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Emerging role of small ribonucleic acids in gastrointestinal tumors

Accepted 27 January 2010. published online 08 February 2010.
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Abstract

Small regulatory ribonucleic acids (RNAs) are recently recognized as being connected with a growing list of common diseases such as: cancer, heart disease, diabetes and inflammation and to date more than 5,000 publications are recorded on PubMed alone. Specific pathways generate each class of RNAs and their activities converge in the process of silence interference.

In gastrointestinal malignancies microRNAs are deregulated, sometimes found in higher or lower levels depending on the type of malignancy and stage of the disease, functioning either as tumor suppressors or as oncogenes they interact forming regulatory loops with known transcription factors and signaling pathways. MiRNAs extracted from archived tissue biopsies can be used effectively as diagnostic, prognostic tools and molecular markers because they are stable over time and resistant to RNAse degradation. The distinct physiology of small RNAs may translate in more targeted cancer therapies in the near future.

Keywords: RNA interference, MicroRNA, Silence interference, Gastrointestinal malignancy, Non-coding RNA, Esophageal cancer, Gastric cancer, Pancreatic cancer, Antagomirs, Hepato-billiary cancers, Colorectal cancers, Gene silencing, Transcription factors

Article Outline

• Abstract

• 1. Origin of small ribonucleic acids and their functions

• 1.1. Definition of small RNA and their physiology

• 1.2. MicroRNA (miRNA) and short interfering RNA (siRNA) pathways in mammalian cells

• 1.3. miRNA pathway for silencing

• 1.4. siRNA pathway of silencing

• 2. The role microRNAs in gastrointestinal malignancies

• 2.1. Selected microRNA families deregulated in gastrointestinal malignancies

• 2.2. MicroRNA evaluation in gastrointestinal tumors

• 2.2.1. Esophageal tumors

• 2.2.2. Gastric tumors

• 2.2.3. Biliary tumors

• 2.2.4. Pancreatic tumors

• 2.2.5. Hepatocellular tumors

• 2.2.6. Colorectal tumors

• 3. Outlook on potential applications of microRNAs in clinical practice

• 3.1. miRNA diagnostics applications in gastrointestinal tumors

• 3.2. RNA interference as possible therapy of the future for gastrointestinal tumors

•

• References

• Biography

• Copyright

1. Origin of small ribonucleic acids and their functions

1.1. Definition of small RNA and their physiology

The normal function of eukaryotic cells depends on accurate expression of ribonucleic acids both protein coding messenger RNAs (mRNA) and non-coding RNAs.

Non-coding RNAs have many defined functions in the cells: protein translation, RNA processing, telomere maintenance, chromosomal architecture and regulating stem cell renewal [1].

Messenger RNA was discovered over 50 years ago and was defined as the molecule transferring the genetic information from DNA into the cell to be further translated into proteins [2]. Cells carrying identical DNA information had the ability to acquire different traits by regulating messenger RNA in a time and tissue specific manner [3]. In the 1960s it was posited that the regulation of messenger RNA transcription from DNA is controlled by the activity of proteins called transcription factors. Transcription factors either inhibited or promoted access of the RNA polymerase to gene promoter sites [4]. The “central dogma” unidirectional flow of genetic information (DNA transcribed to messenger RNA and mRNA translated to protein) and the cardinal role of gene regulation by transcription factors were able to explain the biology of malignancy for the past four decades until new discoveries in molecular biology revealed additional alternative pathways of gene regulation [5].

Some examples of alternative pathways of gene regulation outside transcription factors are: (1) Xist-a large non-coding RNA-functioning in X-chromosome inactivation in mammals [6]; (2) DNA regulation by direct retroviral transduction of 4 genes (c-MYC, SOX2, OCT3/4 and KLF4) implicated in transforming somatic cells into pluripotent stem cells [7]; (3) genome regulation by small RNAs such as microRNAs (miRNAs) [regulators of endogenous genes] or short interfering RNAs (siRNAs) [defenders of genome integrity in response to foreign or invasive nucleic acids such as viruses, transposons and transgenes] [8]. The mechanism of gene silencing by small RNAs was studied in nematode worms by Andrew Fire and Craig Mello. In 1998 they discovered that injecting double stranded RNA (dsRNA) molecules into Caenorhabditis elegans worms will silence the expression of the target gene whose sequence was complementary to the injected dsRNA and the potency of silencing was at least 10-fold higher than with single-stranded complementary RNA [9]. RNA interference (RNAi) was recognized as a potent repressor of messenger RNA translation and an alternative way of gene expression regulation [9]. RNAi is an evolutionary conserved mechanism with similar roles in plants, insects and animals [10]. These small RNAs play a fundamental role in regulating gene expression in multi-cellular eukaryotes [11], [12], [13].

1.2. MicroRNA (miRNA) and short interfering RNA (siRNA) pathways in mammalian cells

The end result of RNA interference process either via siRNA or via miRNA is to degrade the messenger RNA (mRNA) of the target gene. The target gene has complementary sequences with miRNA or siRNA. This mechanism is post-transcriptional and not dependent on the rate of messenger RNA synthesis. The silencing machinery operates only with gene components resulting in messenger RNA (exons). Introns and promoter sequences of the genes are ineffective as silencing triggers [9], [12].

1.3. miRNA pathway for silencing

MiRNAs are encoded in the genomic DNA by specific miRNA genes. They are transcribed by RNA polymerase II [14] into stem-loop structures, the primary transcript, called pre-miRNA of approximately 500–3,000 bases long. These pri-miRNAs are processed further by the enzyme Drosha in the nucleus into the hairpin shaped precursor miRNA (pre-miRNA) of approximately 60–70 nucleotides (Fig. 2). In contrast to siRNA most miRNA are processed to have exact ends, Drosha carries this out by binding to DiGeorge syndrome critical region 8 (DGCR8) that serves as molecular anchor to properly position the Drosha's catalytic site to the correct distance for the stem-flank junction [14]. The precise processing of pre-miRNA is important since the lead determinant of silencing will be the “seed” region on the miRNA product. The “seed” region is defined as the nucleotides at position 2–7 on the anti-sense miRNA strand complementary to target regions on messenger RNA (Fig. 3). Pre-miRNA is exported into the cytoplasm through the nuclear pore complex with the help of Exportin 5 (Fig. 2). In the cytoplasm the double stranded pre-miRNA is further processed by the DICER (an RNase III enzyme) to release 2 complementary 22-base nucleotides (nt) products.

Following enzymatic processing by DICER, the anti-sense miRNA strand complementary to target regions on messenger RNA (mRNA), binds the Argonaut (Ago-1) protein complex, protein that guides it to target sequences on mRNA and incorporates it into the RNA-induced silencing complex (RISC) [15], [16]. Mature miRNA bind the 3′ untranslated region (UTR) of the target messenger RNA through their seed region via complementary Watson–Crick base paring, the miRNA–mRNA interaction will results in mRNA cleavage and thus down-regulation of protein expression by translational repression [16], [17], [18], [19] (Fig. 1, Fig. 2, Fig. 3) [20].

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Fig. 1. Short interfering RNA and microRNA pathways in mammalian cells. MicroRNA pathway: microRNA gene is transcribed into primary transcript pri-miRNA, (2) Pri-miRNA is cleaved by Drosha to a hairpin pre-miRNA, (3) pre-miRNA is transported out of the nucleus by exportin-5, (4) pre-miRNA is cleaved by DICER to form short double stranded miRNA duplex, (5) miRNA duplex separates into single-stranded mature miRNAs and complexes with a RNA-induced silencing complex (RISC), (6) mRNA binds with miRNA programmed RISC complex in the 3′-UTR of the mRNA, and (7) mRNA is translationally repressed or directed to the P-body where it is degraded. siRNA pathway: double stranded RNA (dsRNA) enters the cell, (2) dsRNA is cleaved by DICER to form a short double stranded siRNA duplex, (3) siRNA duplex separates into single-stranded siRNA and complexes with RISC structure, and (4) mRNA is degraded by siRNA programmed RISC. Ago=argonaute protein part of the RISC complex, Ago1 binds miRNA and guides the strand to the RISC complex, Ago2 binds siRNA. Piwi member of Argonaute family is expressed in germline and defends against transposable elements.

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Fig. 2. microRNA processing. Pri-miRNA is transcribed from the genomic DNA. In the nucleus Drosha cleaves it and pre-miRNA is generated. Pre-miRNA complex is exported into the cytoplasm by Exportin 5. In the cytoplasm Dicer cleaved pre-miRNA and miRNA is generated.

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Fig. 3. miRNA–mRNA hybrid region of Let-7a miRNA: miRNA consists of two parts: 5′ region called the seed region (GAGGUA) which has the target recognition specificity and the other miRNA region (GUAGGUUGUAUAGUU) which is able to tolerate mismatches. All members of the Let-7 miRNA family (7a, 7b, 7c, 7d, 7e, 7f, 7g and 7i) have the unique conserved seed sequence (GAGGUA). The binding of an miRNA to a specific 3′ untranslated region is critically dependent on 5′ nucleotides (nt) 2–7 of the mature miRNA. Polymorphism in the miRNA seed regions are extremely rare and when it happens they have the potential to affect hundreds of target genes, the 3′ mismatch tolerant region of miRNA may tolerate to a certain extent. Polymorphisms in the target mRNA seed region affects the regulation of individual target genes.

MiRNAs are almost completely conserved among various (human, mouse, rat and primates) species [21] and have 2 parts (Fig. 3): (1) 5′ region containing nucleotides (nt) 2–7 of the mature miRNA is called the seed region which has the target recognition specificity and has to form complementary Watson–Crick base pair and (2) the other miRNA region and the 3′ mismatch tolerant region (MTR) [22]. The binding of a miRNA to a specific 3′ untranslated region is critically dependent on seed region of the mature miRNA, this sequence also classifies miRNAs into families. Polymorphism in the miRNA seed regions are extremely rare and when this happens it affects hundreds of target genes and usually result in disease, the 3′ mismatch tolerant region of miRNA has the ability to tolerate polymorphism [22] (Fig. 3).

The mechanism of action of miRNA is to inhibit the translation of mRNA into protein and destabilize the target RNA [23], [24]. To date about 50% of known human miRNA are located in fragile sites of genome [25].

1.4. siRNA pathway of silencing

Sources of double stranded DNA are either viruses or genomic DNA (from the transcription of pseudogenes, centromeres and other repetitive sequences) [26].

When double stranded RNA reaches the cytoplasm it is cleaved by the DICER (an RNase III endonuclease) enzyme to form a short double stranded RNA duplex siRNA that will separate into single-stranded 21-nucleotides long siRNA. This strand will bind Ago 2 protein that will guide it to the RNA-induced silencing complex (RISC) and directs target recognition by Watson–Crick base pairing, whereas the other strand of the original duplex is discarded [27] (Fig. 1).

Dicer enzymes are evolutionary conserved proteins capable to recognize and process dsRNA into siRNAs of species-specific size [28]. There is only one Dicer gene in mammals and the resulting enzyme functions in miRNA and siRNA processing [29].

RISC contains two main components the first is the small RNA which triggers its activation and the second is the Argonaute proteins (Ago) which is the catalytic engine driving its activity [30]. Mammals have four Argonaute subfamily members, Ago1,2,3,4 [31]. Ago1 preferentially associate with miRNA and Ago2 with siRNA [32], they are the catalytic components of RISC performing the “slicing” function that cleaves homologous messenger RNA [30] (Fig. 1).

2. The role microRNAs in gastrointestinal malignancies

2.1. Selected microRNA families deregulated in gastrointestinal malignancies

In a variety of cancers cells miRNA appear in abnormal concentrations relative to normal adjacent cells. There are over 1,000 microRNAs listed in the crucial miRBase resource (http://www.microrna.sanger.ac.uk) and more than one-third of all human genes are believed to be regulated by these microRNAs [33]. MicroRNAs function by preventing translation of mRNAs into proteins and/or by triggering degradation of these mRNAs. To date, up- or down-regulated expression of microRNAs has been implicated in several disease states. MicroRNAs are expressed in a time and tissue specific manner, and in cancer they behave either as oncogenes or tumor suppressors [25], [34], [35], [36].

Early estimates grouped microRNA into 48 families based on their similarities in nucleotides 2–8 at their 5′ end, referred as the “seed sequence” [37], [38] (Fig. 3). MiRNA genes can be expressed as single genes (moncistronic) or as cluster of miRNA from within one locus (polycistronic) and closely linked miRNAs can be from different families. They can be transcribed from their own promoters or they are able to “borrow” the promoters of nearby genes [39].

Table 1 illustrates a brief description of known human microRNA families known to be deregulated in gastrointestinal malignancies. Table 2 shows the individual miRNA that are upregulated while Table 3 lists miRNA down-regulated in gastrointestinal malignancies. Listed for each miRNA are some of their known target genes.

Table 1.

Brief description of known human microRNA families known to be up- or down-regulated in gastrointestinal malignancies.


Human microRNA	Genome location	Deregulated in gastrointestinal malignancies	Molecular mechanism	Targets	Potential applications: diagnostic/prognostic marker
let-7 family (12 members [i.e. paralogous]: let7a to let 7i and miR-98)	let-7a1: 9q22.32; let-7b: 22q13.33; let7c: 21q21.1; let 7d: 9q22.32; let7e: 19q13.33; let 7f-1: 9q22.32; let 7i: 12q14.1; miR-98: Xp11.22	Gastric and colon cancer	let-7 family downregulates RAS gene [111]; downregulates HMGA2 [52], [112], is absent in embyonic tissues and highly expressed in mature tissues [51]	CCND1, CDK6, DICER, HMGA2, MYC, RAS, TLR4,	Low expression in poorly differentiated tumors high expression in well differentiated tumors [51] let-7f promotes angiogenesis [113]
miR-17-92 cluster (6 members: miR-17, -18a, -19a, -20a, -19b-1, -92)	13q31.3, intron 3 C13orf25	Over-expressed in colon cancer	Increases tumor growth and vascularization [114], [115], [116] miR-20a is anti-apoptotic [117]	CDKN1A, E2F1, E2F2, E2F3, HIF-1A, PTEN, TGFBR2, TSP1, RB2/P130	High plasma level of miR-92 differentiates colorectal from normal tissues [118]
miR-106b-93-25 cluster	7q22.1	Over-expressed in gastric and colon cancer	Decreases apoptotic resposes via BIM after TGF-b stimulation	CDKN1A, E2F1, BIM
miR-21	17q23.1	Over-expressed in esophageal, gastric, cholangiocarcinoma pancreatic and colon cancer	Induces invasion and metastasis in colon cancer [119]	PDCD4	Predicts poor prognosis in pancreatic cancer [120] high expression associated with poor survival in stage II and III colon cancer treated with chemotherapy [121] inhibition of miR-21 increased cholangiocarcinoma sensitivity to gemcitabine [69]
miR-29 family (miR-29a, -29b-1, -29b-2, -29c)	29a: 7q32.3; 29b-1:7q32.3; 29b-2: 1q32.2; 29c: 1q32.2	Down-regulated in cholangiocarcinoma and colon cancer	Induces aberant methylation via DNMT3A, B, induces apoptosis via p53 and MCL1	DNMT3A, B, MCL1, TCL1
miR-34 family (miR-34a, miR-34b, miR-34c)	34a:1p36.23; 34b:11q23.1 intergenic; 34c:11q23.1 intergenic	Down-regulated in pancreatic cancer and hypermethylated in colon cancer	Induces upregulation of p53 and down-regulation of E2F in colon cancer [122]	BCL2, CCND1, CCNE2, CDK4, CDK6, MYC, DLL1, Notch1, N-MYC, MET, HMGA2, SIRT1	Ectopic expression leads to cell cycle arrest and apoptosis, possible therapeutic implications [123]
miR-101 family (101-1, 101-2)	miR-101-1:1p31.3; miR-101-2: 9p24.1	Down-regulated hepatocellular carcinoma	Alters chromatin structure by repressing EZH2	COX2, EZH2, MCL1
miR-122a	18q21.31 intergenic	Down-regulated hepatocellular carcinoma	Increased Cyclin G1 [88]	CAT-1
miR-124a family (miR124-1; miR124-2; miR124-3)	miR124-1: 8p23.1; 124-2: 8q12.3; 124-3: 20q13.33	Hypermethylated in colon and gastric cancer	Hypermethylation of tumor suppressor genes	CDK6, RB
miR-143, miR-145 cluster	5q32 intergenic	Down-regulated in colon cancer	miR-143 and -145 precursor sequences are abnormally processed in colon cancer	MYC, ERK5, KRAS, PARP8
miR-155	21q21.3 exon 3 ncRNA BIC	Over-expressed in colon and pancreatic cancer		AID, AGTR1
miR-221, miR-222 family	Xp11.3 intergenic	Over-expressed in pancreatic cancer	Promotes cancer cell proliferation	p27kip1	Differential expression between normal and cancerous pancreatic cells possible diagnostic applications [77]

Table 2.

miRNA upregulated in gastrointestinal malignancies: Listed for some miRNA are some of their target genes, regulatory connections were demonstrated between miRNA and messenger RNA for their target transcription factors.


Malignancy	miR	Target Genes	Reference
Chlolangiocarcinoma	miR-21	PTEN	[69]
Cholangiocarcinoma	miR-141	PTEN	[69]
Cholangiocarcinoma	miR-200b	PTEN	[124]
Cholangiocarcinoma	Let-7	NF2; STAT3	[75]
Colorectal cancer	miR-31	FOXC2; FOXP3	[96]
Colorectal cancer	miR-96	CHES1	[96]
Colorectal cancer	miR-135b	CHES1	[96]
Colorectal cancer	miR-19a	THBS1	[114]
Colorectal cancer	miR-21	PDCD4	[119]
Colorectal cancer	miR-18	CTGF	[114]
Colorectal cancer	miR-200b	CHES1	[96]
Colorectal cancer	miR-17-92	MYC	[114]
Colorectal cancer	miR-183	CHES1, FOXO3A	[96]
Esophageal	miR-25		[41]
Esophageal	miR-151		[41]
Esophageal	miR-424		[41]
Esophageal	miR-103	YWHAH; TGFBR3 AXIN2	[41]
Esophageal	miR-21	PDCD4	[42]
Esophageal	miR-107	TAF5; CAPZA2	[41]
Hepatocellular Cancer	miR-21	PTEN	[124]
Hepatocellular Cancer	miR-221	Cyclin G1	[88]
Pancreatic Cancer	miR-21		[76]
Pancreatic Cancer	miR-103		[76]

Abbreviations: PTEN, protein and tensin homolog; NF2, neurofibromin 2 (merlin); STAT3, signal transducer and activator of transcription 3; FOXC2, Forkhead transcription factor C2; FOXP3, Forkhead transcription factor P3; CHES1, check point suppressor 1; THBS1, thrombospondin 1; PDCD4, programmed cell death 4; CTGF, connective tissue growth factor; MYC, myelocytomatosis viral oncogene; FOXO3A, Forkhead transcription factor O3A; YWHAH, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein; TGFBR3, transforming growth factor beta-receptor type 3; AXIN2, axin-related protein; TAF5, TATA box binding protein (TBP)-associated factor; CAPZA2, capping protein (actin filament) muscle Z-line, alpha 2; Cyclin G1, involved in cell cycle.

Table 3.

miRNA down-regulated in gastrointestinal malignancies: Listed for each miRNA are some of their target genes.


Malignancy	miRNA	Target genes	Reference
Cholangiocarcinoma	miR-29	Mcl-1	[125]
Colorectal cancer	miR-133b	KRAS	[96]
Colorectal cancer	miR-145	YES; MAP3K3	[96]
Colorectal cancer	miR-124a	Cdk6	[46]
Colorectal cancer	miR-143	ERK-5	[97]
Colorectal cancer	miR-145	MAP3K	[126]
Colorectal cancer	let-7a	RAS and MYC	[97]
Esophageal	miR-99a		[41]
Esophageal	miR-29c		[41]
Esophageal	miR-100		[41]
Gastric adenocarcinoma	miR-124a	LOX	[49]
Gastric adenocarcinoma	miR-34	bcl-2	[68]
Gastric adenocarcinoma	let-7a, -7b, -7c	HMGA2	[50]
Hepatocellular Ca.	let-7	MYC	[95]
Pancreatic cancer	miR-155		[76]
Pancreatic cancer	miR-217		[103]
Pancreatic cancer	miR-375		[127]

Abbreviations: Mcl-1, myeloid cell leukemia sequence 1; KRAS, Kirsten rat sarcoma viral oncogene homolog; YES, avian sarcoma virus Y73 homolog; MAP3K3, mitogen-activated protein kinase 3; MYC, myelocytomatosis viral oncogene; LOX, lysyl oxidase; bcl-2, B-cell/CLL lymphoma 2; HMGA2, high-mobility group AT-hook 2.

2.2. MicroRNA evaluation in gastrointestinal tumors

2.2.1. Esophageal tumors

In esophageal malignancies the current the tumor-node-metastasis (TNM) staging used to asses patients need for multimodality therapy is not an accurate prognostic indicator of patients who have high risk of early relapse, nor could precisely identify patients who lacked benefit from combined modality therapy due to the aggressive biology of their disease [40].

Genome-wide miRNA expression profile done on 31 fresh frozen pairs of primary esophageal squamous cell carcinoma paired with adjacent normal esophageal tissues distinguished expression signature differences in malignant and normal cells. In malignant cells miR-25, -424, and -151 were upregulated and miR-99a, -29c and -100 were down-regulated when compared to normal esophageal cells. High levels of miR-103 and miR-107 correlated with 2.6-fold decreased survival regardless of patients age, TNM, alcohol history, smoking history and gender p=0.041 [41].

MiR-21 over-expression is also important in solid tumor progression. Hiyoshi et al. examined paraffin embedded tissue samples from 20 patients with squamous-esophageal cancer and adjacent normal esophagus. The relationship between patient prognosis and target miRNA genes was investigated. MiR-21 levels determined by Taq-Man RT-PCR were up to 40-fold higher in esophageal squamous cancer cells compared to normal controls or normal stroma. Using anti-miR-21 transfection in squamous cell esophageal cell lines (TE-8, TE-6, TE-10, TE-11) the authors showed that such treated cells had half the ability to proliferate and a 50% decrease in their invasiveness potential as assessed by Matrigel invasion chamber. The target gene for miR-21 is programmed cell death 4 (PDCD4), the levels of PDCD4 were inversely related to the level of miR-21 when analyzed by Western blot [42].

2.2.2. Gastric tumors

Helicobacter pylori infection, a major carcinogen for gastric cancer, is proven to induce a potent abnormal DNA methylation in gastric epithelial cells [43]. Aberrant DNA methylation of the CpG—islands next to promoters of tumor suppressor genes such as CDKN2A, MLH1, CDH1, LOX and APC have been involved in development and progression of gastric cancer [43], [44], [45]. Similarly DNA methylation of tumor suppressing pri-miRNAs genes may also lead to silencing and down-regulation of miRNAs and cancer progression and reports of miRNA down-regulation through epigenetic silencing have been published for miR-124a [46], miR-137 [47], miR-193a [47] and mir-127 [48]. Ando et al. analyzed fresh frozen normal antral tissues from 56 healthy individuals obtained during endoscopic antral biopsies and gastric cancer tissues from 45 patients who underwent curative resection. H. pylori status was determined in all patients by serology and rapid urease test. H. pylori positive healthy individuals has 7.8–46.7-fold higher methylation levels of miR-124a-1, -124a-2, -125a-3 tumor suppressor microRNA compared to those values in healthy individuals negative for H. pylori. Healthy individuals previously infected with H. pylori, treated successfully, having completely eradicated infections failed to change their methylation status of the miR-124a-1, -124a-2, -125a-3 suggesting that these changes in methylation are long-lasting and could explain the concept of field cancerization defect seen in gastric cancer patients. Of H. pylori negative patients, those with gastric cancer had 7.2–24.7-fold higher methylation levels of miR-124a-1, -124a-2, -125a-3 compared to gastric mucosa from H. pylori negative healthy individuals [49].

One hundred and ten fresh frozen gastric adenocarcinoma tumor samples and matched control (epithelial gastric mucosa) were obtained and analyzed for levels of HMGA2 protein and let-7 miRNA. The data was correlated with clinical patients’ outcomes. HMGA2 messenger RNA level was 7–35 times higher in the tumors compared to normal epithelium p<0.05. The patients overall survival was 4 times lower in the high HMGA2 group compared with low-undetectable HMGA2 group. Patients with high HMGA2 levels had higher incidence of serosal invasion and lymphovascular permeation than those with undetectable levels. At 10 years completely resected patients with undetectable HMGA2 levels in their tumors had 60% survival regardless of TNM stage, age, sex, histology (signet ring, mucinous, poorly differentiated) and lymph node metastasis [50]. The levels of let-7a, let-7b and let-7c was 4-fold higher in patients with low-undetectable HMGA2 levels than in those with high protein levels [50], consistent with previously demonstrated HMGA2 mRNA degradation via RNA let-7 RNA interference [51], [52]. The high-mobility group A 2 (HMGA2) is a small non-histone chromosomal protein with no intrinsic transcriptional ability but capable of altering chromatine architecture [53].

HMGA2 is abundantly expressed during embryogenesis and largely undetectable in adult tissues, playing a critical role in cell development and differentiation during embryogenesis [54]. Knocking out the HMGA2 protein expression in developing mice leads to pigmy phenotype with hypoplasia of mesenchymal tissues [55]. Located on chromosome 12q13-15, HMGA2 over-expression is a hallmark of various benign and malignant mesenchymal tumors such as lipoma, uterine leiomyoma, pulmonary chondroid hamartoma [56], pleomorphic salivary gland adenoma [57], and endometrial polyps [58]. Besides gastric cancer the HMGA2 protein is also elevated in non-small-cell carcinoma of the lung where it was associated with high cell proliferation and poor survival [59]. The HMGA2 gene is selectively amplified and over-expressed in 10% of liposarcomas [60], breast cancer [61], [62], pancreatic cancer [63], [64], head and neck cancer [65], thyroid [66] and ovarian cancer [51]. Like in gastric cancer the HMGA2 protein is negatively regulated by the let-7 miRNAs in head and neck cancer [67], lung cancer cell lines [52] and ovarian cancers [51].

Human gastric cell line Kato III was evaluated for the expression of miRNAs and their respective target genes. Kato III with a mutant p-53 has very low levels of miR-34 and high levels of bcl-2. MiR-34 targets the mRNA of bcl-2 genes for degradation. Forced expression of miR-34 in Kato III cells increased chemotherapy mediated apoptosis 3-fold similar to degradation of bcl-2 mRNA by silence interference [68].

2.2.3. Biliary tumors

Using microarray technology miRNA expression in malignant and non-malignant cholangiocytes revealed that miRNA expression was significantly different. Malignant cholagiocytes demonstrated a greater than 5-fold increased expression of miR-21, more than 2-fold increased expression in miR-200b, miR-21, miR-23a, miR-141, and miR-27a and greater than 5-fold decreased expression of let7b, miR-326, miR-351, miR-373, and miR-150. Cholangiocarcinomas are highly chemoresistant tumors, therefore their sensitivity to chemotherapy when deprived of certain miRNA was assessed. Mz-ChA-1 cells (malignant cholangiocytes derived from metastatic gallbladder cancer) were transfected with miRNA-specific anti-sense inhibitors, and their sensitivity to gemcitabine was assessed. Anti-miR-21 and anti-miR-200b both increased gemcitabine-induced cytotoxicity by 12.4% and 14.4%. Cell viability of normal, non-malignant, cholangiocytes (H69-cell line) in response to gemcitabine was also assessed when deprived of certain miRNA. Cell viability increased by 18.7%, 12.0%, and 14.0% of controls in cells transfected with miR-141, miR-21, and miR-200b precursors, respectively. To determine the specific effect of miRNA on apoptosis, the extent of apoptosis was quantitated during incubation of Mz-ChA-1 cells with gemcitabine in the presence or absence of mir-141, miR-200b, or miR-21 inhibitors. Inhibition of miR-21 and miR-220b decreased gemcitabine-induced apoptosis but no effect on apoptosis was noticed when miR-141 was inhibited. This evidence supports the concept that over-expression of miR-21 and miR-200b miRNA may contribute to chemoresistance in cholangiocarcinoma by decreasing chemotherapy-induced apoptosis [69].

Malignant cholangiocarcinoma cell line overexpresses miR-21, and its inhibition increased cell line sensitivity to gemcitabine by PTEN (phosphatase and tensin homolog deleted on chromosome 10) activation of PI3-kinase pathway [69]. Interleukin-6 (IL-6), the inflammation-associated cytokine, participate in tumor growth and resistance to therapy by the activation of survival signals the signal transducers and activators of transcription (Stat) factors or protein kinase cascades [70]. Constitutively activated Stat-3 is observed in many cancers also known is that malignant cells expressing activated Stat-3 are dependent on it for survival, and abrogation of Stat-3 results in the loss of malignant phenotype [71]. Stat-3 is regarded as an oncogene that contributes to tumor growth [72]. Constitutive phosphorylation and activation of Stat-3 in cholangiocarcinoma cells have been shown to be IL-6-dependent [73].

Using malignant cholangiocytes transfected to overexpress IL-6 (Mz-IL-6 cells), Meng et al., asked if high IL-6 levels would also alter the miRNA expression and thus promote cancer cell survival. MiRNA expression was altered by the constant signaling via IL-6. Let-7a was upregulated in the presence of high levels of IL-6. Enhanced let-7a expression resulted in malignant cells survival in the nu/nu mice Mz-IL-6 xenografts while administration of anti-let-7a increased 4-fold caspase activity and cell death in response to gemcitabine (p<0.05) relative to respective controls. Human NF2 mRNA contains seed regions for mature let-7a. NF2 is located on chromosome 22q12.2 and encodes for merlin, a tumor suppressor gene. Merlin has strong binding to hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a potent regulator of receptor tyrosine kinase trafficking, and the interaction of HRS and Merlin can result in inhibition of Stat activation [74]. Forced expression of NF2 was sufficient to overcome the effects of IL-6 on constitutive Stat-3 expression in malignant cholangiocytes and intratumoral administration of anti-let-7a increased NF2 and decreased phospho-Stat-3 expression in xenografts [75].

2.2.4. Pancreatic tumors

A study analyzed frozen samples from primary pancreatic sporadic tumors collected from patients for the miR expression of normal pancreas, benign tumors, acinar pancreatic cancers and endocrine pancreatic cancers. Specific over-expression of miR-103 and lack of expression of miR-155 is characteristic to pancreatic insular and acinar tumors. Insulinomas over-expressed miR-204, its homologous miR-211 and miR-203. High expression of miR-21 was strongly associated with the proliferation index and presence of liver metastasis [76].

Human pancreatic adenocarcinoma specimens and adjacent normal pancreatic tissues obtained from patients who underwent surgery for pancreatic cancer together with human pancreatic cell lines were analyzed using real-time RT-PCR for expression of 95 miRNA chosen for their previously reported function in cancer biology in a recently reported study [77]. The expression of 8 miRNAs were found to be 3.3-fold higher in the pancreatic cancer cells from the patients and pancreatic cancer cell lines compared to normal pancreatic cells: miR-15b, miR-95, miR-186, miR190, miR-196a, miR-200b, miR-221 and miR-222. The biological functions of these miRNAs are not completely understood: miR-15b was previously reported to target BCL2 and mediate multidrug resistance in human gastric cancer cells [78], miR-95 not reported in human cancer, miR-186 has anti-apoptotic activity and downregulates the pro-apoptotic P2X7 mRNA transcription [79], miR-190 is upregulated in hepatocellular cancer [80], miR-196a is involved in pancreatic organ development [81] and high expression of this miR predicted for poor survival in pancreatic cancer [82], miR-200b regulate epithelial to mesenchymal transition by targeting E-cadherin repressors ZEB and SIP transcription factors known as inducers of cell migration and invasiveness [83], [84], miR-221 and miR-222 regulate cell cycle by targeting p27Kip1 [85]. The understanding of differential miRNA expression between normal and carcinoma cell in pancreas may help to design future therapies for this disease.

2.2.5. Hepatocellular tumors

Hepatocellular carcinoma (HCC) accounts for 80–90% of liver cancers [86], cirrhosis is the strongest predisposing factor, as 80% of HCCs develop in cirrhotic livers [87].

Tissues obtained from 60 patients (45 males and 15 females) undergoing liver resection for HCC that developed on cirrhosis were matched HCC-cirrhosis and analyzed for miRNA expression, Northern blot and quantitative real-time reverse transcription-PCR (RT-PCR). MiR-122a, the most abundantly expressed miRNA in human liver, and was significantly decreased in 70% of HCC when compared to adjacent liver cirrhosis tissue.

Tumors, selected for high or low miR-122a expression, were analyzed for cyclin G1 expression by Western blot. In these tumors, there was an inverse relationship between cyclin G1 and miR-122a: when miR-122a was low, cyclin G1 is high and when miR-122a expression was high [88]. Increased cyclin G1 is associated with genomic instability [89] and reducing cyclin G1 is associated with inhibition of tumor growth [90].

The peroxisome proliferator-activated receptors (PPARs) belong to the steroid hormone nuclear receptor (NR) superfamily, three isoforms of PPAR have been identified, PPAR-α, PPARß/δ, and PPARγ, all encoded by separate genes. PPARs are liganded transcription factors that upon activation heterodimerize with retinoid X receptor (RXR) and bind to direct repeat 1 peroxisome proliferator response elements to initiate PPAR-dependent gene transcription. PPAR-α is expressed mainly in the liver, heart, and muscle and is a major regulator of fatty acid transport and catabolism and energy homeostasis [91].

Synthetic lipid-lowering agents such as: fenofibrate (TriCor), gemfibrozil (Gemcor, Lopid), and clofibrate (Atromid-S) lower plasma triglyceride levels by activating PPAR-α [92]. Rats treated with 0.3–1.7 times the human dose developed hepatocellular carcinoma, pancreatic acinar adenomas and Leydig cell tumors via PPAR-α-dependent mechanisms. Therefore in 1998, FDA issued a warning on all three triglyceride lowering agents [93]. When treated with PPAR-α-agonist [94], PPAR-α null mice do not develop liver tumors [94]. PPAR-α is a major regulator of hepatic miRNA expression. The 3′ untranslated region of c-myc mRNA contains seed regions for let-7c. Let-7c expression in mice liver was inhibited by PPAR-α agonist treatment, however, this inhibition was not seen in PPAR-α null mice. Basal-level expression of let-7C inhibits c-myc. PPAR-α agonist treatment, inhibited let-7C expression and increase in c-myc. C-myc activates mir-17-92 polycistronic cluster. This pathway could be the explanation for PPAR-α mediated hepatocellular proliferation and tumorigenesis [95].

2.2.6. Colorectal tumors

In a study, real-time PCR the expression of 156 mature miRNA was analyzed in a panel of 16 colorectal cancer cell lines and 12 matched-pair of tumoral and non-tumoral tissues obtained from patients. The level of miR-31 was correlated with the stage colorectal cancer it was one log higher in stage IV when compared to stage II. In patients’ tumors miR-133b miR-30c, miR-133a and miR-145 were all down-regulated and miR-31, miR-96, miR-135b miR-19a, miR-21, miR-29a, miR-92, miR-148a, miR-200b and miR-183, were upregulated [96].

The let-7 does not appear to be deregulated in human colorectal carcinoma tumors. When let-7 low-expressing DLD-1 human colon cancer cell line was transfected with let-7a-1 precursor miRNA, which is located at chromosome 9q22.3, the cells underwent significant growth suppression, at 48h after transfection there were 8×105 compared to 14×105 in the control group [97].

Lujambio et al. compared miRNA expression profile of the wild-type colon cancer cell line HCT-116 with the same cell line after genetic disruption by homologous recombination of DNA methyltransferase 1 (DNMT1) and DNMT3b (double knockout, DKO) using a miRNA microarray expression profiling method. Cancer cells and normal colon cells were obtained from patients at the time of their surgeries. When tested by bisulfite genomic sequencing miRNA-124a hypermethylation was observed in 75% (42 of 56) of patients compared to their normal colon cells. The target of miR-124a in colon cancer cells appears to be the oncogenic factor cyclin D kinase 6 (CDK6) and the tumor suppressor retinoblastoma (Rb) gene. Over-expression of miR-124a induced a reduction of CDK6 protein level and the miR-124a transfection diminished the phosphorylation of Rb in the residues 807 and 811, the targets of CDK6 [46]. Thus epigenetic changes in miRNA are equally responsible for cancer initiation and progression similar to epigenetic changes in oncogenes and tumor suppressor genes.

3. Outlook on potential applications of microRNAs in clinical practice

3.1. miRNA diagnostics applications in gastrointestinal tumors

Large collections of patients’ paraffin preserved tissue biopsies with associated clinico-pathologic information and disease outcomes exist in clinical laboratories and biobanks, however, in such tissues the messenger RNA is sometimes poorly preserved [98]. Archived specimens may represent a trove of miRNAs that can potentially be used as diagnostic, prognostic tools and molecular markers because they are stable over time and resistant to RNAse degradation [99], [100].

MicroRNA can be isolated and quantified with ease either for formalin-fixed paraffin-embeded specimens that have been processed and stored for many years and the quality is similar to those extracted from fresh or frozen samples [100]. MicroRNAs are expressed in tissue specific manner and reflect their tissue of origin, this characteristic could be used in developing accurate diagnostic tests for cancers of unknown primaries [25], [34], [35], [36], [101].

MiRNA pattern of tumors are specific to the tissue of origin, this characteristic has the potential of developing accurate diagnostic tests for cancers of unknown primaries [101]. With the development of quantitative reverse transcriptase polymerase chain reaction assays (qRT-PCR) methods only minute amounts of miRNA would be needed for diagnosis [99]. A few nanograms of RNA obtained via fine needle aspiration were enough for successful diagnosis of thyroid papillary carcinoma via miRNA measurement by qRT-PCR [102]. MiR-196a is highly expressed in pancreatic ductal adenocarcinoma but its level is low in chronic pancreatitis tissue and in normal pancreas. miR-217 is high in chronic pancreatitis tissue and in normal pancreas and almost undetectable in pancreatic adenocarcinoma. Investigators have demonstrated that the ratio miR-196a/miR-217 calculated by qRT-PCR from minute FNA specimens indicated whether the sample contained or not ductal adenocarcinoma cells [103].

3.2. RNA interference as possible therapy of the future for gastrointestinal tumors

The therapeutic approach for microRNAs whose expression is lost in tumors is to increase (mimic) or agonize their expression, if the miRNA is upregulated in malignancy the approach would be to inhibit it via silence interference pathway. This may, in theory, be accomplished either by delivery of double- or single-stranded microRNA mimics. Over-expression or inhibition of miRNA could be achieved in several ways:

•With synthetic miRs similar to siRNA-like oligoribonucleotide duplexes.

•Chemically modified oligoribonucleotides [104].

•Antagomirs [104].

Antagomirs are modified anti-sense oligonucleotides such as 2′-O-methyl anti-sense oligonucleotide [104] that antagonizes miRNA. They cannot cross the blood–brain barrier, however, they can be safely injected into the CSF and modulate gene expression in situ [104]. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA that influences the plasma cholesterol level. Pre-clinical studies suggest that miR-122 is essential for the replication of hepatitis C virus (HCV). Anti-miR-122 also reduces cholesterol levels in blood and reverses hepatic steatosis (fatty liver) in obese mice. Together, these findings suggest that anti-miR-122 may both reduce HCV infection and, in addition, improve HCV-associated pathologies like steatosis. Plasma cholesterol dropped significantly for 21 days in antagomir-122-treated mice [105].

In a mouse model of liver cancer Kota et al. noticed that miR-26a was 3–8-fold lower in liver cancer cells compared to their adjacent normal liver cells (p<0.02). The authors placed miR-26a pre-miRNA into an adenovirus associated vector (AAV), 90% of the AAV vector localized to the liver. Ten mice received intravenous injections of miR-26a replacement. Eight of 10 mice treated with the miR-26a construct showed 5-fold increase in apoptosis and 3-fold regression of disease 5 days after the treatment [106]. Many individual miRNAs home in on dozens and sometimes hundreds of genes, the report of Kota highlights that changes in the abundance of a single miR is able to reverse disease progression in malignancy. Using adeno-associated virus (AAV) vectors would be limited for human delivery of microRNAs by the early immunologic memory of AAV infections [107].

The effects miRNA gene expression modulation remains known and delivering miRNA into the right cells is challenging. One of the initial clues that manipulating miRNA might lead to significant untoward effects came from experiments in pathways of heart development. miR-1-2 is highly expressed in heart muscle. The researchers knocked out one of the copies of miR-1-2 and decreased its dosage by 50%. This led to a dramatic effect: half of the knockout mice died of cardiac arrhythmias and cardiac wall development defects [108].

Non-specific side effects of such treatments their off-target activity will be as important as their effectiveness and duration of target miRNA suppression or inhibition [109]. High expression of miRNA mimics may interfere with cellular machinery for miRNA processing or action and have deleterious side effect, fatalities were reported in mice as a result of saturation of miRNA pathway [110].

However, despite all these concerns, finding new, more effective venues in the management of malignant disease and more tame means of diagnosing it remains an exciting goal.

Reviewer

Manfred P. Lutz, M.D., Caritasklinik St. Theresia, Rheinstrasse 2, D-66113 Saarbrücken, Germany.

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Dr. Shapira is an attending physician at the Division of Hematology/Oncology at North Shore University Hospital in Manhasset. Dr. Shapira is the Director of Cancer Genetics Service at the Monter Cancer Center. She is board certified in Internal medicine, Hematology and Medical Oncology and assistant professor of Medicine at Hofstra University Medical School. Dr. Shapira is a member of the American Society of Hematology, the American Society of Clinical Oncology, the American Medical Association, International Society of Geriatric Oncology, American Association for Cancer Research and the American College of Physicians. Dr. Shapira has worked on many research projects including translational research of lymphocytes development, clinical research in breast cancer, gastrointestinal malignancies, drug development, multiple myeloma and T-cell lymphoma and has published in these areas as well as in areas of medical ethics, cancer in the elderly and research communications. Dr. Shapira is member of the Research Communication, Ethics and Audit Committee of the Cancer and Leukemia Group B. She serves as Chairwoman for Clinical Competency Committee Hematology–Oncology Fellowship Program of the North Shore University Hospital and Member Grant Award Review Committee for Clinical and Translational Science Center at Hofstra University Medical School.

a Hematology Oncology, Hofstra University School of Medicine, Monter Cancer Center, 450 Lakeville Road, Lake Success, NY 11042, USA

b Hofstra University School of Medicine, Division of Gastroenterology Hepatology and Nutrition, North Shore University Hospital, 300 Community Drive, Manhasset, NY 11030, USA

c Hofstra University School of Medicine, Hematology Oncology, Monter Cancer Center, 450 Lakeville Road, Lake Success, NY 11042, USA

d Hofstra University School of Medicine, Don Monti Division of Oncology, Monter Cancer Center of North Shore University Hospital, 450 Lakeville Road, Lake Success, NY 11042, USA

Corresponding author. Tel.: +1 516 734 8964; fax: +1 516 734 8950.

1 Tel.: +1 516 562 4281; fax: +1 516 562 2683.

2 Tel.: +1 516 734 8963; fax: +1 516 734 8924.

3 Tel.: +1 516 734 8958; fax: +1 516 734 8924.

PII: S1040-8428(10)00028-4

doi:10.1016/j.critrevonc.2010.01.013