Department of Molecular Pathology, Faculty of Medicine, Oita University, Oita, Japan.
Mol Cancer. 2013 Feb 25;12:15. doi: 10.1186/1476-4598-12-15.
Previously, using miRNA microarray, we have found that miR-29c is significantly downregulated in advanced gastric carcinoma. In the present study, we investigated whether miR-29c functions as a tumor-suppressor miRNA in gastric carcinoma cells. For this purpose, we verified the downregulation of miR-29c in gastric carcinoma tissues, and assessed the biological effect of miR-29c on gastric carcinoma cells.
In miR-29c-transfected cells, both proliferation and colony formation ability on soft agar were significantly decreased. Although apoptosis was not induced, BrdU incorporation and the proportion of cells positive for phospho-histone H3 (S10) were significantly decreased in miR-29c-transfected cells, indicating that miR-29c may be involved in the regulation of cell proliferation. To explain the mechanism of growth suppression by miR-29c, we explored differentially expressed genes (>2-fold) in miR-29c-transfected cells in comparison with negative control transfected cells using microarray. RCC2, PPIC and CDK6 were commonly downregulated in miR-29c-transfected MKN45, MKN7 and MKN74 cells, and all of the genes harbored miR-29c target sequences in the 3'-UTR of their mRNA. RCC2 and PPIC were actually upregulated in gastric carcinoma tissues, and therefore both were identified as possible targets of miR-29c in gastric carcinoma. To ascertain whether downregulation of RCC2 and/or PPIC is involved in the growth suppression by miR-29c, we transfected siRNAs against RCC2 and PPIC into MKN45 and determined cell viability, the rate of BrdU incorporation, and caspase activity. We found that RCC2-knockdown decreased both cell viability and BrdU incorporation without any increase of caspase activity, while PPIC-knockdown did not, indicating that downregulation of RCC2 may be at least partly responsible for the growth suppression by miR-29c.
Our findings indicate that miR-29c may have tumor-suppressive functions in gastric carcinoma cells, and that its decreased expression may confer a growth advantage on tumor cells via aberrant expression of RCC2.
此前，我们通过 miRNA 微阵列发现 miR-29c 在晚期胃癌中显著下调。在本研究中，我们研究了 miR-29c 是否在胃癌细胞中作为肿瘤抑制 miRNA 发挥作用。为此，我们验证了 miR-29c 在胃癌组织中的下调，并评估了 miR-29c 对胃癌细胞的生物学效应。
在转染 miR-29c 的细胞中，细胞增殖和软琼脂集落形成能力均显著降低。虽然没有诱导细胞凋亡，但 BrdU 掺入和磷酸化组蛋白 H3（S10）阳性细胞的比例在转染 miR-29c 的细胞中显著降低，表明 miR-29c 可能参与细胞增殖的调节。为了解释 miR-29c 抑制生长的机制，我们使用微阵列比较了 miR-29c 转染细胞与阴性对照转染细胞中差异表达基因（>2 倍）。RCC2、PPIC 和 CDK6 在 miR-29c 转染的 MKN45、MKN7 和 MKN74 细胞中均共同下调，并且所有基因的 mRNA 3'UTR 中都存在 miR-29c 靶序列。RCC2 和 PPIC 在胃癌组织中实际上上调，因此两者均被鉴定为胃癌中 miR-29c 的可能靶标。为了确定 RCC2 和/或 PPIC 的下调是否参与 miR-29c 的生长抑制，我们将针对 RCC2 和 PPIC 的 siRNA 转染到 MKN45 中，并测定细胞活力、BrdU 掺入率和 caspase 活性。我们发现 RCC2 敲低降低了细胞活力和 BrdU 掺入，而没有 caspase 活性的任何增加，而 PPIC 敲低则没有，表明 RCC2 的下调可能至少部分负责 miR-29c 的生长抑制。
我们的研究结果表明，miR-29c 可能在胃癌细胞中具有肿瘤抑制功能，其表达下调可能通过 RCC2 的异常表达赋予肿瘤细胞生长优势。
