miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines.
miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines.
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DOI：
10.1016/j.bbrc.2010.10.130
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发表时间：
2010-12-03
影响因子：
3.1
通讯作者：
Schmittgen TD
中科院分区：
文献类型：
--
作者：
Henry JC;Park JK;Jiang J;Kim JH;Nagorney DM;Roberts LR;Banerjee S;Schmittgen TD
我们和其他人以前的工作报告了miR-199a-3p在肝细胞癌(HCC)组织中的表达低于邻近的良性组织。我们在此报告了在7个肝癌细胞系中miR-199a-3p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miR-199a-3p前寡核苷酸导入肝癌细胞系。仅在两个细胞系(SNU449和SNU423)中，前miR-199a-3p寡核苷酸使细胞增殖抑制约60%，其余5个处理细胞系的增殖与对照寡核苷酸相似。一种miR-199a-3p之前的寡核苷酸，经过化学修饰以增强稳定性，同时保持加工，在SNU449和SNU423中抑制细胞增殖的程度与商业上可获得的miR-199a-3p之前的寡核苷酸相同。此外，只有miR-199a-3p双链寡核苷酸，而不是单独的引导链，才能有效地降低细胞存活率。由于CD44变异体是c-Met信号转导的关键，而c-Met是已知的miR-199a-3p靶标，我们假设miR-199a-3p也可能靶标CD44。免疫印迹法证实，只有对前miR-199a-3p敏感的两个肝癌细胞系为CD44+。通过荧光素酶报告实验和免疫印迹证实miR-199a-3p直接靶向CD44。将miR-199a-3p基因导入SNU449细胞，可降低细胞体外侵袭能力，并使细胞对阿霉素增敏，加入HA后，上述两种作用均增强。在原发性肝癌组织中，miR-199a-3p和CD44蛋白的表达呈负相关。MiR-199a-3p选择性杀伤CD44+肝癌的能力可能是一种有用的靶向治疗CD44+肝癌的方法。
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 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 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.
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