Department of Urology, The Central Hospital of Linyi, Yishui, Shandong, China.
Department of Urology, Yantai Yuhuangding Hospital, Yantai, Shandong, China.
Cancer Biomark. 2018;21(4):915-923. doi: 10.3233/CBM-171018.
Clusterin promotes cell proliferation, motility and invasiveness in human renal cell carcinoma (RCC) cells but the underlying molecular mechanisms of this action are largely unknown. The aim of this study was to investigate the effects of clusterin on cancer cell growth, invasion and S100A4 expression and to determine the effects of clusterin on in vitro cell proliferation and migration and in vivo tumour growth in RCC cells.
We have established stable transfectants of highly invasive Caki-1 human RCC cells with expression of clusterin shRNA targeting clusterin (Caki-1/clusterin shRNA). We also established stable transfectants of 786-O human RCC cells with expression of clusterin cDNA plaismid (786-O/clusterin cDNA). Clusterin and S100A4 expression was detected by reverse transcription (RT) PCR and western blot assay; Caki-1/clusterin shRNA and 786-O/clusterin cDNA clones were subjected to in vitro-invasion assays. Cell viability and cell growth was assessed in MTT and clonogenic assay. Specific small interfering RNA was employed to down-regulate S100A4. The expression plasmid for S100A4 (pCMV-S100A4) was used to upregulate S100A4. Caki-1/clusterin shRNA clones were injected subcutaneously in nude mice to determine tumour growth and cancer cell invasiveness in vivo. Xenograft tumour tissues were assessed by immunohistochemistry and frozen tissues were used for the detection of S100A4 and clusterin.
Overexpression of clusterin increased cell invasiveness; and targeting clusterin reduced cell invasiveness in vitro. This increase in cell invasiveness was mediated by S100A4. Targeting clusterin decreased cell proliferation and down-regulated cellular S100A4 levels in Caki-1 cells; Overexpression of clusterin increased cell proliferation and up-regulated cellular S100A4 levels in 786-O cells; Stable Caki-1/clusterin shRNA transfectants produced smaller xenograft tumours containing reduced S100A4 protein levels in vivo. Stable 786-O/clusterin cDNA transfectants produced larger xenograft tumours containing increased S100A4 protein levels in vivo.
Our results indicate that clusterin promotes growth and invasion in RCC cells in vitro and in vivo through upregulation of S100A4; And targeting clusterin confers growth inhibitory and anti-invasive properties in RCC cells in vitro and in vivo through a down-regulation of S100A4. These findings provide the rationale for future oncostatic strategies aimed at suppressing clusterin-mediated signal transduction pathways as a novel therapeutic approach in human RCC.
簇集蛋白可促进人肾细胞癌（RCC）细胞的增殖、迁移和侵袭，但这种作用的潜在分子机制在很大程度上尚不清楚。本研究旨在探讨簇集蛋白对癌细胞生长、侵袭和 S100A4 表达的影响，并确定簇集蛋白对体外细胞增殖和迁移以及体内 RCC 细胞肿瘤生长的影响。
我们建立了表达簇集蛋白 shRNA 的高侵袭性 Caki-1 人 RCC 细胞的稳定转染子（Caki-1/clusterin shRNA）。我们还建立了表达簇集蛋白 cDNA 质粒的 786-O 人 RCC 细胞的稳定转染子（786-O/clusterin cDNA）。通过逆转录（RT）PCR 和 Western blot 检测簇集蛋白和 S100A4 的表达；对 Caki-1/clusterin shRNA 和 786-O/clusterin cDNA 克隆进行体外侵袭试验。MTT 和集落形成试验评估细胞活力和细胞生长。采用特异性小干扰 RNA 下调 S100A4。S100A4 的表达质粒（pCMV-S100A4）用于上调 S100A4。将 Caki-1/clusterin shRNA 克隆注射到裸鼠皮下以确定体内肿瘤生长和癌细胞侵袭。通过免疫组织化学检测异种移植肿瘤组织，并使用冷冻组织检测 S100A4 和簇集蛋白。
簇集蛋白的过表达增加了细胞侵袭性；靶向簇集蛋白降低了体外细胞侵袭性。这种细胞侵袭性的增加是由 S100A4 介导的。靶向簇集蛋白降低了 Caki-1 细胞的增殖并下调了细胞内 S100A4 水平；簇集蛋白的过表达增加了 786-O 细胞的增殖并上调了细胞内 S100A4 水平；稳定的 Caki-1/clusterin shRNA 转染子在体内产生了较小的异种移植肿瘤，其中 S100A4 蛋白水平降低。稳定的 786-O/clusterin cDNA 转染子在体内产生了较大的异种移植肿瘤，其中 S100A4 蛋白水平升高。
我们的研究结果表明，簇集蛋白通过上调 S100A4 促进 RCC 细胞在体外和体内的生长和侵袭；靶向簇集蛋白通过下调 S100A4 在体外和体内赋予 RCC 细胞生长抑制和抗侵袭特性。这些发现为抑制簇集蛋白介导的信号转导途径提供了依据，作为人类 RCC 的一种新的治疗方法。
