Department of Surgery, Ohio State University Medical Center, Columbus, OH, USA.
Biochem Biophys Res Commun. 2010 Dec 3;403(1):120-5. doi: 10.1016/j.bbrc.2010.10.130. Epub 2010 Nov 3.
Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid (HA) was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC specimens. The ability of miR-199a-3p to selectively kill CD44+ HCC may be a useful targeted therapy for CD44+ HCC.
先前我们和其他研究人员的工作表明，miR-199a-3p 在肝癌 (HCC) 组织中的表达低于相邻良性组织。我们在此报告 miR-199a-3p 在 7 种 HCC 细胞系中的表达显著降低。为了确定 miR-199a-3p 是否具有肿瘤抑制作用，将 pre-miR-199a-3p 寡核苷酸转染到 HCC 细胞系中。与对照寡核苷酸相比，只有两种细胞系 (SNU449 和 SNU423) 的 pre-miR-199a-3p 寡核苷酸将细胞增殖减少了约 60%；其他 5 种处理细胞系的增殖与对照寡核苷酸相似。用化学修饰来增强稳定性同时保留加工的 pre-miR-199a-3p 寡核苷酸，在 SNU449 和 SNU423 中使细胞增殖减少到与市售的 pre-miR-199a-3p 寡核苷酸相同的程度。此外，只有双链 miR-199a-3p 寡核苷酸，而不是单独的引导链，能够有效降低细胞活力。由于 CD44 变体对于 c-Met 信号传导至关重要[V. Orian-Rousseau、L. Chen、J.P. Sleeman、P. Herrlich、H. Ponta，CD44 是 HGF/c-Met 信号传导的两个连续步骤所必需的，基因开发 16(2002)3074-3086]，并且 c-Met 是已知的 miR-199a-3p 靶标，我们假设 miR-199a-3p 也可能靶向 CD44。免疫印迹证实，只有对 pre-miR-199a-3p 的作用敏感的两种 HCC 系是 CD44+。使用荧光素酶报告基因测定和免疫印迹证实 miR-199a-3p 直接靶向 CD44。将 miR-199a-3p 转染到 SNU449 细胞中可降低体外侵袭并使细胞对阿霉素敏感；当向细胞培养物中添加透明质酸 (HA) 时，这两种作用均增强。在原发性 HCC 标本中观察到 miR-199a-3p 的表达与 CD44 蛋白之间存在负相关。miR-199a-3p 选择性杀死 CD44+HCC 的能力可能是针对 CD44+HCC 的有用靶向治疗方法。
