MicroRNA let-7c inhibits migration and invasion of human non-small cell lung cancer by targeting ITGB3 and MAP4K3
Introduction
Lung cancer is the leading cause of cancer mortality worldwide, with nearly 1,400,000 deaths each year [1]. Non-small cell lung cancer (NSCLC) accounts for almost 85% of lung cancer. About 40% stage I and 60% stage II NSCLC patients are dying of distant metastases within 5 years after curative tumor resection [2]. Thus, identification of new molecules involved in tumor metastasis is a critical step toward the development of novel therapeutics. These new agents are expected to prevent distal metastasis, thereby significantly increasing the survival rate.
MicroRNAs (miRNAs) are recognized as important post-transcriptional regulators of gene expression [3]. Mounting evidence suggests that the aberration or alteration of miRNAs is involved in carcinogenesis, progression and metastasis in many human cancers including NSCLC [4]. Yanaihara and colleagues, by comparing 104 NSCLC samples to corresponding normal lung tissues, have demonstrated that 43 miRNAs are differentially expressed in cancer samples [5]. High levels of mir-328 and mir-378 are found to be associated with brain metastasis from NSCLC [6], [7]. Low level of mir-451 expression is reportedly associated with lymph node involvement of NSCLC [8]. Recently, meta-analysis of prospective trials reveals that mir-21 and mir-155 are reliable predictors for recurrence, metastasis and poor survival of NSCLC patients [9], [10]. Thus, the potential role of miRNAs on NSCLC metastasis warrants further investigation.
Let-7 family has been shown to act as tumor suppressors by inhibiting oncogenes and key regulators of mitogenic pathways including RAS, MYC, HMGA2, etc. [11]. As for lung cancer, some let-7 members are reported to be correlated with tumor growth and pathologic classification. Let-7a and let-7f are firstly reported to be down-regulated in human lung cancers and are proved to be correlated with poor prognosis [12]. Ectopic overexpression levels of let-7b and let-7g are able to effectively suppress cancer growth in mouse models of lung cancer [13], [14], [15]. Expression profiles of let-7 members are also a promising method for classification of adenocarcinoma (AD) and squamous cell carcinoma (SCC) [16]. These studies suggest that specific member of let-7 family plays a crucial role in the growth and development of lung cancer. However, the influence of let-7 members on metastasis in lung cancer remains largely undefined. Our previous findings suggest that let-7c is a suppressor in migration and invasion of colorectal cancer (CRC) [17]. Our current study aimed to generate more results consolidating the correlation between expression of let-7 members (let-7a, 7b, 7c) and metastasis of human lung cancer cells. We focused on let-7c because its expression was significantly down-regulated both in the lung cancer cell lines with relatively highly metastatic potential and in tumor tissues with metastasis. The effect of let-7c on migration and invasion of lung cancer cells was directly verified by cell functional assays after overexpression and inhibition of let-7c in cells. To explore the potential mechanism underlying let-7c mediated metastatic suppression in lung cancer cells, a list of candidate genes were predicted by bioinformatics’ software and further confirmed by dual-luciferase reporter system. As a result, both ITGB3 and MAP4K3 are identified as target genes responsible for the functions of let-7c. ITGB3 (integrin β3, also known as CD61), a member of integrin family, has been shown to be involved in cell adhesion and “outside-in” signaling transduction [18]. MAP4K3, a member of the MAP4K family, is an important mediator in different signaling pathways [19]. Our study first demonstrates that let-7c, in addition to its suppressive role in tumor growth, also inhibits tumor metastasis by directly destabilizing the mRNAs of ITGB3 and MAP4K3 in NSCLC.
Section snippets
Cell lines and clinical samples
Human NSCLC cell lines (SKMES-1, H520, H157, A549, GLC82, H1299) and human lung epithelium cell (HLEC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Grand Island, NY) in a humidified atmosphere of 5% CO2 at 37 °C incubator.
The NSCLC specimens (n = 94) without any preoperative radiotherapy or chemotherapy and matched adjacent normal tissues obtained from the patients undergoing surgery at Beijing Cancer
Let-7c down-regulated in human lung cancer cells with highly metastatic ability
The invasive potential of lung cancer cell lines used in this study was firstly tested by transwell assay as described in Supplementary Fig. 1. The expression levels of let-7c in these cell lines were detected to determine the potential effect of let-7c expression on metastasis. Fig. 1A showed that the relative expression level of let-7c was lower in cells with relatively highly metastatic ability (H1299, SKMES-1 and A549) than those with relatively low or no metastatic potential (H157, H520,
Discussion
In this study we firstly determine the expression level of let-7 members in lung cancer cell lines and clinical samples. The decreased expression of let-7c is found to be associated with highly invasive ability in lung cancer cells and metastasis in NSCLC. Then we investigate the mechanism underlying the let-7c-mediated suppression of metastasis. As a result, ITGB3 and MAP4K3 are identified as direct target genes of let-7c. After rescuing expression of ITGB3 and MAP4K3, the suppressive effects
Conflict of Interest statement
None declared.
Acknowledgements
This work was supported partially by Beijing Natural Science Foundation (Grant Nos. 11G2633 and 5122012), National Natural Science Foundation (Grant Nos. 81201964 and 81071733), the National High Technology Research and Development Program of China (863 Program, No. 2012AA02A502), and the National Basic Research Program of China (973 Program, No. 2010CB529402).
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These authors contribute to this work equally.