Microribonucleic acids and gastric cancer

Abstract

Gastric carcinogenesis is a multistep process involving genetic and epigenetic alteration of protein‐coding proto‐oncogenes and tumor‐suppressor genes. Microribonucleic acids (miR) are a recently‐described class of genes encoding small non‐coding RNA molecules, which primarily act by downregulating the translation of target mRNA. It has become apparent that miR are also key factors in cancer, playing both oncogenic and tumor‐suppressing roles in gastric cancer. Recent studies have shown that a substantial number of miR show differential expression in gastric cancer tissues, and they are turning out to be just like any other regulatory gene. In this connection, miR dysregulation are reported to be associated with incidence, early diagnosis and prognosis of gastric cancer. Therefore, investigation of the biological aspects of miR dysregulation might help us better understand the pathogenesis of gastric cancer and promote the development of miR‐directed therapeutics against this deadly disease. The aim of the present review was to describe the mechanisms of several known miR, summarize recent studies on oncogenic miR (e.g. miR‐21, miR‐106a and miR‐17), tumor suppressor miR (e.g. miR‐101, miR‐181, miR‐449, miR‐486, let‐7a) and controversial roles of miR (e.g. miR‐107, miR‐126) for gastric cancer. In addition, their potential clinical applications and prospects in gastric cancer, such as biomarkers and clinical therapy tools, are also briefly discussed. (Cancer Sci 2012; 103: 620–625)

Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer‐related death in the world.^{( 1 )} Clinically, the absence of specific symptoms renders early diagnosis of this deadly disease difficult. Gastrectomy remains the mainstay treatment for GC, but the prognosis for advanced stage patients is still very poor.^{( 2 )} Thus, early diagnosis and prognosis remain the most promising approach to improve the long‐term survival rate. Therefore, novel markers that are more specific and more sensitive to GC are urgently required for establishment of screening strategies. Recently, the findings of microRNA (miR) suggest ideal diagnostic and prognostic markers.

In 1993, Ambros et al. discovered a gene, lin‐4, which affected the growth of Caenorhabditis elegans (C. elegans). They found that the product of lin‐4 was a small non‐protein coding RNA, miR.^{( 3 )} Microribonucleic acids are small, 20–30 nucleotides in length, non‐coding, single‐stranded RNA that regulate gene expression through post‐transcriptional silencing of target genes (Fig. 1). Sequence‐specific base pairing of miR with 3′ untranslated regions of target messenger RNA within the RNA‐induced silencing complex (RISC) results in target messenger RNA degradation or inhibition of translation, which subsequently controls a wide array of biological processes including cell proliferation, apoptosis and differentiation.^{( 4} , ^{5 )}

Figure 1.

 The microRNA (miR) synthesis and maturation pathway. Microribonucleic acid is transcribed as primary miR (pri‐miR), which is processed by Drosha to produce precursor miR (pre‐miR) in the nucleus. Pre‐microribonucleic acid is subsequently exported to the cytoplasm by exportin 5 in a GTP‐binding nuclear protein Ran dependent manner, and is further processed by Dicer to form the miR:miR* duplex. The mature miR is incorporated into a RNA‐induced silencing complex (RISC) to induce gene silencing.

Microribonucleic acids are involved in biological and pathological processes, and are emerging as highly tissue‐specific biomarkers with potential clinical applicability to define cancer types and origins.^{( 6 )} Recent evidence has shown that overexpressed or underexpressed miR correlate with various human cancers, which shows that some miR might function as oncogenes or tumor suppressor genes. In this connection, miR genes have been characterized as novel proto‐oncogenes or tumor‐suppressor genes in carcinogenesis, including that of gastric cancer (Fig. 2).^{( 7} , ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² , ^{13 )} In the present review, the function of miR and its relationship to signaling pathways governing cancer‐related phenotypes, such as proliferation, resistance to apoptosis and invasiveness in GC, will be discussed. On the basis of recent data, we also describe the possible use of miR to improve the diagnosis, prognosis and treatment of GC.

Figure 2.

 Cancer development was regulated by microRNA (miR) in gastric cancer. Several miR are shown to directly or indirectly regulate cancer‐related phenotypes.

Oncogenic miR in GC

Although GC is a major health burden throughout the world, its molecular pathology remains poorly understood. Analysis of miR regulation will provide opportunities for us to develop new means for diagnosis and treatment of GC. Dysregulation of miR is often associated with human malignancy.^{( 14} , ^{15 )} By targeting oncogenes and tumor suppressor genes, miR themselves can function in various pathways in the development of a tumor. Identification of oncogenic miR provides a new pathway for anticancer treatment.

Microribonucleic acid‐21 is consistently upregulated in solid human cancers, including GC.^{( 16 )} Proof‐of‐principle studies have shown that miR‐21 was significantly overexpressed in human GC tissues^{( 17} , ^{18 )} and GC cell lines. This forced expression of miR‐21 significantly enhanced proliferation and invasion in GC cells. In contrast, knockdown of miR‐21 by an inhibitor caused a significant reduction in proliferation and increase in apoptosis, thus a significant decrease in GC cell invasion and migration. These results suggested that miR‐21 functions as an oncogene and might serve as a novel therapeutic target. The interaction between Helicobacter pylori (H. pylori) infection and miR might contribute to gastritis and gastric carcinogenesis (Fig. 3).^{( 19} , ²⁰ , ^{21 )} In a recent study, Zhang et al. ^{( 18 )} found that miR‐21 was overexpressed in GC, as well as in chronically H. pylori‐infected gastric epithelium tissue, as opposed to non‐infected tissue. They also found that overexpression of miR‐21 promoted cell proliferation and migration, and inhibited apoptosis in GC. AP‐1, as well as the signal transducer and activator of transcription 3 (STAT3), are able to induce miR‐21.^{( 19} , ^{22 )} Nuclear factor‐kappa B (NF‐κB) activation and interleukin (IL)‐6 secretion in the gastric mucosa, which activate AP‐1 and STAT3 respectively, could explain miR‐21 upregulation during H. pylori infection. Shin et al. ^{( 23 )} confirmed that NF‐κB‐binding sites were located in miR‐21 gene transcriptional elements, and nicotine enhanced the binding of NF‐κB to the promoters of miR‐21, suggesting that miR‐21 is directly regulated by the transcription factor NF‐κB. A novel tumor suppressor protein programmed cell death 4 (PDCD4) was negatively regulated by miR‐21, suggesting that PDCD4 might serve as a target for effective therapy.^{( 24 )} Microribonucleic acid‐21 has been reported to be overexpressed in 92% of the GC samples, showing that it could serve as an efficient diagnostic marker for GC. However, the patients with higher miR‐21 expression did not have a worse prognosis, which showed that miR‐21 does not affect the clinical prognosis of GC patients.^{( 25 )} Recent studies have identified the direct targets of miR‐21. All of the direct targets of miR‐21 were tumor suppressors: the PTEN phosphatase,^{( 10 )} the actin‐binding protein tropomyosin 1^{( 26 )} and the reversion‐inducing‐cysteine‐rich protein with Kazal motifs^{( 27 )} and so on. Thus, the downregulation of this single miR provides a significant survival advantage.

Figure 3.

 Interaction between Helicobacter pylori infection and microRNA (miR). Nuclear factor‐kappa B (NF‐κB), which was activated by H. pylori infection, induced miR through intracellular molecular signals. Some miR (miR‐21) contributed to cancer development, and some (miR‐155, miR‐146a) in turn negatively regulated NF‐κB. AP1, activator protein 1; Fas, factor associated suicide; IRAK1, interleukin‐1 receptor‐associated kinase 1; Smad2, SMAD family member 2; STAT3, signal transducer and activator of transcription 3; TRAF6, tumor necrosis factor receptor‐associated factor 6.

Other oncogenic miR were also reported to be associated with gastric cancer,^{( 8} , ¹⁷ , ²⁸ , ^{29 )} and their proved target genes were also listed (Table 1).^{( 30} , ³¹ , ³² , ^{33 )} Microribonucleic acid‐106a, a member of the miR‐106a‐92 cluster located at Xq26.2, is significantly associated with tumor stage, size and differentiation, lymphatic and distant metastasis, and invasion in GC.^{( 29 )} Microribonucleic acid‐17, a member of the miR‐17‐92 cluster located at 13q31.3, shows oncogenic activity.^{( 34 )} In fact, the miR‐17‐92, miR‐106b‐25 and miR‐106a‐92 clusters show high homology in their gene structures and have oncogenic potential.^{( 29} , ³⁵ , ^{36 )} Another report showed that miR‐106b cluster and miR‐222 cluster modulate cell cycle by targeting the Cip/Kip family proteins (p21^Cip1, p27^Kip1 and p57^Kip2), showing that they exert their oncogenic functions by cooperatively suppressing three related Cdk inhibitors (p57, p21 and p27).^{( 8 )}

Table 1.

 Oncogenic microRNA associated with gastric cancer

MicroRNA	Methodology (Ref)	Proposed target(s) (Ref)	Function
miR‐20b	Microarray analysis( 17 )	HIF‐1, STAT3( 30 )	Anti‐apoptosis
miR‐20a		APP( 31 )	Promote proliferation and invasion
miR‐106a
miR‐21		PTEN( 10 ), PDCD4( 24 ), TPM1( 26 )	Promote proliferation, migration and anti‐apoptosis
miR‐18b
miR‐421		CBX7, RBMXL1( 32 )	Promote growth in early stage
miR‐340*
miR‐19a		TNF‐α( 33 )	Anti‐apoptosis
miR‐658
miR‐106a	qRT–PCR( 29 )		Diagnosis of GC
miR‐106b	Microarray analysis, qRT–PCR, Xenograft model( 8 )	Cip/Kip( 8 )	Cell cycle progression
miR‐222
miR‐25
miR‐192	Microarray analysis, qRT–PCR( 28 )	ALCAM( 28 )	Promote GC cell growth and migration
miR‐215

* represents the miRNA expressed at low levels relative to the miRNA in the opposite arm of a hairpin when relative expression levels are known. ALCAM, activated leukocyte cell adhesion molecule; APP, amyloid precursor protein; CBX, chromobox homolog 7; Cip/Kip, CDK interacting protein/kinase inhibitory protein; HIF‐1, hypoxia inducible factor‐1; PDCD4, programmed cell death 4; PTEN, phosphatase and tensin homolog; qRT–PCR, quantitative reverse transcription polymerase chain reactions; RBMXL1, RNA binding motif protein, X‐linked 1; STAT3, signal transducer and activator of transcription 3; TNF‐α, tumor necrosis factor‐α; TPM1, tropomyosin 1.

Tumor suppressor miR in GC

To clarify the role of miR in gastric carcinogenesis, Hashimoto et al. ^{( 37 )} carried out miR microarray analysis and investigated expression changes of miR in a 5‐aza‐2′‐deoxycytidine (5‐aza‐CdR)‐treated GC cell line. They found that miR‐181c and miR‐432AS were upregulated after treatment with 5‐aza‐CdR, and that transfection of the precursor miR‐181c molecule induced decreased growth of GC cells. The results showed that miR‐181c can be silenced through methylation and that it plays important roles in gastric carcinogenesis through its target genes, such as NOTCH4 and KRAS. Zhu found that miR‐181b was downregulated in multidrug‐resistant (MDR) human GC cell line SGC7901/vincristine (VCR), and this downregulation was concurrent with the upregulation of BCL2 protein.^{( 38 )} These findings suggested that miR‐181b could play a role in the development of MDR GC cell lines, at least in part, by modulation of apoptosis through targeting BCL2.

It has been reported that miR‐451 functions as either a tumor suppressor or an oncogene.^{( 39} , ^{40 )} Recent studies have highlighted the tumor suppressing aspect of miR‐451 in gastric cancer. Bandres et al. ^{( 41 )} showed that miR‐451 was downregulated in gastric and colorectal cancer. Overexpression of miR‐451 in gastric and colorectal cancer cells reduced cell proliferation and increased sensitivity to radiotherapy.

Preliminary studies in our laboratory showed that the expression of miR‐101 was downregulated in GC tissues and cells. Ectopic expression of miR‐101 significantly inhibited cellular proliferation, migration and invasion of GC cells by targeting EZH2, Cox‐2, Mcl‐1 and Fos in vitro, and reducing xenograft tumor growth in vivo. The studies suggested that miR‐101 might function as a tumor suppressor in GC.^{( 42 )}

Yang et al. ^{( 43 )} showed that the expression of let‐7a was at a low level in GC. Increased expression of let‐7a suppressed cell growth in vitro and tumor growth in vivo. Furthermore, they showed that RAB40C was regulated directly by let‐7a and played an essential role as a mediator of the biological effects of let‐7a in gastric tumorigenesis.

Microribonucleic acid microarrays showed that miR‐486 was significantly downregulated in primary GC and GC cell lines. Ectopic expression of miR‐486 caused suppression of several pro‐oncogenic traits, whereas inhibiting miR‐486 expression enhanced cellular proliferation. It is supposed that miR‐486 might function as a novel tumor suppressor miR in GC. Furthermore, bioinformatic analysis and luciferase reporter also confirmed that anti‐oncogenic activity of miR‐486 might involve the direct targeting and inhibition of OLFM4.^{( 44 )}

Bou Kheir et al. used quantitative polymerase chain reaction (qPCR) analysis to show a loss of miR‐449 expression in human GC.^{( 45 )} More importantly, cell cycle analysis by FACS showed that miR‐449 overexpressing cells showed a significant increase in the sub‐G1 fraction indicative of apoptosis. β‐Gal assays also showed a senescent phenotype of gastric cell lines with overexpressed miR‐449. This might explain the reasons for misexpression of miR‐449 in GC. Furthermore, miR‐449 overexpression also activated p53 and its downstream target p21, as well as the apoptosis markers cleaved CASP3 and PARP. It suggested that miR‐449 was downregulated in GC and served as tumor suppressor miR to inhibit cell proliferation.

Controversial roles of miR in GC

In previous studies,^{( 16} , ^{36 )} miR‐107 was reported to be significantly overexpressed in GC tissues compared with the matched normal tissues. Li et al. ^{( 46 )} confirmed that miR‐107 was upregulated in GC tissues by using 50 GC cases and the matched normal tissues. Furthermore, high miR‐107 levels correlated strongly with tumor metastasis and worse prognosis. Silencing the expression of miR‐107 by inducing DRCE1 expression could inhibit GC cell migration and invasion in vitro and in vivo, which suggested that miR‐107, an oncogene miR, promotes GC metastasis through downregulation of DRCE1. However, another study reported that miR‐107 expression decreased significantly in GC by using real‐time PCR in 15 patients, and re‐expression of miR‐107 in GC cells significantly reduced proliferation.^{( 47 )} The contradiction in results might be associated with race and the number of specimens. Therefore, biological impact of miR‐107 expression on GC formation in  vivo and in  vitro still needs to be further investigated.

Microribonucleic acid‐126 is known as an endothelium‐specific miR, and has been reported to promote angiogenesis by targeting SPRED1 and PIK3R2, which normally inhibit vascular endothelial cell growth factor (VEGF) signaling.^{( 48} , ⁴⁹ , ^{50 )} Microribonucleic acid‐126 has also been reported to be a tumor suppressive miR, inhibiting tumor cell growth through targeting p85‐β and IRS‐1 in colon cancer cell lines, and in HEK293 and MCF‐7 cells, respectively.^{( 51} , ^{52 )} However, in another study, Otsubo et al. ^{( 53 )} showed that miR‐126 acts as an oncogene by targeting SOX2 in GC cells. They used gain‐ and loss‐of‐function experiments, and luciferase assays to show that miR‐126 inhibited SOX2 expression and played important roles in growth inhibition through cell cycle arrest and apoptosis. Also, an inverse correlation between miR‐126 and SOX2 expression in some cultured and primary GC cells suggested that aberrant overexpression of miR‐126 and consequent SOX2 downregulation might contribute to gastric carcinogenesis.^{( 53 )} However, Feng et al. found that miR‐126 was significantly downregulated in GC tissues compared with matched normal tissues. This downregulation was associated with clinicopathological features, including tumor size, lymph node metastasis, local invasion and TNM stage. Ectopic expression of miR‐126 in SGC‐7901 GC cells potently inhibited cell growth, migration and invasion in vitro, as well as tumorigenicity and metastasis in vivo.^{( 54 )} Therefore, it is controversial as to whether miR‐126 is a tumor suppressive or oncogenic miR. These functional differences in oncogenesis might be explained by “lineage‐dependency model for cancer,” that is, developmentally important genes also have crucial roles during tumor progression in lineage‐specific manners.^{( 55 )} Considering the differences in findings, further studies including a meta analysis are still required to clarify the biological roles of miR‐126 in gastric carcinogenesis and other tissues.

Circulating miR in GC: a new prospect of study

From a clinical point of view, the detection of circulating tumor cells (CTC) might aid the prognosis and thus therapeutic decisions for cancer patients. Tumor‐specific mRNA, which have been used as biomarkers for detecting CTC,^{( 56} , ^{57 )} lack sufficient sensitivity and specificity to facilitate early detection of cancer. Furthermore, there are no other less invasive diagnostic tests for GC, unlike fecal occult blood tests for colon cancer. Recently, several studies have shown that miR, which are involved in tumorgenesis and the development of various cancers, are stably detectable in plasma/serum.^{( 14} , ^{58 )} Microribonucleic acid expression profiling might hold greater promise than mRNA profiling when discriminating between tumor types.^{( 59 )} Thus, the detection of miR in CTC might be a better choice for clinical diagnosis.

To analyze RNA, one should always consider their stability from degradation by RNase. Reports implied that circulating miR could be stably detected, as miR are packaged by exosomes, thus protected from RNA degradation. Tumor miR might be present as a result of death and lysis of tumor cells or tumor cells releasing exosomes that contain miR. Tumor‐derived exosomes are small membrane vesicles of endocytic origin released by the tumor, which might play important roles in cell–cell communication.^{( 60 )} Recent findings have also shown endogenous plasma miR in blood samples are stably detectable in a form that is resistant to RNase activity,^{( 61 )} evidenced by identification of miR in body fluids (Fig. 4).^{( 61} , ⁶² , ⁶³ , ⁶⁴ , ⁶⁵ , ^{66 )} More and more studies have shown that circulating miR were associated with cancer (Table 2).^{( 67} , ⁶⁸ , ⁶⁹ , ^{70 )} Non‐invasive tests are a trend for disease diagnosis, and serum/plasma miR were identified as stable blood‐based markers for cancer detection.^{( 58} , ⁶¹ , ^{69 )}

Figure 4.

 Microribonucleic acids (miR) in human body fluids are stable detectable markers for cancers.

Table 2.

 Circulating microRNA associated with cancer

Cancer	Sample size	MicroRNA name	Expression in cancer	Clinical significance	Study
DLBCL	60 Patients and 43 healthy controls	miR‐155	Elevated	Diagnostic markers for DLBCL	Lawrie et al. ( 67 )
		miR‐210
		miR‐21
CRC	25 Patients and 20 healthy controls	miR‐17‐3p	Elevated	Non‐invasive molecular marker for CRC screening	Ng et al. ( 68 )
		miR‐92
Ovarian cancer	28 Patients and 15 healthy controls	miR‐21	Elevated	Therapeutic and biomarker potential in ovarian cancer	Resnick et al. ( 69 )
		miR‐92
		miR‐93
		miR‐126
		miR‐29a
		miR‐155	Decreased
		miR‐127
		miR‐99b
Tongue SCC	30 Patients and 38 healthy controls	miR‐184	Elevated	Oncogenic role of tongue SCC	Wong et al. ( 70 )

CRC, colorectal cancer; DLBCL, diffuse large B‐cell lymphoma; SCC, squamous cell carcinoma.

The comparison between expressions of miR in plasma and primary tumor tissues showed that plasma and primary GC tissues showed similar tendencies concerning the expression of miR in almost all cases.^{( 66 )} In addition, the plasma concentrations of miR (miR‐17‐5p, miR‐21, miR‐106a, miR‐106b) were significantly higher in GC patients than controls, whereas let‐7a was lower in GC patients, showing that plasma miR might be available as a new marker for GC.

However, whether the expression patterns in serum/plasma are consistent with those in cancer tissues is still a hot topic. Chim et al. ^{( 71 )} first detected placental miR in maternal plasma and found that the expression levels of 17 miR were higher in placentas than those in maternal blood cells. Lodes et al. discovered that miR expression patterns in serum were not identical to those seen from miR taken directly from cancer cell lines. They suggested that tumor cell lysis might be the most obvious source of serum or plasma miR. Alternatively, miR might be produced during the active transport forming exosomes.^{( 72 )}

Recently, the detection of occult cancer cells in peripheral blood or bone marrow has received attention for predicting postoperative cancer recurrence and novel strategies of adjuvant therapy.^{( 57 )} Zhou et al. reported that the miR levels in the peripheral blood of GC groups were found to be significantly higher than those of the healthy volunteer group. In preoperative and postoperative patient groups, miR‐106a and miR‐17 levels were significantly higher than those in controls. The aforementioned results showed that the detection of miR in peripheral blood might be a novel tool for monitoring CTC in patients with GC.^{( 73 )} However, further clinical trials using a variety of plasma miR should be carried out to define the usefulness of the assay.

Concluding remarks and future perspectives

Discovered approximately 10 years ago, miR are currently considered as crucial post‐transcriptional regulators of gene expression. Their roles in cell development, proliferation and differentiation are widely recognized, as is their importance in the regulation of immune responses.^{( 74 )} Furthermore, miR are frequently altered in cancer cells and function as either oncogenes or tumor suppressors. The diverse and fundamental role of miR in cellular mechanisms suggests that proper control of these regulatory elements is essential for the maintenance of a non‐pathological state.

Dysregulation of miR occurs in gastric cancer, as well as other malignant diseases. The mechanisms by which miR take part in tumor promotion and progression are complex. As the progression of GC is rather the combination of changes in expression of oncogenes and tumor suppressors, discovery of miR marks a step forward to providing a novel regulatory mechanism for GC. More and more miR are reported to be involved in GC up to now; however, little is known about their functions and their target mRNA. Thus, further investigations on the complicated interaction between the multiple miR and multiple target mRNA are required.

From the recent publications on miR in GC, we suppose that miR are turning out to be similar to any other regulatory genes, except that they do not encode protein; some promote oncogenesis, some prevent it, but most are not directly involved. It is anticipated that, with a more comprehensive understanding of miR dysregulation and the associated abnormalities in cellular signaling in GC, the unique mode and action of miR biosynthesis will provide new possible therapeutic approaches.

Disclosure statement

The authors have no conflict of interest.