滋养干细胞转录因子叉头蛋白D3 过表达在人绒癌细胞株JAR的恶性生物学特性中的作用研究

doi:10.3969/j.issn.2095-4344.2016.10.006

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (10): 1409-1418.doi: 10.3969/j.issn.2095-4344.2016.10.006

• 肿瘤干细胞 cancer stem cells • 上一篇下一篇

滋养干细胞转录因子叉头蛋白D3 过表达在人绒癌细胞株JAR的恶性生物学特性中的作用研究

刘媛¹，王亚欣¹，吴维宾¹，王玉东²，张慧娟¹

上海交通大学医学院附属国际和平妇幼保健院，¹病理科&生物样本库，²妇科，上海市 200030

收稿日期:2015-12-24 出版日期:2016-03-04 发布日期:2016-03-04
通讯作者: 张慧娟，博士，主任医师，上海交通大学医学院附属国际和平妇幼保健院病理科&生物样本库，上海市 200030
作者简介:刘媛，女，1982年生，安徽省池州市人，汉族，2008年复旦大学上海医学院毕业，硕士，住院医师，主要从事妇产科病理基础和临床研究。共同第一作者：王亚欣，女，1988年生，甘肃省张掖市人，汉族，2015年上海交通大学医学院毕业，硕士，主要从事妊娠滋养细胞疾病基础研究。
基金资助:
国家自然科学基金(81072140，81172477)，上海交通大学医工交叉项目(YG2013MS67，YG2014QN11)

Overexpression of trophoblastic stem cell transcription factor, forkhead box D3, contributes to malignancy of human choriocarcinoma JAR cells

Liu Yuan¹, Wang Ya-xin¹, Wu Wei-bin¹, Wang Yu-dong², Zhang Hui-juan¹

1 Department of Pathology and Bio-Bank, International Peace Maternity and Child Health Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China

2 Department of Gynecology, International Peace Maternity and Child Health Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200030, China

Received:2015-12-24 Online:2016-03-04 Published:2016-03-04
Contact: Zhang Hui-juan, M.D., Chief physician, Department of Pathology and Bio-Bank, International Peace Maternity and Child Health Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China
About author:Liu Yuan, Master, Physician, Department of Pathology and Bio-Bank, International Peace Maternity and Child Health Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China Wang Ya-xin, Master, Department of Pathology and Bio-Bank, International Peace Maternity and Child Health Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China Liu Yuan and Wang Ya-xin contributed equally to this work.
Supported by:
the National Natural Science Foundation of China, No. 81072140, 81172477; the Medical Engineering Interdisciplinary Project of Shanghai Jiao Tong University, No. YG2013MS67, YG2014QN11

摘要/Abstract

摘要：

文章快速阅读：

文题释义：

滋养干细胞：在胚胎发育成桑椹胚后桑椹胚进一步发育，细胞开始出现分化，聚集在胚胎的一端，个体较大的细胞，被称为滋养干细胞，是胎盘细胞的前体细胞，具有发育全能性，将来发育成胎儿的各种组织。

滋养干细胞转录因子叉头蛋白D3过表达：叉头蛋白D3是叉头蛋白家族中的一个转录调控因子，在胚胎发育和维持胚胎干细胞多能性方面具有重要作用。过表达的叉头蛋白D3通过调控局部黏附分子表达和局部黏附激酶活性，有增强滋养干细胞癌变的可能。

背景：绒毛膜细胞癌是一种具有高度侵袭能力的滋养细胞肿瘤。叉头蛋白D3作为胚胎干细胞和滋养干细胞的转录因子，在胚胎发生、肿瘤发生和进展等很多生理、病理状态下发挥着重要作用。

目的：研究叉头蛋白D3在绒毛膜细胞癌恶性生物学特性中的作用及可能作用机制。

方法：利用短发夹RNA干扰叉头蛋白D3在人绒毛膜细胞癌细胞株JAR中的表达，体外细胞增殖、迁移/侵袭实验检测JAR的细胞功能学改变，体内动物成瘤实验检测肿瘤生长情况。Agilent转录表达芯片初筛叉头蛋白D3调控下游基因并通过定量实时PCR验证。

结果与结论：与原代培养的正常妊娠滋养细胞相比，绒癌细胞株JAR中叉头蛋白D3的转录和蛋白水平表达均显著增高。短发夹RNA干扰叉头蛋白D3表达抑制了体外JAR的细胞增殖、迁移和侵袭功能，同时抑制了裸鼠体内肿瘤的生长，降低了肿瘤细胞β-人绒毛膜促性腺激素分泌。通过初筛和后期验证，发现7个受叉头蛋白D3调控的局部黏附分子(ITGA5, ITGB6, THBS4, COL6A3, VTN, NRXN3和NLGN1)，同时短发夹RNA干扰叉头蛋白D3表达后，JAR细胞内局部黏附激酶活性降低。提示过表达的叉头蛋白D3通过调控局部黏附分子表达和局部黏附激酶活性进而增强JAR的恶性生物学特性。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

ORCID: 0000-0002-9548-1702(Zhang Hui-juan)

关键词: 干细胞, 肿瘤干细胞, 绒毛膜细胞癌, JAR, 叉头蛋白D3, 增殖, 迁移, 侵袭, 黏附分子, 黏附激酶, 短发夹RNA干扰, 基因芯片, 国家自然科学基金

Abstract:

BACKGROUND: Choriocarcinoma is a kind of trophoblastic neoplasm with highly aggressive phenotypes. Forkhead box D3 (FoxD3) is an embryonic and trophoblastic stem cell transcription factor. It plays important roles in different physical and pathological situations such as embryogenesis, carcinogenesis and tumor progression.

OBJECTIVE: To investigate the role of FoxD3 in choriocarcinoma malignancy and the possible mechanism.

METHODS: The human choriocarcinoma JAR cell line was employed in this study. The mRNA and protein expressions of genes were measured by quantitative RT-PCR (qRT-PCR) and western blot, respectively. The FoxD3 specific short hair RNA was applied to down-regulate gene expression. The cell proliferation was evaluated in vitro by cell counting assay and in vivo by tumor growth. The migration/invasion was determined by transwell assay. The profile of FoxD3 targeted genes was investigated with an Agilent microarray and verified by qRT-PCR.

RESULTS AND CONCLUSION: The FoxD3 mRNA and protein expressions in JAR cells were significantly higher than those in primarily cultured normal trophoblastic cells. Knockdown of FoxD3 by short hair RNA in JAR cells could inhibit cell proliferation and migration/invasion in vitro, and suppress the tumor growth with decreased β-human chorionic gonadotropin secretion in vivo. A profile of seven focal adhesion molecules (ITGA5, ITGB6, THBS4, COL6A3, VTN, NRXN3 and NLGN1) was verified to be targeted by FoxD3. Furthermore, knockdown of FoxD3 by short hair RNA could decrease the activation of focal adhesion kinase. All these findings suggest the overexpression of FoxD3 can contribute to the aggressive phenotype of choriocarcinoma JAR cells by regulating the profile of focal adhesion molecules and focal adhesion kinase.

刘媛，王亚欣，吴维宾，王玉东，张慧娟. 滋养干细胞转录因子叉头蛋白D3 过表达在人绒癌细胞株JAR的恶性生物学特性中的作用研究[J]. 中国组织工程研究, 2016, 20(10): 1409-1418.

Liu Yuan, Wang Ya-xin, Wu Wei-bin, Wang Yu-dong, Zhang Hui-juan. Overexpression of trophoblastic stem cell transcription factor, forkhead box D3, contributes to malignancy of human choriocarcinoma JAR cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(10): 1409-1418.

图/表（结果） 4

The FoxD3 expression in JAR cells was significantly higher than that in PHT cells

We found that the endogenous FoxD3 expression was found in normal PHT cells. The mRNA expression of FoxD3 in JAR cells was significantly higher than that in normal PHT cells (P < 0.05; Figure 1A). The protein expression of FoxD3 showed the similar pattern as mRNA (Figure 1B).

ShFoxD3 in JAR cells impaired cell functions in vitro and suppressed tumor growth with decreased β-hCG secretion in vivo

To better understand the role of FoxD3 in the pathogenesis of choriocarcinoma, the functional changes were explored in JAR cells with FoxD3 knockdown. In vitro, compared with control cells, shFoxD3-transfected JAR cells demonstrated significantly retarded proliferation (P < 0.001; Figure 2A, B), markedly impeded migratory and invasive capacities (P < 0.001; Figure 2C, D). In vivo, significantly decreased tumor volume was observed in mice of the shFoxD3 group compared to the control (P < 0.001; Figure 2E, F). Additionally, the serum β-hCG level in shFoxD3 group mice was significantly lower than that in control group (P <0.001; Figure 2G).

FoxD3 related gene profile screened by microarray and validated by qRT-PCR

To get an overall view of genes affected by FoxD3 in JAR cells, the mRNA microarray analysis was performed on JAR cells with/without shFoxD3 treatment. A total of 42 400 genes were screened for mRNA expression, of which 2 315 genes were found to be differentially expressed with 982 genes upregulated and 1 333 genes downregulated after knockdown of FoxD3 (Figure 3A). The very close relationship between FoxD3 and focal adhesion was disclosed by pathway analysis (Figure 3B), biological process analysis (Figure 3C) and cellular component analysis (Figure 3D). We further fixed 11 genes which were commonly demonstrated in aforementioned three analysis results, and 7 genes of them were validated by qRT-PCR to be indeed changed after FoxD3 knockdown (Figure 3E). ITGA5, ITGB6, THBS4, COL6A3 and VTN genes were demonstrated to be significantly up-regulated while NRXN 3 and NLGN1 were down-regulated (Figure 3F).

Activation of focal adhesion kinase (FAK) was markedly down-regulated after FoxD3 knockdown in JAR cells

FAK is crucial for cellular focal adhesion. By responding to transmembrane integrins, the activation of FAK has been reported to promote cell phenotype of invasion and movement through motility-associated signaling pathways^[21-22].

To further make clear the effect of FoxD3 on focal adhesion microenvironment, the association between FoxD3 and FAK was also investigated in the current study. Consistently, we demonstrated the activated

FAK (pFAKTyr397) was substantially decreased in shFoxD3 transfected JAR cells compared with control cells while the total FAK level kept unchanged (Figure 4).

参考文献

[1] Huang CY, Chen CA, Hsieh CY, et al. Intracerebral hemorrhage as initial presentation of gestational choriocarcinoma: a case report and literature review. Int J Gynecol Cancer. 2007;17(5):1166-1171.

[2] Hensley JG, Shviraga BA. Metastastic choriocarcinoma in a term pregnancy: a case study. MCN Am J Matern Child Nurs. 2014;39(1):8-15; quiz 16-17.

[3] McDonald TW, Ruffolo EH. Modern management of gestational trophoblastic disease. Obstet Gynecol Surv. 1983;38(2):67-83.

[4] Altieri A, Franceschi S, Ferlay J, et al. Epidemiology and aetiology of gestational trophoblastic diseases. Lancet Oncol. 2003;4(11):670-678.

[5] Soper JT. Gestational trophoblastic disease. Obstet Gynecol. 2006;108(1):176-187.

[6] Kohorn EI. Worldwide survey of the results of treating gestational trophoblastic disease. J Reprod Med. 2014; 59(3-4):145-153.

[7] Sutton J, Costa R, Klug M, et al. Genesis, a winged helix transcriptional repressor with expression restricted to embryonic stem cells. J Biol Chem. 1996;271(38):23126-23133.

[8] Plank JL, Suflita MT, Galindo CL, et al. Transcriptional targets of Foxd3 in murine ES cells. Stem Cell Res. 2014;12(1):233-240.

[9] Yaklichkin S, Steiner AB, Lu Q, et al. FoxD3 and Grg4 physically interact to repress transcription and induce mesoderm in Xenopus. J Biol Chem. 2007;282(4): 2548-2557.

[10] Douglas GC, VandeVoort CA, Kumar P, et al. Trophoblast stem cells: models for investigating trophectoderm differentiation and placental development. Endocr Rev. 2009;30(3):228-240.

[11] Pan G, Li J, Zhou Y, et al. A negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal. FASEB J. 2006;20(10):1730-1732.

[12] Tompers DM, Foreman RK, Wang Q, et al. Foxd3 is required in the trophoblast progenitor cell lineage of the mouse embryo. Dev Biol. 2005;285(1):126-137.

[13] Liu Y, Labosky PA. Regulation of embryonic stem cell self-renewal and pluripotency by Foxd3. Stem Cells. 2008;26(10):2475-2484.

[14] Chu TL, Zhao HM, Li Y, et al. FoxD3 deficiency promotes breast cancer progression by induction of epithelial-mesenchymal transition. Biochem Biophys Res Commun. 2014;446(2):580-584.

[15] Liu LL, Lu SX, Li M, et al. FoxD3-regulated microRNA-137 suppresses tumour growth and metastasis in human hepatocellular carcinoma by targeting AKT2. Oncotarget. 2014;5(13):5113-5124.

[16] Katiyar P, Aplin AE. FOXD3 regulates migration properties and Rnd3 expression in melanoma cells. Mol Cancer Res. 2011;9(5):545-552.

[17] Zhang H, Hou L, Li CM, et al. The chemokine CXCL6 restricts human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester. Hum Reprod. 2013;28(9):2350-2362.

[18] Liu Y, Wu J, Wu H, et al. UCH-L1 expression of podocytes in diseased glomeruli and in vitro. J Pathol. 2009;217(5):642-653.

[19] Zhang HJ, Siu MK, Wong ES, et al. Oct4 is epigenetically regulated by methylation in normal placenta and gestational trophoblastic disease. Placenta. 2008;29(6):549-554.

[20] Holmberg H, Kiersgaard MK, Mikkelsen LF, et al. Impact of blood sampling technique on blood quality and animal welfare in haemophilic mice. Lab Anim. 2011;45(2): 114-120.

[21] Schlaepfer DD, Mitra SK, Ilic D. Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochim Biophys Acta. 2004;1692(2-3):77-102.

[22] Schaller MD. Cellular functions of FAK kinases: insight into molecular mechanisms and novel functions. J Cell Sci. 2010;123(Pt 7):1007-1013.

[23] Perera HK, Caldwell ME, Hayes-Patterson D, et al. Expression and shifting subcellular localization of the transcription factor, Foxd3, in embryonic and adult pancreas. Gene Expr Patterns. 2006;6(8):971-977.

[24] Plank JL, Suflita MT, Galindo CL, et al. Transcriptional targets of Foxd3 in murine ES cells. Stem Cell Res. 2014;12(1):233-240.

[25] Stewart RA, Arduini BL, Berghmans S, et al. Zebrafish foxd3 is selectively required for neural crest specification, migration and survival. Dev Biol. 2006; 292(1):174-188.

[26] Liu LL, Lu SX, Li M, et al. FoxD3-regulated microRNA-137 suppresses tumour growth and metastasis in human hepatocellular carcinoma by targeting AKT2. Oncotarget. 2014;5(13):5113-5124.

[27] Schmid CA, Müller A. FoxD3 is a novel, epigenetically regulated tumor suppressor in gastric carcinogenesis. Gastroenterology. 2013;144(1):22-25.

[28] Siu MK, Wong ES, Chan HY, et al. Overexpression of NANOG in gestational trophoblastic diseases: effect on apoptosis, cell invasion, and clinical outcome. Am J Pathol. 2008;173(4):1165-1172.

[29] Cheung M, Chaboissier MC, Mynett A, et al. The transcriptional control of trunk neural crest induction, survival, and delamination. Dev Cell. 2005;8(2): 179-192.

[30] Förster S, Gretschel S, Jöns T, et al. THBS4, a novel stromal molecule of diffuse-type gastric adenocarcinomas, identified by transcriptome-wide expression profiling. Mod Pathol. 2011;24(10): 1390-1403.

[31] Goel HL, Li J, Kogan S, et al. Integrins in prostate cancer progression. Endocr Relat Cancer. 2008;15(3): 657-664.

[32] Greco SA, Chia J, Inglis KJ, et al. Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation. BMC Cancer. 2010;10:494.

[33] Xiong G, Deng L, Zhu J, et al. Prolyl-4-hydroxylase α subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition. BMC Cancer. 2014;14:1.

[34] Pirazzoli V, Ferraris GM, Sidenius N. Direct evidence of the importance of vitronectin and its interaction with the urokinase receptor in tumor growth. Blood. 2013; 121(12): 2316-2323.

[35] Goel HL, Li J, Kogan S, et al. Integrins in prostate cancer progression. Endocr Relat Cancer. 2008;15(3): 657-664.

[36] Wang Y, Shenouda S, Baranwal S, et al. Integrin subunits alpha5 and alpha6 regulate cell cycle by modulating the chk1 and Rb/E2F pathways to affect breast cancer metastasis. Mol Cancer. 2011;10:84.

[37] Bot N, Schweizer C, Ben Halima S, et al. Processing of the synaptic cell adhesion molecule neurexin-3beta by Alzheimer disease alpha- and gamma-secretases. J Biol Chem. 2011;286(4):2762-2773.

[38] Golubovskaya VM. Targeting focal adhesion kinase in cancer-part I. Anticancer Agents Med Chem. 2010; 10(10):713.

[39] Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol. 2005;6(1):56-68.

[40] Ili? D, Genbacev O, Jin F, et al. Plasma membrane-associated pY397FAK is a marker of cytotrophoblast invasion in vivo and in vitro. Am J Pathol. 2001;159(1):93-108.

引言

Choriocarcinoma is a highly aggressive gestational trophoblastic neoplasm with the features of strong destruction to local tissues and metastasis to distant organs such as the lung, liver, and brain^[1-2]. The incidence of choriocarcinoma varies from 2 to 202 in 100 000 pregnancies, which is higher in Orientals than in Whites^[3-4]. Gestational choriocarcinomas are diagnosed after any kinds of pregnancies, 50% after term pregnancies and 25% after molar pregnancies^[5]. Although choriocarcinoma is a highly curable tumor, a mortality rate of 13.4% has been reported in post-term choriocarcinoma ^[6]. Understanding the

exact mechanisms that govern the pathogenesis of choriocarcinoma is thus very important for discovering new molecular therapeutic targets.

Forkhead box D3 (FoxD3), also termed Genesis and HFH2, belongs to the forkhead transcription factor family^[7]. It is located on chromosome 1p31.3 in human (NM_012183).

FoxD3 functions either as a transcription activator or as a transcription repressor in the different cell lineage progenitors of early embryos^[8-11]. Murine FoxD3^-/- embryo dies early because of placenta defects^[12]. Specifically, FoxD3 determines the fate of trophoblast cell lineage and maintains the pluripotency and renewal of trophoblastic stem cells^[12-13]. Research on FoxD3 in the aspect of tumourigenesis and tumor progress is becoming hot in recent years^[14-16]. However, the role of FoxD3 in trophoblastic tumor has never been investigated.

In the current study, a human placenta-derived choriocarcinoma JAR cell line was manipulated to detect FoxD3 expression, and to investigate its function as well as associated downstream effectors via short hair RNA (shRNA) knockdown.

材料方法

Design

Cytological test in vitro and randomized controlled animal experiment.

Time and setting

Experiments were performed in the Central Laboratory of International Peace Maternity and Child Health Hospital, China and the Animal Experimental Center of Tongji University from September 2012 to July 2014.

Materials

Human choriocarcinoma JAR cell line was obtained from Shanghai Cell Bank of Chinese Academy of Sciences, Shanghai, China. The primary human trophoblast-12 gestational weeks) with signed consent. SPF BALB/c nude female mice at 5 weeks of age were purchased from Slac Laboratory Animal Co., Ltd., Shanghai, China and maintained in a SPF environment with free access to water and food (animal license No. SYXK (Hu) 2014-0026). (PHT) cells were from normal first trimester placentas (7

Methods

Cell lines and stable transfection of plasmid-shRNA

Human choriocarcinoma JAR cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 ℃ in 5% CO₂. Plasmid-based anti-FoxD3 shRNA (shFoxD3) with a GFP tag (pGFP-V-RS-shFoxD3, OriGene Technologies, Rockville, MD, USA) was constructed. One day before transfection, JAR cells were seeded at 90% confluence on 24-well plates. The plasmid-shRNA was added with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) to each well according to manufacturer’s recommendation. In the next 1-2 months, shRNA-positive cells were recognized by GFP in fluorescence and selected using puromycin (Sigma-Aldrich, St. Louis, MO, USA) at an optimized concentration of 1 μg/mL and stable shRNA cell clones were established. JAR cells transfected with scramble shRNA plasmids served as controls.

Isolation and culture of normal PHT cells

The normal PHT cells were isolated and cultured according to a previous report with some modification^[17]. Briefly, placental pieces were digested with 0.25% trypsin (Invitrogen) and

200 IU/L DNase I (Sigma-Aldrich) for 15 minutes at 37 ℃ with agitation. The resuspended and pooled cell suspension was layered over a discontinuous Percoll (GE Healthcare, Piscataway, NJ, USA) gradient (15%, 25%, 30%, 40%, 50%, 60% and 70%) and centrifuged at 1 100×g for 20 minutes. The 25%–40% layer was collected for trophoblast isolation. The isolated PHT cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 IU/mL penicillin and 100 mg/mL streptomycin at 37 ℃

in 95% air, 5% CO₂.

Quantitative real time-PCR (qRT-PCR)

Total RNA was isolated by 500 μL Trizol reagent (Invitrogen) per 6-cm dish. The first strand cDNA was synthesized using M-MLV RNaseH-deficient reverse transcriptase (Takara Biotechnology, Dalian, China). 2 μL cDNA dilution (1:4) was used for qRT-PCR with SYBR Green PCR Master Mix (Takara) by ABI Prism 7700 Sequence Detector (Life Technologies, Gaithersburg, MD, USA).

PCR reactions were performed in triplicate. Primers used for qRT-PCR are listed in Table 1. Ct values of the sample were calculated and transcript levels were analyzed by 2^-ΔΔCt method.

Microarray experiment

RNA extracted from JAR stably transfected by either scramble shRNA or FoxD3 shRNA plasmid was transferred to CapitalBio Corporation in Beijing (http://bioservice.capitalbio.com) for labeling and hybridization with Agilent SurePrint G3 Human Gene Expression 8x60K genechip according to the manufacturer’s instruction. Data-mining analyses such as pathway and gene ontology analyses were performed. The database used by these analyses tools is Kyoto Encyclopedia of Genes and Genomes (KEGG).

Western blot assay

Briefly, cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer (Beyotime, Shanghai, China) adding a 1% dilution of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) (Beyotime)^[18]. The supernatant was separated by electrophoresis in 8% SDS-polyacrylamide gel.

Cell proliferation assay

The cell counting kit-8 (CCK-8, Dojindo, Japan) colorimetric assay was used to measure cell proliferation in triplicate experiments. First, 5×10³ cells per well were seeded in a 96-well plate. The number of viable cells was then assessed using this kit after culture for 0, 24, 48, 72 and 96 hours, and the absorbance at 450 nm was measured with a Model 680 plate reader (Bio-Rad Laboratories, CA, USA).

The boyden chamber technique (transwell analysis) was performed^[19]. Briefly, 6.5 mm transwell chambers with 8 μm pores (Corning, Inc., Corning, NY, USA) were coated with matrigel or not. 1×10⁵ cells/well were added to the upper chambers and 600 μL DMEM with 10% fetal bovine serum to the lower ones, and then incubated for 48 hours. The remaining cells attached to the upper surface of the inserts were removed with cotton swabs. Migrated cells in the lower surface were stained with 0.1% crystal violet for 10 minutes at room temperature. The migration ability was presented as mean percentage of cell number counted in five fields by LEICA DMI 3000 light microscopy (×200 magnification) compared to control as 100%.

Tumor formation in nude mice

JAR and stably transfected JAR cells with shFoxD3 were suspended in physiological saline with a dilution of 1×10⁸ cells/mL. Twelve BALB/c female mice at 5 weeks of age were divided equally into two groups (control group and shFoxD3 group). Both hindlegs of each mouse were injected with 0.1mL of cell solution subcutaneously. The tumor volume defined as (length×width²)/2 was measured every 2-3 days until the mice were sacrificed after 23 days. The average tumor volumes between the two groups were statistically analyzed. All in vivo experiments were performed in accordance with the institutional animal experiment guidelines.

ELISA assay

The human chorionic gonadotropin (β unit, β-hCG) concentration in mice serum collected by retrobulbar puncture was detected using β-hCG testing kit with electro-chemiluminescence immunoassay (Roche, Mannheim, Germany)^[20].

Main outcome measures

In vitro, cytological functional detection including proliferation, migration and invasion, and expressional detection in JAR at mRNA and protein levels. In vivo, tumor detection in tumor formation test of nude mice.

Statistical analysis

Data are expressed as mean±SD and were statistically analyzed using the Statistical Package for Social Science 15.1 for Windows (SPSS Inc., Chicago, USA) and GraphPad Prism 3.02 for Windows (GraphPad Software, San Diego, CA). Independent t-test and two-way analysis of variance was used for continuous data and Mann-Whitney U test for comparison between two groups of non-parametric data. P < 0.05 was defined as statistical significance.

讨论

FoxD3, as an embryonic and trophoblastic stem cell transcription factor, has been extensively explored in the fields of embryogenesis, specification of diversity cell lineages and development of specific organs^{[12, 23-25]}. In recent years, FoxD3 related research has been broadened to tumorigenesis, and mainly focused on somatic cancers such as hepatic cancer, breast cancer and gastric cancer^{[14, 26-27]}. In our current study on JAR cells, we originally discovered that FoxD3 virtually plays an oncogenic role in trophoblastic tumors. Overexpression of FoxD3 in JAR cells promotes aberrant proliferation, migration and invasion. Considering FoxD3 is a positive activator of another embryonic stem cell transcription factor Nanog by binding to the Nanog promoter^[11], our results were logically in accordance with a previous study which demonstrated that Nanog is responsible for the aggressive phenotype of choriocarcinoma cells and clinical outcomes of gestational trophoblastic disease^[28].

The defective placentas in FoxD3^-/- mice give the fact that FoxD3 is critically required in the trophoblastic cell lineage^[12]. The maintenance of stemness by FoxD3 in mouse trophoblastic cells suggests FoxD3 is important to the expansion of trophoblastic cell population in the development of placenta^[13]. However, overexpression of FoxD3 can lead to aberrant cell proliferation and tumor growth as demonstrated in our study. Research has also revealed FoxD3 is pivotal to the migration of neural crest cells by regulating cell adhesion molecules such as N-cadherin, laminin and intergrin-β1^[29]. Consistently, we found knockdown of FoxD3 in JAR cells were closely bound up with dysregulated expression of focal adhesion factors. By microarray analysis and qRT-PCR, we identified seven FoxD3 related focal adhesion genes, ITGA5, ITGB6, THBS4, COL6A3, VTN, NRXN3 and NLGN1. ITGA5 and ITGB6, code integrin α5 and β6 subunits while THBS4, COL6A3 and VTN genes code ligands thrombospondin-4, collagen VI and vitronectin proteins, respectively^[30-31]. Accumulating evidences have approved the dysbalanced integrin-ligand network plays important roles in carcinogenesis. Integrins/ligands have variable functions either as tumor suppressors or as oncoproteins in tumourigenesis and metastasis^[32-36]. Our current study indicated that FoxD3 inhibited the expressions of integrins and their ligands in JAR cells, which might make the cells easily detached from surrounding extracellular matrix to move and metastasis. As another pair of transmembrane receptor and ligand, the NRXN/NLGN complex is crucial for synaptic cell adhesion^[37]. The high expression of NRXN/NLGN induced by FoxD3 in JAR cells may promote the migration and invasion in an unclear mechanism, which need to be studied further.

滋养干细胞转录因子叉头蛋白D3 过表达在人绒癌细胞株JAR的恶性生物学特性中的作用研究

Overexpression of trophoblastic stem cell transcription factor, forkhead box D3, contributes to malignancy of human choriocarcinoma JAR cells

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