Yan Xin, Chen Xi, Liang Hongwei, Deng Ting, Chen Weixu, Zhang Suyang, Liu Minghui, Gao Xiujuan, Liu Yanqing, Zhao Chihao, Wang Xueliang, Wang Nan, Li Jialu, Liu Rui, Zen Ke, Zhang Chen-Yu, Liu Baorui, Ba Yi
Department of Gastrointestinal Medical Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy of Tianjin, Huanhuxi Road, Tiyuanbei, Tianjin 300060, China.
Mol Cancer. 2014 Sep 24;13:220. doi: 10.1186/1476-4598-13-220.
ERBB3, one of the four members of the ErbB family of receptor tyrosine kinases, plays an important role in breast cancer etiology and progression. In the present study, we aimed to identify novel miRNAs that can potentially target ERBB3 and their biological functions.
The expression levels of miR-143/145 and target mRNA were examined by relative quantification RT-PCR, and the expression levels of target protein were detected by Western blot. We used bioinformatic analyses to search for miRNAs that can potentially target ERBB3. Luciferase reporter plasmids were constructed to confirm direct targeting. Furthermore, the biological consequences of the targeting of ERBB3 by miR-143/145 were examined by cell proliferation and invasion assays in vitro and by the mouse xenograft tumor model in vivo.
We identified an inverse correlation between miR-143/145 levels and ERBB3 protein levels, but not between miR-143/145 levels and ERBB3 mRNA levels, in breast cancer tissue samples. We identified specific targeting sites for miR-143 and miR-145 (miR-143/145) in the 3'-untranslated region (3'-UTR) of the ERBB3 gene and regulate ERBB3 expression. We demonstrated that the repression of ERBB3 by miR-143/145 suppressed the proliferation and invasion of breast cancer cells, and that miR-143/145 showed an anti-tumor effect by negatively regulating ERBB3 in the xenograft mouse model. Interestingly, miR-143 and miR-145 showed a cooperative repression of ERBB3 expression and cell proliferation and invasion in breast cancer cells, such that the effects of the two miRNAs were greater than with either miR-143 or miR-145 alone.
Taken together, our findings provide the first clues regarding the role of the miR-143/145 cluster as a tumor suppressor in breast cancer through the inhibition of ERBB3 translation. These results also support the idea that different miRNAs in a cluster can synergistically repress a given target mRNA.
ERBB3是受体酪氨酸激酶ErbB家族的四个成员之一,在乳腺癌的病因学和进展中起重要作用。在本研究中,我们旨在鉴定可能靶向ERBB3的新型微小RNA(miRNA)及其生物学功能。
通过相对定量逆转录-聚合酶链反应(RT-PCR)检测miR-143/145和靶mRNA的表达水平,通过蛋白质免疫印迹法检测靶蛋白的表达水平。我们使用生物信息学分析来寻找可能靶向ERBB3的miRNA。构建荧光素酶报告质粒以确认直接靶向作用。此外,通过体外细胞增殖和侵袭试验以及体内小鼠异种移植肿瘤模型,研究miR-143/145靶向ERBB3的生物学后果。
在乳腺癌组织样本中,我们发现miR-143/145水平与ERBB3蛋白水平呈负相关,但miR-143/145水平与ERBB3 mRNA水平之间无相关性。我们在ERBB3基因的3'-非翻译区(3'-UTR)中鉴定出miR-143和miR-145(miR-143/145)的特异性靶向位点,并调节ERBB3的表达。我们证明,miR-143/145对ERBB3的抑制作用抑制了乳腺癌细胞的增殖和侵袭,并且在异种移植小鼠模型中,miR-143/145通过负调节ERBB3显示出抗肿瘤作用。有趣的是,miR-143和miR-145在乳腺癌细胞中对ERBB3表达以及细胞增殖和侵袭表现出协同抑制作用,使得这两种miRNA的作用大于单独使用miR-143或miR-145时的作用。
综上所述,我们的研究结果首次揭示了miR-143/145簇通过抑制ERBB3翻译在乳腺癌中作为肿瘤抑制因子的作用。这些结果也支持了一个观点,即簇中的不同miRNA可以协同抑制给定的靶mRNA。