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Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7.

ABSTRACT: MicroRNAs are endogenous small noncoding RNAs that are functionally involved in numerous critical cellular processes including tumorigenesis. Data mining using a microRNA array database suggested that let-7d microRNA may be associated with renal cell carcinoma (RCC) malignant progression. Here, we performed further analyses to determine whether let-7d is functionally linked to RCC malignancy.Quantitative real-time PCR was used to determine the level of mature let-7d in RCC clinical specimens and its correlation with clinicopathological data. Immunohistochemical staining was conducted to characterize the stroma of RCC. Let-7d overexpressing RCC cell lines combined with mouse models bearing cell-derived xenografts and patient-derived xenografts were used to assess the functional role of let-7d in vitro and in vivo.Downregulation of let-7d in clinical RCC samples was associated with advanced tumor grade and T stage and increased vascular invasion. An inverse relationship between let-7d expression and macrophage infiltration was found in clinical RCC samples. Functional studies indicated that ectopic expression of let-7d significantly inhibited RCC cell proliferation, migration, and peripheral blood monocyte (PBMC) recruitment in vitro, as well as tumor growth, metastasis, and tumor macrophage infiltration in vivo. In silico analysis and subsequent experimental validation confirmed collagen, type III, alpha 1 (COL3A1) and C-C subfamily chemokine member CCL7 as direct let-7d target genes. The addition of COL3A1 and CCL7 counteracted the inhibitory effects of let-7d on RCC cell proliferation, migration, and PBMC recruitment. The inhibition of let-7d increased cell proliferation, migration, and PBMC recruitment by the enhanced expression of COL3A1 and CCL7 genes in vitro. The mRNA levels of COL3A1 and CCL7 were inversely correlated with let-7d level in RCC clinical specimens.These results suggest that let-7d may suppress RCC growth, metastasis, and tumor macrophage infiltration at least partially through targeting COL3A1 and CCL7.

SUBMITTER: Su B

PROVIDER: S-EPMC4168121 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7.

Su Boxing B Zhao Wei W Shi Bentao B Zhang Zhongyuan Z Yu Xi X Xie Feng F Guo Zhongqiang Z Zhang Xiaoyu X Liu Jin J Shen Qi Q Wang Jinghua J Li Xuesong X Zhang Zhiqian Z Zhou Liqun L

Molecular cancer 20140906

<h4>Background</h4>MicroRNAs are endogenous small noncoding RNAs that are functionally involved in numerous critical cellular processes including tumorigenesis. Data mining using a microRNA array database suggested that let-7d microRNA may be associated with renal cell carcinoma (RCC) malignant progression. Here, we performed further analyses to determine whether let-7d is functionally linked to RCC malignancy.<h4>Methods</h4>Quantitative real-time PCR was used to determine the level of mature l ...[more]

PMID: 25193015

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Project description:BackgroundMetastasis is the primary cause of tumor death in renal cell carcinoma (RCC). Improved diagnostic markers of metastasis are critically needed for RCC. MicoRNAs are demonstrated to be stable and significant biomarkers for several malignancies. In this study, we aimed to explore the metastasis related microRNAs and its mechanism in RCC.MethodsThe relationship between microRNAs expression and prognosis and metastasis of RCC patients were explored by data mining through expression profiles from The Cancer Genome Atlas (TCGA). A total of 80 RCC tissues and adjacent normal kidney tissues were obtained from Department of Urology, Peking University First Hospital. Expression of microRNA-200b (miR-200b) in RCC tissues and cell lines were determined by bioinformatic data mining and quantitative real-time PCR (qRT-PCR). The effects of miR-200b on cell proliferation, migration and invasion were determined by cell counting kit-8 and colony formation assay, wound healing assay and Boyden chamber assay. Mouse cell-derived xenograft and patient-derived xenograft model were also performed to evaluate the effects of miR-200b on tumor growth and metastasis in vivo. The molecular mechanism of miR-200b function was investigated using bioinformatic target predication and high-throughput cDNA sequencing (RNA-seq) and validated by luciferase reporter assay, qRT-PCR, Western blot and immunostaining in vitro and in vivo.FindingsOur findings indicates that miR-200b is frequently downregulated and have potential utility as a biomarker of metastasis and prognosis in RCC. Interestingly, ectopic expression of miR-200b in the Caki-1 and OSRC-2 cell lines suppresses cell migration and invasion in vitro as well as tumor metastases in vivo. However, miR-200b has no effect on cell proliferation in vitro and tumor growth in vivo. In addition, bioinformatics target predication and RNA-seq results reveals that Laminin subunit alpha 4 (LAMA4) is one target of miR-200b and significantly inhibited by miR-200b in vitro and in vivo.InterpretationThese results demonstrate a previously undescribed role of miR-200b as a suppressor of tumor metastasis in RCC by directly destabilizing LAMA4 mRNA.

Dataset Information

Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7.

Publications

Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7.

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