Wang Changlin, Cai Licheng, Liu Jing, Wang Gang, Li Haoming, Wang Xiaoxiong, Xu Wanhai, Ren Minghua, Feng Li, Liu Pinghuang, Zhang Cheng
Department of Urology, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Department of Neurosurgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Cell Physiol Biochem. 2017;43(6):2405-2419. doi: 10.1159/000484394. Epub 2017 Oct 27.
BACKGROUND/AIMS: MiR-30a-5p, a member of the microRNA-30 family (miR-30), is known to function as a tumor suppressor in several different cancers. However, the expression levels, biological function, and underlying mechanisms of miR-30a-5p in renal cell carcinoma (RCC) remain unclear. Glucose-regulated protein78 (GRP78) is a common cancer biomarker and promotes the growth and survival of cancer cells. The expression of GRP78 has been reported to be modulated by miR-30a in neurons. In this study, the expression profile of miR-30a-5p in clear cell renal cell carcinoma (ccRCC) and its effect on ccRCC through regulating GRP78 expression was investigated.
MiR-30a-5p expression was analyzed using bioinformatic software on open microarray datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), and confirmed by quantitative RT-PCR (qRT-PCR) in ccRCC cell lines. Cell proliferation was investigated using CCK-8 and cell count assays. Western blotting, immunohistochemistry, luciferase reporter assays, and flow cytometry were employed to investigate the mechanisms of the effect of miR-30a-5p on ccRCC Results: MiR-30a-5p was down-regulated in ccRCC and related to the clinicopathological factors and prognosis of ccRCC. MiR-30a-5p was found to both suppress the growth of ccRCC cells and promote apoptosis of ccRCC cells in vitro. GRP78 was the direct target gene of miR-30a-5p, and the GRP78 expression was inversely correlated with the expression of miR-30a-5p in vivo and in vitro. The functional studies of GRP78 overexpression or knockdown demonstrated that GRP78 promoted proliferation and anti-apoptosis of ccRCC cells, and the oncogenic activity of GRP78 resulting in by miR-30a-5p overexpression.
MiR-30a-5p is a bona fide negative regulator of GRP78 expression, and the anti-tumor activity of miR-30a-5p in ccRCC is due at least in part to down-regulating GRP78 expression and modulating the unfolded protein response (UPR) pathway. Thus, miR-30-GRP78 interaction provides a novel therapeutic candidate target in ccRCC treatment.
背景/目的：微小RNA-30家族（miR-30）成员之一的miR-30a-5p在多种不同癌症中发挥肿瘤抑制作用。然而，miR-30a-5p在肾细胞癌（RCC）中的表达水平、生物学功能及潜在机制仍不清楚。葡萄糖调节蛋白78（GRP78）是一种常见的癌症生物标志物，可促进癌细胞的生长和存活。据报道，miR-30a可调节神经元中GRP78的表达。在本研究中，我们调查了miR-30a-5p在透明细胞肾细胞癌（ccRCC）中的表达谱及其通过调节GRP78表达对ccRCC的影响。
使用生物信息学软件分析来自癌症基因组图谱（TCGA）和基因表达综合数据库（GEO）的开放微阵列数据集，以分析miR-30a-5p的表达，并通过定量逆转录聚合酶链反应（qRT-PCR）在ccRCC细胞系中进行验证。使用CCK-8和细胞计数试验研究细胞增殖情况。采用蛋白质免疫印迹法、免疫组织化学法、荧光素酶报告基因试验和流式细胞术研究miR-30a-5p对ccRCC作用的机制。结果：miR-30a-5p在ccRCC中表达下调，且与ccRCC的临床病理因素及预后相关。发现miR-30a-5p在体外既能抑制ccRCC细胞的生长，又能促进ccRCC细胞的凋亡。GRP78是miR-30a-5p的直接靶基因，在体内和体外，GRP78的表达与miR-30a-5p的表达呈负相关。GRP78过表达或敲低的功能研究表明，GRP78促进ccRCC细胞的增殖和抗凋亡，且GRP78的致癌活性是由miR-30a-5p过表达导致的。
miR-30a-5p是GRP78表达的真正负调节因子，miR-30a-5p在ccRCC中的抗肿瘤活性至少部分归因于下调GRP78表达并调节未折叠蛋白反应（UPR）途径。因此，miR-30-GRP78相互作用为ccRCC治疗提供了一个新的治疗候选靶点。
