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（英文）

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

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。

BACKGROUND： Choriocarcinoma is a kind of trophoblastic neoplasm with highly aggressive phenotypes. Forkhead boxD3（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 mR NA 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 FoxD 3 targeted genes was investigated with an Agilent microarray and verified by qR T-PCR. RESULTS AND CONCLUSION： The FoxD3 m RNA 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.