌的预后呈负相关。与其他癌症相比,uPA在卵巢癌组织和腹水中明显的高表达。
uPA既可通过催化纤溶酶原转化成纤溶酶导致基底膜的降解,从而加速癌细胞的侵袭和转移,也可通过与其特异性受体(urokinase plasminogen activatorreceptor,uPAR)结合刺激细胞的迁移和增殖。研究显示:LPA能够上调卵巢癌细胞uPA的分泌而不影响uPAR的表达,抑制uPA蛋白表达可以明显降低LPA诱导的卵巢癌细胞的侵袭,因此针对LPA及其与uPA关系的研究将有助于我们更好的了解LPA在卵巢癌侵袭、转移中的作用。
RNA干扰技术作为生物学领域的一种全新的技术,越来越受到广大研究者的重视并在肿瘤基因治疗中有着巨大的发展潜力。选择对肿瘤有治疗作用的靶基因是利用RNA干扰技术进行基因治疗的关键所在。在LPA诱导的肿瘤细胞侵袭转移的研究中发现,尽管其他受体也起作用,但LPA受体2(LPA_2)在LPA诱导的侵袭相关因子的分泌中起关键作用。故本研究选择LPA_2作为RNA干扰基因治疗的靶点。
基于以上报道,本研究首先观察LPA对人卵巢癌SKOV-3细胞uPA蛋白分泌和细胞侵袭能力的影响;其次,通过脂质体介导法将针对LPA_2的小干扰RNA转染人卵巢癌SKOV-3细胞,观察转染后卵巢癌SKOV-3细胞中LPA_2 mRNA和蛋白的表达情况;同时研究LPA_2受体抑制后对LPA诱导的uPA蛋白分泌、细胞侵袭和迁移能力的影响,探讨LPA_2受体在LPA诱导的卵巢癌侵袭转移中的作用。
第一部分溶血磷脂酸对卵巢癌SKOV-3细胞uPA分泌的影响
目的:研究发现,在卵巢癌患者的血浆和腹水中LPA的水平明显升高,且其水平与卵巢癌的生物学行为有一定的相关性。本研究旨在探讨溶血磷脂酸(lysophosphatidic acid,LPA)对人卵巢癌SKOV-3细胞尿激酶型纤溶酶原激活剂(uPA)分泌和细胞侵袭能力的影响。
方法:
1.常规培养的人卵巢癌SKOV-3细胞,加入不同浓度的LPA(0、2、20、40、80μmol/L)作用24小时后,收集细胞上清液,采用酶联免疫吸附试验(ELISA)检测溶血磷脂酸作用后,人卵巢癌SKOV-3细胞上清液中uPA蛋白的表达。
2.常规培养的人卵巢癌SKOV-3细胞,加入80μmol/L LPA,作用0、3、8、12、24小时后,收集细胞上清液,采用酶联免疫吸附试验(ELISA)检测溶血磷脂酸作用后,人卵巢癌SKOV-3细胞上清液中uPA蛋白的表达。
3.常规培养的人卵巢癌SKOV-3细胞,加入0或80μmol/L LPA后,利用Matrigel Transwell小室法检测LPA对SKOV-3细胞体外侵袭能力的影响。
结果:
1.加入不同浓度的LPA,作用24小时后,2μmol/L LPA即可引起uPA分泌增加,但与对照相比无明显的统计学差异(P＞0.05);而加入20、40、80μmol/LLPA后,uPA蛋白分泌量明显增加(P＜0.001)。
2.加入80μmol/L LPA,作用不同时间后,与对照相比,8小时后LPA诱导的uPA产量即明显增加(P＜0.01)。
3.Matrigel Transwell小室检测80μmol/L LPA作用后,穿透人工基底膜的SKOV-3细胞数较无LPA作用的对照组明显增多(219.4±23.6 vs.67±10.9)(P＜0.001)。
结论:
1.低浓度的LPA即可诱导uPA分泌增加,且随着LPA浓度的加大,uPA产量明显增多;二者间呈现明显的浓度依赖性。
2.相同浓度LPA作用于SKOV-3细胞,随着时间的延长uPA分泌量增多,二者间呈现明显的时间依赖性。
3.LPA可以明显增加SKOV-3细胞的体外侵袭能力。
第二部分siRNA沉默LPA_2基因对LPA诱导的卵巢癌SKOV-3细胞uPA产量和细胞侵袭的影响
目的:运用RNA干扰技术抑制卵巢癌SKOV-3细胞中LPA_2基因的表达,观察LPA_2基因沉默对LPA诱导的uPA产量和细胞侵袭、迁移能力的影响,探讨RNA干扰技术在抗肿瘤基因治疗方面的应用前景以及沉默LPA_2基因作为卵巢肿瘤治疗靶点的可能性。
方法:
1.复苏、培养卵巢癌细胞株SKOV-3至对数生长期,将实验分成三组,即正常对照组、阴性对照组和干扰组。
2.经脂质体介导将不同浓度梯度的siRNA转染至SKOV-3细胞中,观察转染效果,筛选出最佳转染浓度。
3.将特异性siRNA及阴性对照siRNA分别转染至干扰组和阴性对照组,正常对照组未作任何siRNA的转染,仅加转染液。
4.转染36小时后采用半定量RT-PCR和Western blot检测LPA_2 mRNA和蛋白的表达情况。
5.采用酶联免疫吸附实验(ELISA)检测干扰前、后LPA诱导的uPA产量的变化,采用Matrigel Transwell小室法和Transwell Chemotaxis实验检测干扰后LPA对卵巢癌细胞体外侵袭和迁移能力的影响。
结果:
1.半定量RT-PCR和Western blot结果显示siRNA转染后,SKOV-3细胞LPA_2 mRNA表达受抑制、蛋白表达水平降低。
2.ELISA结果显示干扰后80μmol/L LPA诱导的uPA产量下降,干扰组uPA蛋白表达(OD值:0.344±0.039)较阴性对照组uPA蛋白表达(OD值:0.746±0.031)明显降低(P＜0.001)。
3.Matrigel Transwell小室实验结果显示,80μmol/L LPA刺激后,干扰组穿过Matrigel膜的细胞数(36.2±3.3),明显低于阴性对照组(178±17.2)(P＜0.001)。
4.Transwell Chemotaxis实验结果显示,80μmol/L LPA刺激后,干扰组进入下室的细胞数(57±7.6)较阴性对照组(220.4±25.5)明显下降(P＜0.001)。
结论:
1.针对LPA_2基因的特异性siRNA成功转染SKOV-3细胞后,能够特异性抑制、降解同源mRNA,有效阻滞LPA_2 mRNA和蛋白的表达,可作为特异性阻断LPA_2基因表达、评价其功能的良好手段。
2.经RNA干扰技术特异性沉默LPA_2基因后,能够明显抑制LPA诱导的SKOV-3细胞uPA的分泌及细胞的侵袭和迁移能力,提示LPA_2可能成为卵巢癌治疗的靶基因。
Background:
Ovarian cancer accounts for more deaths than all other gynecologic malignancies combined.Because of the obscure early symptoms and the absence of effective measures of universal inspection and early diagnosis of ovarian cancer,more than 2/3 of the patients with ovarian cancer have reached an advanced stage when they were diagnosed,with little hope to get good curative effect,and the 5-year survival is only 20%-30%,while the survival rate of patients diagnosed in early stage could reach up to 90%.Although three therapeutic advances of malignant ovarian carcinoma in the late twenty years,which are comprehensive stage laparotomy, cytoreductive surgery and omentectomy,and paclitaxel-cisplatin combined chemotherapy,cannot improve the patients' survival rate in the long run.The invasion and metastasis of ovarian cancer are the most important influencing factors to success or failure of treatment.The metastasis of tumors is a complicated process including multiple steps and factors.So it has an important significance to investigate the metastasis and invasive mechanism of epithelial ovarian cancer,and can provide new methods and potential targets for molecule treatment.
LPA is a small molecular lipid functionally related with growth factors.Its signaling pathways play many biological functions mainly through G protein-coupled receptor.LPA induces proliferative and/or morphological effects.Recent years,LPA and its pathways were found to play important roles in the appearance,development, invasion and transfer of malignant tumors,such as thyroid cancer,pancreatic cancer, gastric cancer,colorectal cancer,ovarian caner and so on.To date,five LPA receptors have been found,namely LPA1/EDG-2,LPA2/EDG-4,LPA3/EDG-7, LPA4/GPR23/p2y9,and LPA5/PPARγ.LPA stimulates the proliferation,adhesion, invasion and metastasis,and the production of various invasion-associated factors through these receptors.In vitro studies have shown that LPA_1 is highly expressed in the normal and immortalized ovarian cells,while LPA_2/EDG4 and LPA_3/EDG7 mRNA are highly expressed in ovarian tissues and tumor cell lines.Thus LPA receptor is hopeful to become a new target for treatment of ovarian carcinoma.
Urokinase plasminogen activator(uPA)is one of the most important enzymes to degrade extracellular matrix(ECM).The function it performs in the invasion and metastasis of tumor cells is also testified,uPA has been linked to the malignant transformation in ovarian,breast and colon cells.In ovarian cancer,cellular uPA levels correlate inversely with prognosis.In comparison with other cancers,uPA is highly expressed in ovarian cancer cell lines and ascites.
uPA contributes to metastasis and migration because it catalyzes the conversion of plasminogen to plasmin,thus leading to degradation of the basement membrane.It also stimulates cellular migration and proliferation pltentially by binding to its specific cell surface receptor(uPAR).Recent research shows that LPA can induce uPA but not uPAR the secretion of uPA not uPAR and accelerate the uPA-induced invasion of tumor cells.Therefore,research on the relationship between LPA and uPA will help us understand the function of LPA in the invasion and metastasis of ovarian cancer.
As a totally new way in biological field RNA interference technology has very huge potential and is getting more and more importance from the investigators.To choose the target gene which has therapeutic action on tumor is the key of the RNA interference.The studies on LPA-induced metastasis of tumor cells show that LPA_2 receptor plays an important part role in the secretion of invasion-associated cell factors in response to LPA,although other LPA receptors could also mediate the response.So we choose LPA_2 receptor to be the target gene to RNA interference.
Based on the above report,this research will firstly observe the role of LPA on the production of uPA in human ovarian cancer cell line SKOV-3.Secondly,LPA_2 siRNA was transfected into SKOV-3 by using Lipofectamine~(TM)2000.The transfection effect was identified through detecting LPA_2 mRNA level by semiquantitative RT-PCR and protein level by Western blot,respectively. Furthermore,the role of inhibiting LPA_2 pathway in LPA-induced uPA regulation was observed,and the function of LPA2 in LPA-induced invasion and metastasis of ovarian cancer would be discussed.
PartⅠ:Effect of lysophosphatidic acid(LPA)on urokinase plasminogen activator(uPA)secretion of ovarian cancer SKOV-3 cells
Objective:To investigate the effect of LPA on the uPA secretion and cell invasion ability of ovarian cancer SKOV-3 cells.
Methods:Human ovarian cancer cell lines were cultured in various concentrations of LPA(0,2,20,40,80μmol/L)for 24 hours and cultured in 80μmol/L LPA for different time-points up to 24 hours(0,3,8,12,24h).Then cell culture supernatant was collected,and LPA-induced uPA expression was analyzed using uPA ELISA.After LPA stimulation with the concentrations of 0 or 80μmol/L, the invasiveness of SKOV-3 cells was analyzed by using Matrigel Transwell Assay.
Results:
1.After 24h of incubation with various concentrations of LPA,2μmol/L of LPA can cause the increase of uPA secretion in comparison with the control group(P＞0.05).While after adding LPA with concentration of 20,40,80μmol/L,the uPA secretion increased obviously(P＜0.001).
2.After incubation with 80μmol/L LPA in different time-points up to 24 hours, the secretion of uPA increased obviously in comparison with the control group after 8 hours(P＜0.01).
3.The number of SKOV-3 cells that penetrated the Matrigel after 80μmol/L LPA stimulation increased obviously in comparison with control,group(219.4±23.6 vs.67±10.9)(P＜0.001).
Conclusions:
1.The low concentration of LPA could induce uPA secretion.With the increase of LPA concentrations,the production of uPA significantly increased,there was obvious dose-dependent manner between LPA and uPA.
2.LPA treatment at 80μmol/L induced considerably uPA secretion in time-dependent manner.
3.LPA could significantly increase the invasion ability of SKOV-3 cells in vitro.
PartⅡ:Effect of inhibition of LPA_2 by siRNA on LPA-induced uPA production and cell invasion of ovarian cancer SKOV-3 cells
Objective:The purpose of this study is to investigat the inhibition effect of RNA interference(RNAi)on the expression of LPA_2 in ovarian cancer SKOV-3 cells,and to observe its impact on the output of LPA-induced uPA and on cell invasion. Meanwhile,we will explore the applying prospects of RNAi technique on anti-tumor genetic treatment and the possibility that knock-down of LPA_2 gene as a treatment target of ovarian cancer.
Methods:
1.Ovarian cancer cell strain SKOV-3 was resuscitated and cultivated until it came to logarithmic growing period,and then they were divided into three groups, which were normal control group,negative control group and siRNA group.
2.The siRNA of different concentrations of grads were transfected into SKOV-3 cells in order to observe the transfection effects,and then the optimal transfection concentration was selected.
3.The specific siRNA and negative control siRNA were transfected into siRNA group and negative control group respectively.The normal control group was not transfected by any siRNA,only transfection liquid.
4.The expression of LPA2 mRNA and protein were detected by using the semi-quantitative RT-PCR and Western blot after 36 hours.Meanwhile,the production of LPA-induced uPA was measured by ELISA analyse before and after the interference.The impacts of LPA on the ex-vivo invasion ability of ovarian cancer using Matrigel invasion assay.
Results:
1.The results of semi-quantitative RT-PCR and Western blot indicated that after transfection of LPA_2-specifical siRNA,the expression of LPA2 mRNA and protein in SKOV-3 cells were obviously reduced.
2.ELISA results indicated that the production of uPA induced by LPA of 80μmol/L after interference decreased,which of siRNA group(uPA OD 450nm: 0.344±0.039)decreases obviously in comparsion with that of negative control group (uPA OD 450nm:0.746±0.031)(P＜0.001).
3.The experiment of Matrigel invasion membrane indicated that the number (36.2±3.3)of cells that penetrated through Marigel chamber in siRNA group after the stimulation of 80μmol/L LPA were lower than that of negative control group (178±17.2)(P＜0.001).
4.The experiment of Transwell Chemotaxis chamber indicated that the number (57±7.6)of cells that entered the lower well in siRNA group after the LPA stimulation decreased obviously than that of negative control group(220.4±25.5)(P＜0.001).
Conclusions:
1.The specific siRNA aiming at LPA2 gene can be successfully transfected into SKOV-3 cells and then inhibites and degrades congenetic mRNA.It can also block the expression of LPA2 mRNA and protein effectively,and can be used as a new tool of blocking LPA2 gene expression and evaluating its biological function.
2.The inhibition of LPA_2 gene by using RNAi can reduce the LPA-induced uPA secretion and the ability cell invasion and migration in SKOV-3 cells,which indicates that LPA2 is probable to work as target gene in the therapy of ovarian cancer.
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