Potential Diagnostic, Prognostic and Therapeutic Targets of MicroRNAs in Human Gastric Cancer

Human gastric cancer (GC) is characterized by a high incidence and mortality rate, largely because it is normally not identified until a relatively advanced stage owing to a lack of early diagnostic biomarkers. Gastroscopy with biopsy is the routine method for screening, and gastrectomy is the major therapeutic strategy for GC. However, in more than 30% of GC surgical patients, cancer has progressed too far for effective medical resection. Thus, useful biomarkers for early screening or detection of GC are essential for improving patients’ survival rate. MicroRNAs (miRNAs) play an important role in tumorigenesis. They contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors. Because of their stability in tissues, serum/plasma and other body fluids, miRNAs have been suggested as novel tumor biomarkers with suitable clinical potential. Recently, aberrantly expressed miRNAs have been identified and tested for clinical application in the management of GC. Aberrant miRNA expression profiles determined with miRNA microarrays, quantitative reverse transcription-polymerase chain reaction and next-generation sequencing approaches could be used to establish sample specificity and to identify tumor type. Here, we provide an up-to-date summary of tissue-based GC-associated miRNAs, describing their involvement and that of their downstream targets in tumorigenic and biological processes. We examine correlations among significant clinical parameters and prognostic indicators, and discuss recurrence monitoring and therapeutic options in GC. We also review plasma/serum-based, GC-associated, circulating miRNAs and their clinical applications, focusing especially on early diagnosis. By providing insights into the mechanisms of miRNA-related tumor progression, this review will hopefully aid in the identification of novel potential therapeutic targets.


Introduction
Gastric cancer (GC), a malignant epithelial cancer disease [1], is associated with a high global incidence of mortality [2,3]. Although surgical resection, together with chemotherapy and radical therapy, shows significant improvement over surgery alone in early-stage GC patients [4,5], GC Table 1. Up-regulated miRNAs in tissues for GC.

Cellular Functions of miRNAs in GC
Aberrantly expressed miRNAs serve oncogenic or tumor-suppressor functions in tumorigenesis. They can regulate cell proliferation, cell cycle progression, apoptosis, angiogenesis, cell migration, cell invasion and/or metastasis in GC (Tables 1 and 2), depending on their target genes. Therefore, a given miRNA may exert dual, opposite functions in GC. Generally, oncogenic miRNAs (oncomiRs) are over-expressed in GC and act to inhibit tumor-suppressor genes. Conversely, tumor-suppressor miRNAs, which inhibit oncogene expression, are usually down-regulated in GC. miRNAs in this category regulate various biological processes to stimulate cancer development.

GC-Related miRNAs in Cell Proliferation, Cell Cycle, and Apoptosis
Accelerated cell proliferation, cell cycle progression or disturbed apoptosis are common features of malignancy that arise through silencing of cell cycle-inhibitory or apoptotic pathway-associated genes. In several malignant tumors, miRNA dysregulation stimulates cell cycle progression by up-regulating cyclin expression or down-regulating the expression of other cell cycle regulators or cyclin-CDK (cyclin-dependent kinase) inhibitors, including members of the p16 family (p15, p16, p18 and p19) and p21 family (p21, p27, p28 and p57) [212][213][214]. Moreover, transforming growth factor (TGF)-β1 has been shown to repress GC cell proliferation through transcriptional up-regulation of p21 [108]. In this context, oncomiR-106b and oncomiR-93 are both up-regulated in GC and target the downstream E2F1 (E2F transcription factor 1) and p21 (cyclin-dependent kinase inhibitor 1A), thereby inhibiting the activity of TGF-β1 [26] and contributing to GC by enhancing cell proliferation.
In summary, the abundance of miRs expression may accelerate cell cycle progression through direct or indirect regulation of CDK inhibitors and several cell cycle regulators. Moreover, the abundance of miRs expression also may influence anti-apoptosis or the pro-survival pathway by targeting apoptosis-associated proteins. They may play an important molecular role in GC progression.

GC-Related miRNAs in Cell Migration, Invasion, and Metastasis
Metastasis, a complex, multistep process that involves cytoskeleton remodeling, matrix metalloproteinases (MMPs), homing receptors and their ligands, intracellular signaling pathways (TGFβ and TGFβ/c-Met) and angiogenesis, is a hallmark of malignant tumors [218,219]. As noted above, Zhang et al. [68] identified RECK as a direct target of the oncomiR miR-21, and also found that oncomiR-21 is up-regulated in Helicobacter pylori-infected GC tissues. RECK might also possess anti-invasion, anti-metastasis and anti-angiogenesis functions through modulation of MMP2, MMP9 and MMP14 expression. In addition, miR-21 targeting of PDCD4 (programmed cell death 4) is associated with lymph node metastasis and venous invasion. Another report indicated that PTEN (phosphatase and tensin homologue) is a target of miR-21 that promotes anoikis through activation of the PI3K/Akt pathway [68,72]. In addition, Tsai et al. [59] have shown that oncomiR-196a/b expression promotes GC cell migration, invasion, and metastasis by increasing radixin (RDX) expression in GC tissues. OncomiR-370 was shown to decrease TGFβ-RII expression and stimulate TGFβ1-induced phosphorylation of Smad3. Thus, oncomiR-370 is capable of triggering cell migration by disturbing the TGFβ signaling pathway [85]. Moreover, oncomiR-215 was shown to target ALCAM (activated leukocyte cell adhesion molecule) and increase GC metastasis [71]. Conversely, the tumor suppressor miR-218 promotes invasion and metastasis by targeting Robo1, and thereby activating the Slit/Robo1 signaling pathway [156][157][158][159][160]. Other tumor suppressors of the let-7 family increase the expression of HMGA2 (high mobility group AT-hook 2), which is associated with tumor invasion and is an independent prognostic factor in GC [107]. Furthermore, members of the miR-200 family increase the epithelial-mesenchymal transition (EMT), and contribute to cell migration by reducing the expression of E-cadherin repressors ZEB1 and ZEB2 (zinc finger E-box binding homeobox 2) [145,146]. Down-regulation of miR-335 was found to be significantly associated with lymph node metastasis, invasion of lymphatic vessels, cell invasion and metastasis through targeting of BCL-w and SP1 (specificity protein 1) [82,83].
Interestingly, the function of miRNAs depends on the expression of their target genes. Previous studies revealed that some miRs could target both oncogenes and tumor-suppressor genes, leading to opposite roles in GC. Accordingly, miR-9 may play dual but opposing roles in GC. Thus, acting as an oncomiR, miR-9 targets CDX2 [102] and increases cell proliferation by facilitating cell cycle progression; conversely, acting as a tumor suppressor, miR-9 targets NF-κB1, cyclin D1, and ETS1 to contribute to anti-proliferation and anti-metastasis [102,[112][113][114]. Nakayama et al. [20] reported that oncomiR-10b targets HOXD10 (homeobox D10) to promote GC metastasis. However, Kim et al. [116] found that miR-10b also represses the expression of MAPRE1 (microtubule associated protein RP/EB family member 1), resulting in the inhibition of colony formation and cell proliferation. Moreover, oncomiR-223 was shown to promote GC invasion and metastasis by targeting EPB41L3 (erythrocyte membrane protein band 4.1-like 3) expression [76]. However, Kang et al. [162,163] reported that miR-223 acted as a tumor suppressor, directly targeting STMN1 (stathmin 1) expression to inhibit cell growth and metastasis. Thus, some miRNAs play dual roles through targeting of different genes during GC progression. Further studies will be required to elucidate the details of these different roles.

GC-Related miRNAs as Diagnostic Biomarkers
Early diagnosis permits effective and radical treatment of GC before it develops to an advanced and metastatic stage. Gastroscopy with biopsy, the current standard clinical practice, is not a good screen for GC on a population basis, and existing biomarkers exhibit poor sensitivity and specificity. Thus, there are currently no reliable diagnostic biomarkers for GC. Multiple or combined biomarker assays are expected to provide more accurate results [220]. Investigators continue their efforts to identify convenient, high-sensitivity, high-specificity, and noninvasive biomarkers for early GC diagnosis [185]. MiRNAs can be released from tumor tissues into bodily fluids, including serum, plasma, urine, tears, amniotic fluid and gastric juice, through the secretion of exosome particles [15,221,222]. Mitchell et al. [15,221,222] demonstrated that circulating miRNAs in plasma/serum from GC patients are consistent with those in tissues; therefore, they could be useful as noninvasive biomarkers for the initial diagnosis of GC and assessment of GC recurrence. The most widely investigated biomarkers have been discovered using newer methods, such as systematic analysis of miRNA profiling, miRNA profiling, microarray profiling, and Q-RT-PCR profiling approaches [223][224][225][226][227]. The major plasma/serum-based, GC-related circulating miRNAs that have been suggested as useful GC biomarkers are listed in Tables 3 and 4. Liu et al. [111] used systematic analysis of miRNA profiling, miRNA profiling to identify a signature of five circulating oncomiRs-miR-1, miR-20a, miR-27a, miR-34 and miR-423-5p-and correlated it with tumor stage. Using receiver-operating characteristic (ROC) curve analyses, these authors evaluated the diagnostic value of this miR signature, showing that it achieved a sensitivity of 80% and a specificity of 81%. They observed that the circulating five-oncomiR signature I exhibited a high diagnostic value, with an area under the ROC curve (AUC) of 0.879. By comparison, the five-oncomiR signature II exhibited an AUC of 0.831, which is higher than that of CEA, with an AUC of 0.503, and CA19-9, with an AUC of 0.6. In a large-scale analysis, four circulating oncomiRs (miR-17-5p, miR-21, miR-106a and miR-106b) significantly distinguished GC patients from healthy controls and pre-operative from post-operative GC patients [185]. Moreover, Valladares-Ayerbes et al. [42], using a Cox multivariate regression model, identified circulating oncomiR-200c as a biomarker for GC diagnosis and as an independent prognostic factor for progression-free survival and overall survival in GC patients. Liu et al. [49] found that oncomiR-378 in the GC patients was significantly higher than that in the healthy controls. OncomiR-378 exhibited a high diagnostic value, with an AUC of 0.861, a sensitivity of 87.5% and a specificity of 70.7%.
Taken together, these findings suggest that circulating miRNAs are useful, noninvasive biomarkers for early diagnosis or monitoring of cancer survivors after treatment of GC. The significance of these biomarkers compares favorably to the use of the traditional biomarkers CEA or CA19.9 alone.

GC-Related miRNAs as Prognostic Biomarkers
To predict GC patient survival time, cancer progression (disease stage), prognostic outcome, lymph node metastasis or response to treatment is challenging. Recurrence is also a key problem leading to the failure of treatments, including radical or chemical treatment and surgical resection. Although the clinical outcome of GC has improved, prognostic indicators capable of predicting recurrence in GC patients after treatment are still lacking. Recently, due to the stability and specificity of expression in tissues and circulation, accumulating evidence has shown that miRNAs can be regarded as novel biomarkers with a potential clinical significance tool for GC patients' outcomes.
In general, the occurrence of a distant metastasis frequently leads to advanced-stage cancer and shorter survival. In this context, it has been shown that oncomiR-10b, miR-21, and miR-212 in GC patients are associated with a high metastasis risk and poor clinical outcomes, including tumor-node-metastasis, tumor size, stage, lymph node metastasis, and five-year survival rate [69,126,228].
Conversely, the tumor suppressors miR-125a and miR146a are significantly correlated with lymph node metastasis, indicating that they could be prognostic factors of overall survival [126,134]. Other study showed that low expression of let-7 is related to tumor invasiveness and prognosis by targeting HMGA2 [231].
Therefore, many potential predictors have been regarded as beneficial for mediating the prognosis of GC patients and are the basis for targeted therapy. In future, these prognostic miRNAs could be useful for making choices concerning treatment.

GC-Related miRNAs as Treatment Biomarkers
One miRNA may regulate multi-target gene expression and multiple pathways, affecting the process of tumor development [232][233][234]. Thus, miRNAs are more effective than coding genes as biological regulation molecules.
The methods of current miRNA-mediated treatment are focused on miRNA knockout or silencing the endogenous oncomiRs, including anti-miRNA oligonucleotides (AMOs) [13], miRNA sponges [235], miR-Mask [236], antagomiRs and miRNA inhibitors [12,237]. For example, Chun et al. [200] transfected AS-miR-221/222 with liposomes into GC cell line SGC7901 to inhibit GC cell growth and invasion. Moreover, high expression of miR-196a/-196b promotes GC cell migration and invasion. Elevated miR-196a/-196b expression results in decreasing target RDX protein in GC cells and vice versa. Similar results were obtained in a mouse model of human GC. Tsai et al. [59], through AMOs, used anti-miR-196a/-196b oligonucleotides or the over-expression of RDX, which may serve a therapeutic purpose to inhibit GC metastasis.
Conversely, forced expression the tumor suppressor miRNAs is used to gain the resolution of tumor treatment. miRNA over-expression is often performed by using an in vivo or in vitro RNA delivery system as in cancer therapeutics, including adeno-associated viruses [238] or nonpathogenic bacteria [239] as a carrier to introduce a specific miRNA or miRNA mimics to up-regulate miRNA [240]. miR-1207-5p and miR-1266 are significantly down-regulated in GC tissues. Over-expression of these two miRs inhibited GC growth through targeting hTERT system in vitro and in vivo. Chen et al. [241] showed a novel therapeutic method for the delivery of these two miRs for GC treatment.
However, some problems must be considered. For example, one miRNA modulates multi-target genes and multiple pathways. Also, the off-target effects are unexpected. Thus, better specificity and an effective miRNA delivery system for a therapeutic strategy must be developed [242,243].

Conclusions
miRNA biomarkers have been found at elevated levels in the blood or tissues of patients with tumors. Changes in different biomarkers during tumor progression can help clinicians monitor cancer status. Although higher levels of a biomarker can potentially predict a tumor, other factors may also account for such elevated levels. Dysregulation of tumor markers can occur in response to the presence of a tumor or a change in status, enabling them to be used for a range of applications, including screening, diagnosis, staging, prognosis, and monitoring of recurrence after treatment. The values of these miRNAs as biomarkers will require further confirmation in human GC patients. In the future, an miRNA or an miRNA signature could be a better diagnostic or therapeutic tool than a single gene. However, the challenge is to develop a standard protocol for collecting large specimens, re-analyzing them in a large independent cohort, and validating their significance in clinical applications.
Moreover, several studies have suggested that tumor-derived circulating miRs might be secreted into circulation. Circulating miRs in the plasma/serum of GC patients can be used as diagnostic biomarkers to compare GC patients with healthy controls [185,244]. Most of these biomarkers are more sensitive and specific than the traditional biomarkers CEA or CA19.9 alone [111,185,190].
These findings provide novel indicators for monitoring GC dynamics and early diagnosis for GC to improve survival.
Currently, biomarkers are only used as a reference and not to diagnose the disease per se. A professional physician will still need to provide a comprehensive judgment, including choice of tumor marker, evaluation of clinical symptoms, assessment of related imaging performance, and other non-specific factors. Ultimately, the personalized management, diagnosis, and prognosis of the disease can be achieved using a panel of miRNAs.