Endoglin (CD105) and SMAD4 regulate spheroid formation and the suppression of the invasive ability of human pancreatic cancer cells
Endoglin (CD105) and SMAD4 regulate spheroid formation and the suppression of the invasive ability of human pancreatic cancer cells
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DOI：
10.3892/ijo.2018.4262
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发表时间：
2018-03-01
影响因子：
5.2
通讯作者：
Imura, Johji
中科院分区：
文献类型：
--
作者：
Kokaji, Eri;Shimomura, Akiko;Imura, Johji
在这项研究中，我们研究了胰腺癌细胞系形成球状体的能力，目的是确定参与细胞侵袭的因素，这是导致胰腺癌预后不良的一种特性。为此目的，在非贴壁培养条件下培养8种来源于人胰腺癌组织的细胞系以形成球状体以及正常单层。观察细胞形态，测量球体直径。在两种培养条件下培养的细胞之间比较mRNA表达。使用siRNA在球状体中进行内皮糖蛋白（ENG）和SMAD 4（转化生长因子-（TGF-）信号传导系统的组分）的基因敲低，以鉴定受影响的蛋白质信号传导因子，确定随时间发生的形态学变化，并测量构成球状体的细胞的侵袭能力。细胞系表现出不同的球体形成能力。SMAD 4和ENG的表达在形成球状体的细胞中伴随增加。SMAD 4在球状体形成时进入细胞核。在SMAD 4被敲低的细胞中，ENG的表达降低;相反，BMP和激活素膜结合抑制剂（BAMBI）和头蛋白（NOG）（TGF-信号传导系统的进一步组分）的表达增加。在ENG被敲低的细胞中，观察到TGF-2型受体（TGFBR 2）和SMAD 9的mRNA表达降低，以及pSMAD 1/5/9表达的变化，以及球状体尺寸减小的趋势。在基质胶上培养的球状体表现出随着时间的推移尺寸减小的趋势，以及侵入基质胶的趋势。特别地，ENG被敲除的细胞表现出尺寸减小的球状体，并且还表现出侵袭性增加和侵袭性降低。因此，我们的数据表明，在胰腺癌细胞中，ENG的表达可能是由SMAD 4介导的途径控制的。此外，ENG被发现与细胞的球体形成能力有关，并参与胰腺癌细胞的侵袭能力。
In this study, we investigated the ability of pancreatic cancer cell lines to form spheroids with the aim of identifying factors involved in cell invasiveness, a property that leads to a poor prognosis in pancreatic cancer. For this purpose, 8 cell lines derived from human pancreatic cancer tissues were cultured in non-adherent culture conditions to form spheroids, as well as normal monolayers. The morphology of the cells was observed and spheroid diameters measured. mRNA expression was compared between cells cultured under both culture conditions. The gene knockdown of endoglin (ENG) and SMAD4, components of the transforming growth factor- (TGF-) signaling system, using siRNAs was conducted in spheroids in order to identify affected protein signaling factors, determine the morphological changes occurring over time and to measure the invasive capacity of the cells constituting spheroids. The cell lines exhibited differences in their spheroid-forming abilities. The expression of SMAD4 and ENG concomitantly increased in the cells that formed spheroids. SMAD4 was transported into the nucleus when spheroids were formed. The expression of ENG was decreased in the cells in which SMAD4 was knocked down; by contrast, the expression of BMP and activin membrane-bound inhibitor (BAMBI) and noggin (NOG), further components of the TGF- signaling system, increased. In the cells in which ENG was knocked down, the decreased mRNA expression of TGF- receptor type 2 (TGFBR2) and SMAD9 was observed, as well as a change in the expression of pSMAD1/5/9, and a tendency of spheroids to decrease in size. Spheroids cultured on Matrigel exhibited a tendency towards a reduction in size over time, as well as a tendency to invade into the Matrigel. In particular, the cells in which ENG was knocked down exhibited spheroids which were reduced in size, and also exhibited an increase in invasiveness, and a decrease in adhesiveness. Thus, our data indicate that in pancreatic cancer cells, the expression of ENG may be controlled by a pathway mediated by SMAD4. In addition, ENG was found to be related to the spheroid-forming ability of cells and to be involved in the invasive capacity of pancreatic cancer cells.