Cancer Letters

Cancer Letters

Volume 369, Issue 1, 1 December 2015, Pages 58-66
Cancer Letters

Original Articles
Identification of miRNomes in human stomach and gastric carcinoma reveals miR-133b/a-3p as therapeutic target for gastric cancer

https://doi.org/10.1016/j.canlet.2015.06.028Get rights and content

Highlights

  • Heatmap analysis of the miRNomes was performed across TCGA datasets.

  • miR-133 is downregulated in gastric cancer.

  • Downregulation of miR-133 is mediated by histone modification.

  • miR-133 promotes gastric cancer cell apoptosis by targeting Mcl-1 and Bcl-xL.

  • Anti-tumor effect of miR-133 was verified in xenograft mice.

Abstract

Gastric cancer (GC) is the fourth most frequent malignant disease and the second leading cause of cancer mortality worldwide, but the molecular mechanisms underlying this clinically heterogeneous disease are complex and remain far from completely understood. Accumulating evidence suggests that abnormal microRNA (miRNA) expression is involved in tumorigenesis. However, their accurate expression pattern, function, and mechanism in GC remain unclear. Here, a heatmap analysis of the miRNomes was performed across TCGA datasets and the expression of miR-133 family was found to be consistently downregulated in GC. This result was confirmed in two GC cell lines and 20 pairs of primary GC tissues, and further study demonstrated that the downregulation of miR-133 was mainly mediated by histone modification within its promoter region. Importantly, restoration of miR-133b/a-3p expression could suppress GC cell proliferation and promote cell apoptosis by targeting anti-apoptotic molecules Mcl-1 and Bcl-xL. Consistent with in vitro results, reintroducing of miR-133b/a-3p expression significantly delayed tumor formation and reduced tumor size of GC cells in xenograft nude mice. And the inverse relationship between miR-133b/a-3p and its targets was verified in xenograft mice. Taken together, our findings suggest that miR-133b/a-3p acts as a tumor suppressor in GC by directly targeting Mcl-1 and Bcl-xL. Revealing novel mechanism for oncogene inhibition by miRNA-mediated pathways offers new avenues for GC treatment.

Introduction

Gastric cancer (GC) is the fourth most frequent malignant disease and the second leading cause of cancer mortality worldwide [1]. Although GC patients benefit from earlier diagnosis, standardized surgical resection and chemotherapy in the past few decades, the overall survival remains far from satisfactory [2]. Currently, GC was recognized as a biologically heterogeneous disease characterized by complex genetic and epigenetic alterations [3], [4], [5]. Therefore, a better understanding of the cellular and molecular mechanisms underlying GC development and progression could provide insights leading to improved diagnosis and therapeutic approaches.

MicroRNAs (miRNAs) represent a large family of endogenous noncoding RNAs involved in a fundamental layer of post-transcriptional regulation of gene expression [6]. They function to regulate gene expression by directly binding to the 3′ untranslational region (3′-UTR) of their target mRNAs. Numerous studies have revealed that miRNAs play crucial roles in a number of biological processes, including development, infection, immune responses, and carcinogenesis [7], [8], [9]. Although accumulating evidence indicates that miRNAs are frequently dysregulated in human cancers [10], [11], the molecular mechanisms by which miRNAs modulate the process of carcinogenesis and the behavior of cancer cells remain largely unknown. In the past few years, a wide variety of miRNAs have been found to participate in carcinogenesis via altering expressions of oncogenes and tumor suppressor genes to affect cell proliferation, apoptosis, motility and invasion [12], [13]. Meanwhile, some miRNAs are closely associated with malignant phenotypes, tumor stages, or patient survival, making them potential diagnostic or prognostic markers [14].

The sequence of miR-133 was first experimentally characterized in mice [15]. In the human genome there are three known miR-133 genes: miR-133a-1, miR-133a-2 and miR-133b, which are found on chromosomes 18, 20 and 6 respectively [16]. The mature sequences of miR-133a-3p and miR-133b were excised from the3′ arm of the pri-miRNA hairpin, but miR-133b does not have 3′ arm excised segment. The mature sequences of miR-133a-3p and miR-133b are highly homologous and only showed single base difference at 3′ end [17]. Previously, miR-133 was believed to be muscle-specific and plays a critical role in the regulation of muscle development. Therefore, its aberrant expression is linked to many diseases in skeletal muscle and cardiac muscle [18]. Beyond these observations, studies examining the expression of miRNA in clinical samples have established a link between miR-133 function and carcinogenesis, including bladder cancer [19], prostate cancer [20], and GC [21]. However, the accurate molecular mechanisms and therapeutic roles of miR-133 in GC are largely unknown.

In the present study, we performed a heatmap analysis of the miRNomes across TCGA datasets and found that the expression of miR-133b/a-3p was significantly downregulated in GC. We systemically validated the tumor suppressive role of miR-133b/a-3p in a series of in vitro experiments and confirmed that the downregulation of miR-133 was mainly mediated by histone modification within its promoter region. Importantly, we showed that restoration of miR-133b/a-3p expression could suppress GC cell proliferation and promoted cell apoptosis by targeting anti-apoptotic molecules Mcl-1 and Bcl-xL. Meanwhile, since miRNAs are highly stable and compatible with tissues, we explored the feasibility of using miR-133b/a-3p as a potential therapeutic approach to treat GC in a nude mice xenograft model.

Section snippets

Patients and tissue specimens

Tumor and corresponding normal stomach mucosa tissues were collected from patients with GC who underwent surgical resection at Department of Gastrointestinal Surgery, Changzheng Hospital, Second Military Medical University. None of these patients had a history of chemotherapy or radiotherapy before sampling. Individuals with multi-primary cancers were excluded. The clinical stages of tumors were determined according to the TNM classification system of the International Union Against Cancer (7th

Identification of miR-133b/a-3p as a dysregulated miRNA in GC

We firstly sought to address the expression pattern of miRNAs in GC tissues. MiRNA-Seq data that comprise 14 GC samples were downloaded from the Cancer Genome Atlas (TCGA) datasets and a heatmap analysis of the miRNomes was undertaken. Results showed that miR-133a-1, miR-133a-2 and miR-133b were significantly downregulated in tumor tissues compared with control normal stomach tissues (Fig. 1A). Then, the expression miR-133b/a-3p, which is the mature miR-133, was determined by qRT-PCR analysis

Discussion

Herein, we performed a heatmap analysis of the miRNomes across TCGA datasets to identify miR-133b/a-3p as a GC-specific miRNA. Thereafter, using a series of in vitro and in vivo assays, we demonstrated that miR-133b/a-3p acted as a critical tumor suppressor in human stomach tissue. Although decreased miR-133b/a-3p expression has been reported previously in human cancers, the present study provided a novel and comprehensive insight into the functional role and molecular mechanisms of

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81402359 and No. 81372670), and Initiation Foundation For Young Scientist of Changzheng Hospital (No. 2013CZQN05).

Conflict of interest

The authors have no conflict of interest to declare.

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