Chen Gang, He Mingfeng, Yin Yin, Yan Ting, Cheng Wenfang, Huang Zebo, Zhang Lan, Zhang Huo, Liu Ping, Zhu Wei, Zhu Yichao
Department of Oncology, The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, 210029 People's Republic of China.
Department of Oncology, First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029 People's Republic of China.
Cancer Cell Int. 2017 Oct 24;17:95. doi: 10.1186/s12935-017-0466-y. eCollection 2017.
The tumor suppressive role of miR-1296 is observed in triple negative breast cancer (TNBC). However, the effect of miR-1296-5p in ERBB2-positive breast cancers remains obscure.
Whether was the target gene of the miR-1296-5p was predicted by online software, and determined by dual-luciferase activity assay. miR-1296-5p expression levels were determined in breast cancer samples (114 breast cancer tissues and 30 adjacent normal tissues) by using qRT-PCR. The effect of miR-1296-5p and inhibition of ERBB2/mTORC1 signaling on the downstream target was assessed by Western blot. SK-BR-3 and BT-474 breast cancer cell line was transfected with miR-1296-5p mimic after which cell proliferation and apoptosis were determined by the clonogenic assay and the flow cytometry system, respectively. In addition, the chemotherapeutic drug sensitivity of SK-BR-3 and BT-474 cells transfected with miR-1296-5p mimic were determined by MTT assay.
The luciferase assay carrying ERBB2 3'-untranslated region-based reporters expressed in SK-BR-3 and BT-474 cells suggested that was the target gene of miR-1296-5p. MiR-1296-5p was significantly decreased in breast cancer tissues compared to adjacent normal tissues. Moreover, it was declined in ERBB2-positive breast cancer samples compared with that in ERBB2-negative breast cancer tissues. Overexpressed miR-1296-5p reduced its target protein level and mTORC1/S6 activation, inhibited the proliferation of ERBB2-positive breast cancer cells and sensitized these cells to cisplatin and 5-fluorouracil-induced apoptosis.
Our findings suggest that miR-1296-5p is involved in the regulation of proliferation in breast cancer cells via targeting ERBB2/mTORC1 signaling pathway.
在三阴性乳腺癌（TNBC）中观察到miR - 1296具有肿瘤抑制作用。然而，miR - 1296 - 5p在ERBB2阳性乳腺癌中的作用仍不清楚。
通过在线软件预测 是否为miR - 1296 - 5p的靶基因，并通过双荧光素酶活性测定进行确定。使用qRT - PCR检测乳腺癌样本（114例乳腺癌组织和30例癌旁正常组织）中miR - 1296 - 5p的表达水平。通过蛋白质印迹法评估miR - 1296 - 5p和抑制ERBB2/mTORC1信号对下游靶点的影响。用miR - 1296 - 5p模拟物转染SK - BR - 3和BT - 474乳腺癌细胞系，之后分别通过克隆形成试验和流式细胞术系统检测细胞增殖和凋亡情况。此外，通过MTT试验测定转染miR - 1296 - 5p模拟物的SK - BR - 3和BT - 474细胞的化疗药物敏感性。
在SK - BR - 3和BT - 474细胞中进行的携带基于ERBB2 3'非翻译区报告基因的荧光素酶试验表明 是miR - 1296 - 5p的靶基因。与癌旁正常组织相比，miR - 1296 - 5p在乳腺癌组织中显著降低。此外，与ERBB2阴性乳腺癌组织相比，其在ERBB2阳性乳腺癌样本中也降低。过表达miR - 1296 - 5p降低了其靶蛋白水平和mTORC1/S6激活，抑制了ERBB2阳性乳腺癌细胞的增殖，并使这些细胞对顺铂和5 - 氟尿嘧啶诱导的凋亡敏感。
我们的研究结果表明，miR - 1296 - 5p通过靶向ERBB2/mTORC1信号通路参与调节乳腺癌细胞的增殖。
