RNA interference targeting of A1 receptor-overexpressing breast carcinoma cells leads to diminished rates of cell proliferation and induction of apoptosis
RNA interference targeting of A1 receptor-overexpressing breast carcinoma cells leads to diminished rates of cell proliferation and induction of apoptosis
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DOI：
10.4161/cbt.4.12.2196
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发表时间：
2005-12-01
影响因子：
3.6
通讯作者：
Liu, SX
中科院分区：
文献类型：
--
作者：
Mirza, A;Basso, A;Liu, SX
为了确定A1腺苷受体是否介导乳腺肿瘤发生，我们使用定量RT-PCR和Western印迹分析评估了人肿瘤细胞系和人原发性乳腺肿瘤组织中A1受体的表达。通过定量RT-PCR分析确定，与正常乳腺上皮细胞/细胞系（n = 3）相比，在检查的所有乳腺肿瘤细胞系（n = 7）中A1受体mRNA表达上调。Western blot分析表明，24例乳腺癌组织中有15例（62.5%）A1腺苷受体蛋白表达高于正常乳腺组织。为了探索其细胞功能，A1腺苷受体在MDA-MB-468人乳腺肿瘤细胞中被小干扰RNA（siRNA）耗尽。MDA-MB-468乳腺肿瘤细胞中A1受体的消耗分别通过细胞数计数和[C-14]-胸苷掺入来测量，从而减弱细胞生长和细胞增殖。细胞周期分析表明，siRNA去除A1受体后，G1检查点被破坏，导致细胞明显聚集在G（2）/M期，这与siRNA抑制细胞增殖的作用一致。进一步支持这一发现的是，对不同细胞周期阶段的Hela细胞的同步化研究表明，G（2）/M细胞中A1受体表达受到抑制，并且通过siRNA耗尽A1受体表达产生了几种关键细胞周期调节因子的差异表达，即，细胞周期蛋白依赖性激酶抑制剂p27的积累伴随着CDK 4和细胞周期蛋白E蛋白的减少。除了对细胞周期进程的影响外，siRNA对A1受体的消耗导致大量细胞死亡和凋亡，如通过FACS分析和膜联蛋白V染色方法所确定的。总之，这些发现表明，A1腺苷受体可能有助于乳腺肿瘤细胞的生长和存活。
To determine if A1 adenosine receptors mediate breast tumorigenesis, we evaluated A1 receptor expression in human tumor cell lines and human primary breast tumor tissues using both quantitative RT-PCR and Western blot analysis. A1 receptor mRNA expression is upregulated in all breast tumor cell lines examined (n = 7) compared to normal mammary epithelial cells/cell lines (n = 3) as determined by quantitative RT-PCR analysis. Western blot analysis indicates that protein expression of A1 adenosine receptor is higher in 15 (62.5%) of 24 human primary breast tumor tissues than in matched normal breast tissue. To explore its cellular function, the A1 adenosine receptor was depleted by small interfering RNA (siRNA) in MDA-MB-468 human breast tumor cells. Depletion of A1 receptors in MDA-MB-468 breast tumor cells attenuated both cell growth and cell proliferation as measured by cell number counts and [C-14]-thymidine incorporation, respectively. Cell cycle analysis indicated that depletion of A1 receptors by siRNA impairs G1 checkpoint, leading to marked accumulation of cells in G(2)/M phase, in agreement with the inhibitory effect on cell proliferation. Further supporting this finding, synchronization studies of Hela cells in various cell cycle phases suggest that A1 receptor expression is suppressed in G(2)/M cells and depletion of A1 receptor expression by siRNA produced differential expression of several key cell cycle regulators, i.e., accumulation of the cyclin-dependent kinase inhibitor p27 with concomitant reduction of CDK4 and cyclin E proteins. In addition to the impact on cell cycle progression, depletion of A1 receptors by siRNA results in substantial cell death and apoptosis as determined by FACS analysis and annexin V staining method. Together these findings suggest that the A1 adenosine receptor may contribute to tumor cell growth and survival in breast tumor cells.