Pan Cuicui, Gao Hua, Zheng Ni, Gao Qi, Si Yuanquan, Zhao Yueran
Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China.
Central Laboratory, Provincial Hospital Affiliated to Shandong University, 544 Jingwu Road, Jinan, 250021, China.
Biol Res. 2017 Sep 21;50(1):31. doi: 10.1186/s40659-017-0137-4.
MiR-320 is downregulated in multiple cancers, including glioma and acts as tumor suppressor through inhibiting tumor cells proliferation and inducing apoptosis. PBX3 (Pre-B cell leukemia homeobox 3), a putative target gene of miR-320, has been reported to be upregulated in various tumors and promote tumor cell growth through regulating MAKP/ERK pathway. This study aimed to verify whether miR-320 influences glioma cells growth through regulating PBX3.
Twenty-four human glioma and paired adjacent nontumorous tissues were collected for determination of miR-320 and PBX3 expression using RT-qPCR and western blot assays. Luciferase reporter assay was performed to verify the interaction between miR-320 and its targeting sequence in the 3' UTR of PBX3 in glioma cells U87 and U251. Increased miR-320 level in U87 and U251 cells was achieved through miR-320 mimic transfection and the effect of which on glioma cells growth, proliferation, cell cycle, apoptosis and activation of Raf-1/MAPK pathway was determined using MTT, colony formation, flow cytometry and western blot assays. PBX3 knockdown was performed using shPBX3 and the influence on MAPK pathway activation was evaluated.
MiR-320 downregulation and PBX3 upregulation was found in glioma tissues. Luciferase reporter assays identified miR-320 directly blinds to the 3' UTR of PBX3 in glioma cells. MiR-320 mimic transfection suppressed glioma cells proliferation, and induced cell cycle arrest and apoptosis. Both miR-320 overexpression and PBX3 knockdown inhibited Raf-1/MAPK activation.
MiR-320 may suppress glioma cells growth and induced apoptosis through the PBX3/Raf-1/MAPK axis, and miR-320 oligonucleotides may be a potential cancer therapeutic for glioma.
miR-320在包括胶质瘤在内的多种癌症中表达下调，并通过抑制肿瘤细胞增殖和诱导凋亡发挥肿瘤抑制作用。PBX3（前B细胞白血病同源盒3）是miR-320的一个假定靶基因，据报道在各种肿瘤中上调，并通过调节MAKP/ERK途径促进肿瘤细胞生长。本研究旨在验证miR-320是否通过调节PBX3影响胶质瘤细胞生长。
收集24例人脑胶质瘤组织及配对的相邻非肿瘤组织，采用RT-qPCR和蛋白质免疫印迹法检测miR-320和PBX3的表达。进行荧光素酶报告基因检测，以验证miR-320与其在胶质瘤细胞U87和U251中PBX3的3'UTR靶向序列之间的相互作用。通过转染miR-320模拟物提高U87和U251细胞中miR-320水平，并采用MTT法、集落形成实验、流式细胞术和蛋白质免疫印迹法检测其对胶质瘤细胞生长、增殖、细胞周期、凋亡及Raf-1/MAPK途径激活的影响。使用shPBX3敲低PBX3，并评估其对MAPK途径激活的影响。
在胶质瘤组织中发现miR-320下调和PBX3上调。荧光素酶报告基因检测确定miR-32 在胶质瘤细胞中直接与PBX3的3'UTR结合。转染miR-320模拟物可抑制胶质瘤细胞增殖，并诱导细胞周期阻滞和凋亡。miR-320过表达和PBX3敲低均抑制Raf-1/MAPK激活。
miR-320可能通过PBX3/Raf-1/MAPK轴抑制胶质瘤细胞生长并诱导凋亡，miR-320寡核苷酸可能是一种潜在的胶质瘤癌症治疗药物。
