[小干扰RNA沉默缺氧诱导因子-2α基因抑制缺氧微环境下裸鼠乳腺球细胞生长]
[Silencing hypoxia inducible factor-2α gene by small interference RNA inhibits the growth of mammosphere cells in nude mice under hypoxic microenvironment].
作者信息
Qu Hong-bo, Fan Yuan-ming, Han Ming-li, Zeng Ni, Zhu Zhi-kun, Liu Hong, Xie Jia, Wu Cheng-yi, Tang Wei-xue
机构信息
Department of Endocrine Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
出版信息
Zhonghua Yi Xue Za Zhi. 2013 Apr 16;93(15):1182-7.
OBJECTIVE
To explore the effects of silencing hypoxia inducible factor-2α (HIF-2α) by small interference RNA on the growth of mammosphere cells in nude mice under hypoxic microenvironment.
METHODS
The empty and interference vectors were transfected into MCF-7 cell. Then G418 was added to screen the positive cells to obtain stable cell line. The empty and interference vectors were inoculated subcutaneously into left and right back near hind limb of nude mice. The volume and weight of tumors were calculated respectively. The expressions of HIF-2α, CD44, OCT-4 and KLF-4 protein in xenograft tumor tissues were detected by Western blot.
RESULTS
The expression vector of HIF-2α-siRNA was successfully established. The formation of mammosphere was lowered by silencing HIF-2α gene expression. In contract to empty vector group cell, there were obvious decreases in the volumes and weights of tumors in interference group (P < 0.05). The expression of HIF-2α protein of interference group (0.42 ± 0.01) was much lower than that of the empty vector group (0.89 ± 0.03, P < 0.05), the expression of CD44 protein of interference group (0.21 ± 0.01) was much lower than the empty vector group (0.78 ± 0.03, P < 0.05), the expression of OCT-4 protein of interference group (0.42 ± 0.01)was much lower than the empty vector group (0.68 ± 0.03, P < 0.05) and the expression of KLF-4 protein of interference group (0.34 ± 0.01) was much lower than the empty vector group (0.72 ± 0.03, P < 0.05).
CONCLUSION
Silencing HIF-2α gene can effectively inhibit the growth of breast cancer stem cells in nude mice under hypoxic microenvironment. Its mechanism may be through inhibited capacity for self-renewal and proliferation of breast cancer stem cells in vivo through the down-regulated expressions of markers associated with stem cells. HIF-2α is expected to become a new target for gene therapy of breast cancer.
目的
探讨小干扰RNA沉默缺氧诱导因子-2α（HIF-2α）对缺氧微环境下裸鼠乳腺球细胞生长的影响。
方法
将空载体和干扰载体转染至MCF-7细胞。然后加入G418筛选阳性细胞以获得稳定细胞系。将空载体和干扰载体分别皮下接种于裸鼠后肢附近的左右背部。分别计算肿瘤的体积和重量。采用蛋白质免疫印迹法检测异种移植瘤组织中HIF-2α、CD44、OCT-4和KLF-4蛋白的表达。
结果
成功构建了HIF-2α-siRNA表达载体。沉默HIF-2α基因表达可降低乳腺球的形成。与空载体组细胞相比，干扰组肿瘤的体积和重量明显减小（P<0.05）。干扰组HIF-2α蛋白表达（0.42±0.01）明显低于空载体组（0.89±0.03，P<0.05），干扰组CD44蛋白表达（0.21±0.01）明显低于空载体组（0.78±0.03，P<0.05），干扰组OCT-4蛋白表达（0.42±0.01）明显低于空载体组（0.68±0.03，P<0.05），干扰组KLF-4蛋白表达（0.34±0.01）明显低于空载体组（0.72±0.03，P<0.05）。
结论
沉默HIF-2α基因可有效抑制缺氧微环境下裸鼠乳腺癌干细胞的生长。其机制可能是通过下调与干细胞相关的标志物表达，抑制体内乳腺癌干细胞的自我更新和增殖能力。HIF-2α有望成为乳腺癌基因治疗的新靶点。
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