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MicroRNA-107, an oncogene microRNA that regulates tumour invasion and metastasis by targeting DICER1 in gastric cancer

Abstract MicroRNAs are small non-coding RNA molecules that control expression of target genes. Previous studies showed that microRNA-107 (miR-107) is overexpressed in gastric cancer tissues compared with the matched normal tissues. However, it remains largely unclear as to how miR-107 exerts its function and modulates the malignant phenotypes of gastric cancer, because our understanding of miR-107 signalling pathways is limited. In this study, we demonstrate that miR-107 is frequently up-regulated in gastric cancers and its overexpression is significantly associated with gastric cancer metastasis. Furthermore, silencing the expression of miR-107 could inhibit gastric cancer cell migration and invasion in vitro and in vivo. Subsequent investigation characterized DICER1 as a direct target of miR-107. Up-regulation of DICER1 resulted in a dramatic reduction of in vitro migration, invasion, in vivo liver metastasis of nude mice, which is similar to that occurs with the silencing of miR-107, indicating that DICER1 functions as a metastasis suppressor in gastric cancer. Furthermore, the restoration of DICER1 can inhibit miR-107-induced gastric cancer cell invasion and metastasis. In conclusion, our results suggested that miR-107, an oncogene miRNA promoting gastric cancer metastasis through down-regulation of DICER1. Inhibition of miR-107 or restoration of DICER1 may represent a new potential therapeutic target for gastric cancer treatment.


Introduction
Gastric cancer is one of the most common malignancies in tumours in East Asian countries [1][2]. Like other solid human cancers, the biggest obstacle for the treatment of gastric cancer is recurrence and metastasis. So, seeking for new therapeutic modalities to prevent the metastasis of gastric cancer becomes an urgent issue. But the precise mechanism of this process has not yet been defined, which has confined the improvement of therapeutic strategies.
MicroRNAs (miRNAs) are small (20-24 nucleotides [nt]) noncoding RNA gene products that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs [3]. Currently, more than several hundred unique mature human miRNAs are known (http://microrna. sanger.ac.uk). Aberrant expression of miRNAs has been reported to be involved in tumourigenesis, acting, as one might expect, variously as either oncogenes [4] or tumour suppressors [5]. Previously, altered miRNA expression signature is observed in gastric cancers that have been collected from different study cohorts [6][7] and miR-107 was found significantly up-regulated in gastric cancers, compared with the matched normal tissues.
In this study, we demonstrated that the overexpression of miR-107 is significantly correlated with metastasis of gastric cancer. We comprehensively investigate the biological functions and underlying molecular mechanism of miR-107 in gastric carcinogenesis performed with both in vitro and in vivo models. Our results showed that knockdown of miR-107 could inhibit gastric cancer cell invasion and migration in vitro and in vivo. Furthermore, DICER1, which is a putative metastasis suppressor in gastric cancer, is characterized as a bona fide direct and functional target of miR-107, which provides a possible regulation pathway for DICER1 and a candidate target for gastric cancer treatment. #

RNA extraction and quantitative real-time polymerase chain reaction
Stem-loop reverse transcription for mature miRNA was performed as described previously [8]. All reagents were obtained from Applied Biosystems (Foster City, CA, USA). For miRNA detection, mature miR-107 was reverse-transcribed with specific RT primers, quantified with a TaqMan probe. The small nuclear U6 RNA was used as an internal control for normalization and quantification the miRNAs expression as previously reported [9].

Plasmid construction and cell transfection
Transient CMV-driven miRNA sponge backbones and a control sponge containing tandem non-targeting binding sites was identical to that utilized previously [9]. The miR-107 sponge was constructed by annealing, purifying and cloning oligonucleotides containing seven tandem 'bulged' (at positions 9-12) miR-107 binding motifs into the XhoI and ApaI sites of the CMV sponge backbone. The miR-107 sponge contained 'bulged' sites of sequence is CGCCAAUAUUCUAUGCUGCUA. For stable expression studies, the fragments of the miR-107 and control CMV sponge constructs were subcloned into the pBABE-puro retroviral vector. All stable cell lines were generated via retroviral infection performed with HEK293T cells, as has been previously described [10].

In vitro migration and invasion assays
For transwell migration assays, 1ϫ10 5

MiR-107 is up-regulated in gastric cancer and associated with tumour metastasis and worse prognosis
In previous studies [5][6]

MiR-107 promotes gastric cancer cell invasion and metastasis in vitro and in vivo
To determine whether miR-107 has a causal function on gastric cancer metastasis, we first examined the expression of mature miR-107 in various gastric originated cell lines and found that miR-107 was significantly up-regulated in gastric cancer cell lines ( Fig. 2A), especially in SGC-7901 and MKN-45, which were demonstrated to have invasive ability to penetrate Matrigel-coated transwell in vitro invasion assay and metastatic ability to metastasize mainly to liver instead of lung and other organs in vivo tail vein metastatic assay [11]. We silenced the expression of miR-107 with retroviral miRNA sponge [9] in SGC-7901 and MKN-45 cells.
We found that miR-107 sponge specifically inhibited miR-107 function by nearly Ͼ3.0-fold, when compared with the control sponge in SGC-7901 and MKN-45 cells (Fig. 2B). Given that expression of miR-107 is highly associated with the metastasis and prognosis of gastric cancer, we wondered whether miR-107 could play an important part in gastric cancer cell invasion and metastasis. Migration and invasion assays showed that down-regulation of miR-107 could significantly inhibit migration and invasion of SGC-7901 and MKN-45 cells, when compared with their control groups (Fig. 2C, D). Tail vein metastatic assay in nude mice was further adopted to examine the in vivo metastatic ability of SGC-7901-miR-107-sponge and MKN-45-miR-107-sponge.  (Fig. 2E, both P Ͻ 0.05). Taken together, these observations suggest that miR-107 is a positive metastatic regulator for gastric cancer.

MiR-107 post-transcriptionally down-regulates DICER1 expression by directly targeting its 3-UTR
To understand the mechanisms by which miR-107 mediate tumour invasion and metastasis, the target prediction program TargetScan [12], PicTar [13] or miRanda (microrna.org and miRBase) was used to search for predicted direct target genes of miR-107. Among the hundreds targets of miR-107, three genes-CHRD, NF2 and DICER1-were previously implicated as tumour suppressor genes [14][15][16][17][18][19][20]. DICER1 was of particular interest, because its expression has been found to be progressively lost in gastric cancers showing increasing degrees of malignancy [20]. TargetScan and PicTar bioinformatics analysis of the 3Ј-UTRs of DICER1 revealed one putative binding site for miR-107, which is highly conserved across various species (Fig. 3A).
To investigate the potential interaction experimentally, the human DICER1 3Ј-UTR was subcloned after the firefly luciferase open reading frame (ORF) and co-transfected into HEK 293T cells with the miR -107-expressing vector (pcDNA-3.1-miR-107). An analogous reporter with point substitutions disrupting the target

Fig. 2 MiR-107 promotes gastric cancer cell invasion and metastasis in vitro and in vivo. (A) The expression level of mature miR-107 in various gastric cancer cell lines was determined using quantitative PCR. (B) MiR-107 sponge inhibition function was assayed in SGC-7901 or MKN-45 cells. Luciferase reporter assays were used to examine the sponge inhibition efficiency. (C, D) Transwell migration assay (C) and Matrigel invasion assay (D) of SGC-7901 or MKN-45 cells transfected with miR-107 sponge or its control retrovirus. Magnification in D2, 200ϫ. (E) Mice were injected with 1ϫ10 6 cells through tail vein. Four weeks later, the mice were sacrificed. The liver tissues were observed with naked eyes and the number of visible tumours in liver surface was counted (E1). The liver tissues were made serial sections before being haematoxylin and eosin dyed and observed under a light microscope (E2). N ϭ 3; error bars represent S.E.M. P-values obtained using a one-sided Student's t-test. P Ͻ 0.05 versus cells transfected with control retrovirus.
sites (Fig. 3B) was also co-transfected with pcDNA-3.1-miR107. Our results showed that pcDNA-3.1-miR-107 produced a 60% decrease in relative luciferase activity compared with pcDNA3.1transfected cells, when the full-length wild-type 3Ј-UTR of DICER1 was present (Fig. 3C). However, the relative luciferase activity did not drop as sharply in UTRs that contained mutant binding sites as in those that contained wild-type binding sites, which indicates that this reduction was sequence-specific (Fig. 3C).
To demonstrate that inhibited expression of miR-107 has a direct effect on DICER1 target. We assessed DICER1 protein expression levels after miR-107 inhibition by Western blot. We found that inhibition of miR-107 expression in SGC-7901 and MKN-45 cells increased the protein level of DICER1 (Fig. 3D). Those results really demonstrated that the endogenous DICER1 is direct target of miR-107.
To exclude the possibility that DICER1 is regulated by other predicted miRNAs in parallel with miR-107, we used TargetScan, PicTar and miRanda (miRBase) to search all miRNAs predicted to target DICER1. Besides miR-107, six candidate mature miRNAs (miR-103, miR-29a, miR-29b, miR-29c, miR-196a, miR-196b) were commonly predicted to target DICER1 by all of the three algorithms. We then test the expression of six candidate miRNAs (miR-103, miR-29a, miR-29b, miR-29c, miR-196a, miR-196b) in gastric cancer tissues and their paired adjacent non-tumour gastric cancer tissues, our results showed that miR-29b, and miR-29c were down-regulated in gastric cancer tissues compared with their paired adjacent non-tumour gastric cancer tissues. However, we did not find any expression differences of miR-103, miR-29a, miR-196a and miR-196b in gastric cancer tissues compared with their paired adjacent non-tumour gastric cancer tissues (Fig. S1). In conclusion, our results indicated that endogenous DICER1 is direct target of miR-107 in gastric carcinoma.  progressive gastric cancers, which indicating DICER1 may play an important role in the development of cancer and the epigenetical regulation involved [20]. However, the biopathological significance of DICER1 in human malignancies, especially in gastric cancer, is still unknown. To better understand the potential role of DICER1 in miR-107-mediated tumour invasion and metastasis, SGC-7901 cells were stable infected with pcDNA-3.1-DICER1 or pcDNA-3.1 vector alone (Fig. 4A). Remarkably, overexpression of DICER1 can strongly inhibit gastric cancer cell migration and invasion in vitro (Fig. 4B, C). Tail vein metastatic assay in nude mice also showed that ectopic expression of DICER1 in SGC-7901 cells led to significantly less visible tumours in liver surface (Fig. 4D, both P Ͻ 0.05), compared with control cells. This phenotype was similar to the one which was induced by the inhibition of miR-107.  Western blot analysis (Fig. 5A). Importantly, transwell assays indicated that the knockdown of DICER1 significantly prompted miR-107-knockdown SGC-7901 cells migration and invasion in vitro (Fig. 5B, C) and in vivo (Fig. 5D), which were inhibited by miR-107 deletion, suggesting that DICER1 is a direct and functional target for miR-107.

Discussion
Metastasis, the spread and growth of tumour cells to distant organs, represents the most devastating attribute of cancer. A notable feature of this process is the variation in metastatic tissue tropism displayed by different types of cancer [21]. In the case of gastric cancer, metastasis is the most frequent pattern of recurrence in patients with gastric carcinoma [22]. About half of patients with serosa-invasive gastric carcinoma develop recurrence and die of this disease during the first 2 years, even if curative resection is performed [23]. The 5-year survival rate of patients with metastasis and recurrence is only 2% [24]. An incomplete understanding of the molecular and cellular mechanisms underlying metastasis hinders the development of effective therapies that would eliminate or ameliorate this condition.
Recently, miRNAs have been shown to be potential tools for cancer progression, diagnosis and prognosis [8]. With the advent of miRNA expression profiles, significant efforts have been made to correlate miRNA expressions with tumour development [25][26][27]. Because single miRNA can regulate hundreds of downstream genes, the information gained from miRNAs may be complementary to that from the expression profiling of protein-coding genes. Thus, identification of the actions of miRNAs could add new layers of complexity to our understanding of cancer dynamic.
In this study, we demonstrated that miR-107 is commonly upregulated in gastric cancers and can promote gastric cancer cell migration, invasion and metastasis both in vitro and in vivo. A direct and functional target of miR-107 was also identified. The target gene, DICER1, is frequently repressed and functions as a  Recent findings have shown that miR-107 is involved in various biological and pathological processes. For example, miR-107 expression is reduced in Alzheimer's disease brain neocortex and tends to be significantly relative to AD progresses [28]. Nelson et al. also reported that the expression of miR-107 was decreases early in Alzheimer's disease and may accelerate disease progression through regulation of ␤-site amyloid precursor protein-cleaving enzyme 1 [29]. Yamakuchi [19]. We report for the first time that the enhanced expression of DICER1 could inhibit gastric cancer cell migration and invasion. Moreover, we also demonstrated that miR-107 can bind to one site of the 3Ј-UTR of DICER1 and dramatically decrease the level of DICER1 protein expression, which provides the first line of demonstrated in support of a mechanism for DICER1 regulation at the posttranscriptional level.
In summary, our results showed that miR-107, acts as an oncogene miRNA, can significantly promote gastric cancer cell invasion and metastasis through up-regulation its functional target of DICER1, which is down-regulated in gastric cancer and can inhibit gastric cancer cell migration and invasion. The newly identified miR-107/DICER1 axis provides a new insight into the pathogenesis of gastric cancer, especially with respect to invasion and metastasis, and represents a new, prognosis marker and potential therapeutic target for gastric cancer.

Conflict of interest
The authors confirm that there are no conflicts of interest.

Supporting information
Additional Supporting Information may be found in the online version of this article: