MicroRNAs in colorectal cancer: Small molecules with big functions

Cancer Letters 360 (2015) 89–105

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Mini-review

MicroRNAs in colorectal cancer: Small molecules with big functions Yu Xuan a,b,1, Huiliang Yang b,1, Linjie Zhao b,1, Wayne Bond Lau c,1, Bonnie Lau d, Ning Ren e, Yuehong Hu a, Tao Yi a, Xia Zhao a, Shengtao Zhou a,*, Yuquan Wei b a Department of Gynecology and Obstetrics, Key Laboratory of Obstetrics & Gynecologic and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second Hospital, Sichuan University, Chengdu 610041, China b The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China c Department of Emergency Medicine, Thomas Jefferson University Hospital, USA d Department of Surgery, Emergency Medicine, Kaiser Santa Clara Medial Center, Affiliate of Stanford University, USA e College of Biological Sciences, Sichuan University, Chengdu 610041, China

A R T I C L E

I N F O

Article history: Received 28 August 2014 Received in revised form 19 November 2014 Accepted 20 November 2014 Keywords: Colorectal cancer MicroRNA Tumor suppressor Oncogene

A B S T R A C T

Colorectal cancer (CRC) is the third most lethal malignancy, with pathogenesis intricately dependent upon microRNAs (miRNAs). miRNAs are short, non-protein coding RNAs, targeting the 3′-untranslated regions (3′-UTR) of certain mRNAs. They usually serve as tumor suppressors or oncogenes, and participate in tumor phenotype maintenance. Therefore, miRNAs consequently regulate CRC carcinogenesis and other biological functions, including apoptosis, development, angiogenesis, migration, and proliferation. Due to its differential expression and distinct stability, miRNAs are regarded as molecular biomarkers (for diagnosis/prognosis) and therapeutic targets for CRC. Recently, a remarkable number of miRNAs have been discovered with implications via incompletely understood mechanisms in CRC. As further study of relevant miRNAs continues, it is hopeful that novel miRNA-based therapeutic strategies may be available for CRC patients in the future. © 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Colorectal cancer (CRC) carries one of the highest morbidity and mortality rates worldwide, in both sexes. Despite great success in the treatment of CRC, the prognosis of CRC patients remains poor. Thus, understanding its underlined molecular genesis is fundamental for its diagnosis, treatment, and prognosis [1]. MicroRNAs (miRNAs) are short (19–25 nucleotides, nt), nonprotein coding RNAs. Accounting for 2–5% of the whole human genome, miRNAs number approximately one thousand, and regulate the expression of at least 20% of human genes [2]. They are translated by Poly II (or rarely Poly III) to form pri-miRNA, composed of hundreds of nt, including a 33 bp stem and a terminal loop structure with flanking segments. Subsequently, pri-miRNA is transformed into pre-miRNA by a protein complex including Drosha (a highly conserved 160 kDa protein containing two RNAse III domains and one double-strand RNA-binding domain) to generate a 60–70 nt long hairpin RNA with 2 nt overhangs at its 3′-end. Apart from this classical processing pathway, an alternative miRNA biogenesis pathway also exists, in which regulatory RNAs (called miRtrons) form pre-miRNAs by splicing without Drosha involvement [3]. Pre-

* Corresponding author. Tel.: +86 13551070137; fax: +86 28 85164046. E-mail address: [email protected] (S. Zhou). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2014.11.051 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.

miRNA is transported by exportin-5 from the nucleus to the cytoplasm, and combines with Dicer, a cytoplasmic endonuclease RNAse III. The miRNA duplex now contains the mature miRNA guide and its complementary passenger strand (miRNA*). Per the widely accepted model [3], miRNA* is degraded, although evidence suggests miRNA* may also be functional as well, albeit for uncertain purposes [4]. The single remaining strand integrates into the RNAinduced silencing complex (RISC), a ribonucleoprotein effector containing a catalytic endonuclease core, Ago2, and binds the site of 3′-untranslated regions (3′-UTR) in target mRNA. In turn, the “seed region” (the 5′-end region of mature miRNA, which includes 2–8 nt) binds the 3′-UTR of target mRNA. According to their match level, the target mRNA will degrade or decrease its expression level. Most animal miRNAs are imperfectly complementary, resulting in translation inhibition of target mRNA rather than full blockage [5–7] (Fig. 1). Accordingly, miRNAs exert momentous impacts upon different cellular functions, such as development, metabolism, and carcinogenesis. miRNAs are particularly influential in pathologic processes like malignancy, where proliferation, differentiation, apoptosis, invasion, and metastasis are all regulated via gene expression manipulation. miRNAs may either promote or suppress carcinogenesis. To date, many miRNAs have been confirmed to act as tumor suppressors or oncogenes (or rarely, both), with promising clinical practice applications. Many dysregulated miRNAs play a role in various cancers, such as chronic lymphocytic leukemia (CLL) [8], breast cancer [9], cholangiocarcinoma [10], hepatocellular

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Fig. 1. The general processing pathway/function pattern of miRNAs.

carcinoma, and gastric cancer [11–13]. Many functional miRNAs have been discovered pertaining to CRC in recent years, and many will be discussed elaborately in this review. However, the majority of miRNAs await future investigation. Herein, we summarize the recent advancements on miRNAs, emphasizing their alterations and roles in CRC pathogenesis, and comment upon their potential utility as disease biomarkers or novel therapeutic targets in colorectal cancer patients. Involvement of miRNAs in colorectal cancer CRC is the third most lethal malignancy. However, despite its 5% overall incidence and 40–60% 5-year survival rate, CRC remains curable by surgical resection, if diagnosed at an early stage [14]. The CRC genome involves many genetic alterations, including activating mutations of proto-oncogenes (such as K-ras and B-raf) and inactivating mutations of tumor suppressor genes (such as adenomatous polyposis coli (APC) and tTP53) [15]. Each of these processes is variably dependent upon miRNAs. miRNAs as tumor suppressors As expressions of the let-7 family (including let-7a, let-7b, let7c, let-7d, let-7e, let-7f, let-7g, let-7i, and miR-98), miR-133b, miR34 family, miR-126, miR-143, and miR-145 are down-regulated in CRC tissues, these miRNAs are considered tumor-suppressors in CRC (Table 1) [84]. As tumor suppressors, the let-7 family targets oncogenes such as K-ras, c-myc, CDC34, CDC25A, CDK6, HMGA2, Lin28, and Lin28B. Additionally, cell-cycle regulators [85], Bcl-2 [86], signal transducer and activator of transcription 3 (STAT3), and integrins are also negatively regulated by the let-7 family. Among these oncogenes, K-ras (whose translated product is RAS protein) is a promising therapeutic target. K-ras is the most commonly dysregulated protooncogene in CRC patients. Certain K-ras-controlled genes regulate miRNA expression level to promote cellular proliferation, tumor survival, angiogenesis, and metastasis. Therefore, K-ras mutations, casually occurring in CRC patients, may lead to uncontrolled pro-

liferation of tumor cells. For metastatic CRC patients harboring K-ras mutations, conventional anti-EGFR therapeutics is useless, rendering analysis of K-ras mutational status clinically mandatory [87]. To optimize the selection of patients with metastatic CRC eligible for anti-EGFR therapy, assessment of K-ras copy number aberration and miRNAs targeting K-ras is necessary. Tumors harboring K-ras codon 12 or 13 mutations are frequently resistant to anti-EGFR therapy. A B-raf oncogene mutation in the background of K-ras wild-type tumor (occurring in approximately 10% of metastatic CRC patients) negatively predicts anti-EGFR therapy success, and carries strong negative prognostic value [88]. In most cases, miRNAs localized within the same narrow genomic region (0.1–1 kb) have similar expression trends. Different let-7 family members exert similar effects upon CRC cellular proliferation in a cell-type or tissuespecific manner. The potent growth inhibitor Let-7a frequently serves as the let-7 family representative during experiments [89,90]. Upregulated let-7a (associated with increased survival) directly targets K-ras mutations via Ras family regulation, and portends a favorable outcome with EGFR inhibition therapy. Furthermore, downregulation of the let-7 family and miR-18a in CRC accelerates tumorigenesis by reversing K-ras suppression, while elevated levels of miR-200b and miR-21 portend good prognosis. Interestingly, decreased miR-143 expression is related with improved survival, contradictory with the widely accepted tumor suppressor role that miR-143 plays, an observation requiring further study and validation. Collectively, increased levels of let-7 family, miR-18a, miR200b, and miR-21, and decreased levels of miR-143 level and K-ras copy number increase the sensitivity to anti-EGFR therapy in patients harboring mutated K-ras tumors. Consistently, copy number gains in wild-type K-ras patients prompt resistance to conventional anti-EGFR therapy (such as cetuximab and panitumumab) [91]. In addition, T to G base change in the let-7 complementary site (LCS6) within the 3′UTR of K-ras attenuates the capability of mature let-7 binding to target K-ras mRNA [87], although the LCS6 variant allele is not an apparent risk factor for CRC development [92]. However, the presence of decreased let-7 and increased K-ras levels portend a worse prognosis in advanced (but not early) stage CRC patients harboring the K-ras-LCS6 variant. Perplexingly, K-ras

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Table 1 miRNAs as tumor suppressors in CRC. miRNA

Targets

Biological functions

Expression level

References

let-7 family

K-ras , c-Myc, CDC34, CDC25A, CDK6, HMGA2, Lin28 and Lin28B, Bcl-2, STAT3, mRNA of integrin, vimentin and E-cadherin K-ras

Suppression of migration and invasion, promotion of angiogenesis

↓

[42–44,84–88,91]

Suppression of cell proliferation, tumor survival, angiogenesis and metastasis Blocking constitutive phosphorylation of MAPK, promotion of apoptosis, sensitization of colon cancer cells to 5-FU, induction of cell cycle arrest, suppression of cell growth, proliferation, angiogenesis and metastasis, promotion of malignant transformation Suppression of the metastasis, involvement in stem cell-like properties inhibition and inflammation Activation of K-ras, promotion of CRC proliferation Regulation of metastasis Regulation of metastasis Suppression of cell cycle progression and cell proliferation, desensitization of colon cancer cells to 5-FU Inhibition of tumor formation and metastasis, inhibition of TICs or CSCs, enhancement of chemotherapy and radiotherapy sensitivities Promotion of senescence, induction of apoptosis,sensitization of colon cancer cells to 5-FU, suppression of proliferation, motility and metastasis Suppression of proliferation, induction of cell cycle arrest

↓

[87,89,90]

↓

[45,70,84,91, 100–104]

↓

[7,43,44,49,50,64]

↓

[91] [41,43,44,122] [41,43,44,122,150] [39,124,125]

↓

[7,84]

Induction of cell cycle arrest, suppression of cell growth, proliferation, invasion and metastasis, regulation of apoptosis, inhibition of angiogenesis

↓

[38,46,47,52,70,84, 100,105–115,144, 158]

Suppression of CRC growth Promotion of apoptosis Induction of apoptosis Regulation of apoptosis, involvement in cellular proliferation, differentiation, and survival Suppression of growth, migration and invasion, promotion of apoptosis Inhibit ion of angiogenesis Maintenance of genetic integrity Suppression of tumor cell progression Induction of cell cycle arrest, inhibition of cell proliferation and invasion Suppression of metastasis Induction of cell cycle arrest, promotion of apoptosis Inhibition of cell cycle progression and promotion of apoptosis Promotion of cell proliferation, invasion, lymph node metastasis, induction of apoptosis Suppression of angiogenesis Suppression of angiogenesis Suppression of angiogenesis

↓ ↓ ↓ ↓

[82,84,116] [127] [128,169,170] [30,130]

↓

[20]

↓ ↓ ↓ ↓

[65,66,183] [67,68] [40,143] [115,126]

↓ ↓ ↓ ↓

[7] [119] [14,117] [118]

↓ ↓ ↓

[131] [30,132] [35,132]

let-7a miR-143

ERK5, DNMT3A, Bcl-2,NF-κB, K-ras, ELK1

miR-200 family

ZEB1,ZEB2,TGFβ2,Sox2,Klf2

miR-200b miR-141 miR-200c miR-215

TGFβ2 ZEB1 DHFR

miR-34 family

c-Met

miR-34a

miR-34b and miR-34c miR-145

miR-126 miR-195 miR-491 miR-133 miR-101

E2F1/3, Bcl-2, CD24 and Src, CD44

IRS-1, IGF-IR, YES, FLI1, FSCN1, BIRC2, VANGL1, DFF45, HIF1a,VEGF,c-myc, MUC1, OCT4, SOX2, KLF4, ADAM17, CCND2, p70S6K1 p85β regulatory subunit Bcl-2 Bcl-xL c-Met, K-ras

miR-107 miR-155 miR-137 miR-342

COX-2, PTGER4/EP4, EZH2, N-Myc, Mcl-1 VEGF hMSH6, hMSH2 and hMLH1 LSD-1 DNMT1

miR-30 family miR-30a-5p miR-365 miR-345

DTL Cyclin D1, Bcl-2, Mybl2 BAG3

miR-214 miR-16 miR-424

mRNA of Quaking VEGF,VEGFR2,FGFR1 VEGF,VEGFR2,FGFR1

mutations contribute to significantly increased survival in earlystage CRC. In contrast, in K-ras/B-raf-mutated CRC patients, latestage G-allele carriers exhibit reduced survival rate [93] and an altered response to cetuximab [93,94] compared to early-stage G-allele carriers. The underlying mechanisms of this divergent phenomenon remain unclear. CpG island hyper-methylation of tumor suppressor gene promoters has been considered the main mechanism of transcriptional silencing. Therefore, its exploitation may predict the putative promoter regions of certain genes like miRNAs [76,95]. However, it is noteworthy that miRNAs without promoter CpG islands are also potential targets of epigenetic silencing in CRC, likely via miRNAinduced secondary effects of DNA demethylation (such as transcription factor up-regulation) [96]. The tumor suppressor miR34 family inhibits tumor sphere growth in vitro and tumor formation in vivo. Additionally, the miR-34 family inhibits growth of tumorinitiating cells (TICs) or cancer stem cells (CSCs) [7]. Unfortunately, in CRC, miR-34b and miR-34c are frequently silenced by hypermethylation of neighboring CpG islands, repressing the expression of B-cell translocation gene 4 (BTG4, another tumor suppressor

[36,37,98,99,178]

[97]

exerting anti-proliferative effect as well as inducing cell cycle arrest [97], Fig. 3). miR-34 increases chemotherapy and radiotherapy sensitivity of cancer cells [7]. E2F1/3, a member of the E2F transcription factor family, is one of the targets of miR-34a. The induction of E2F1/3 in CRC is due to CpG methylation of the miR-34a promoter [98]. The expression and senescence-promoting function of miR-34a are both relevant to p53. Sirtuin1, an enzyme responsible for p53 deacetylation/inactivation, is also the target of miR-34a [99]. Atop its apoptotic induction effects, miR-34a also represses expression of Bcl-2, and sensitizes colon cancer cells to 5-fluorouracil (5-FU) treatment [36]. Additionally, miR-34a suppresses migration and invasion of CRC by directly targeting CD44-encoded mRNA [37]. In addition, miR-143 and miR-145 are both down-regulated in colorectal tumors [100]. miR-143 directly targets Bcl-2 and restores miR-143 in CRC cell lines, markedly decreasing cellular viability and increasing sensitivity to 5-FU-induced cell death [70,101]. Chen et al. reported comparatively low miR-143 expression in HCT116 human CRC cell lines. Nevertheless, once exposed to 5-FU, miR143 expression is stably induced, contributing to down-regulation of extracellular-regulated protein kinase 5, NF-κB and Bcl-2 protein

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expression, and approximately 60% cellular viability reduction. Protein kinase 5 and NF-κB increase sensitivity of colon cancer cells to 5-FU. Moreover, NF-κB activates extracellular signal-regulated kinase 5 (ERK5), a mitogen-activated protein kinase. ERK5 promotes cellular transformation, and is critical for G2–M cell cycle progression and timely mitotic entry. NF-κB suppresses apoptosis and stimulates cell growth, tumor promotion, angiogenesis and metastasis. The direct targets of miR-143 include the mRNA of K-ras [102], ERK5, DNA methyltransferase 3A (DNMT3A) [103], and ELK1, which participate in proliferation (Fig. 3) and apoptosis regulation. Oncogene K-ras, a direct target of miR-143, usually stimulated by EGFR signals, activates RAS/RAF/MEK/ERK signaling [104], contributing to cell proliferation enhancement (Fig. 3), cell cycle dysregulation, and ultimately malignant transformation of normal cells. The phosphorylation level of ERK1/2 is decreased by treatment with pre-miR-143 in CRC cell lines. Moreover, miR-143 targets ERK5 and DNMT3A, thus blocking constitutive MAPK phosphorylation [70,102]. Additionally, miR-143 acts as a fundamental differentiation promoter in adipocytes, a process requiring further characterization [102]. miR-145 is also reduced in CRC, usually at the adenomatous and cancer stages [70]. Recently, Xu et al. reported miR-145 expression is markedly decreased in CRC, which acts as a pivotal tumor suppressor [105]. miR-145 [106] targets insulin receptor substrate 1 (IRS-1), YES [107], friend leukemia virus integration 1 (FLI1) [108], FSCN1, BIRC2, and VANGL1 [107], with unknown functional interaction. IRS-1 [109], mediating the oncogenic signals transduced by insulin-like growth factor I receptor (IGF-IR) [109,110], activates downstream Ras/Raf/mitogen-activated protein kinase kinase/ ERK and PI3K/Akt cascades, thereby promoting cell proliferation and survival [46]. miR-145 directly targets IRS-1 and IGF-IR, repressing cell growth and proliferation (Fig. 3). IRS-1 rescues colon cancer cells from growth inhibition [109]. In addition, p53 pathway also plays a role in the growth-inhibiting effect of miR-145. p53 induces miR-145 expression by directly binding its promoter transcriptionally [52], thus repressing oncogene c-myc [111], IRS-1, mucin 1(MUC1) [38], OCT4, SOX2, and Krüppel-like factor 4 (KLF4) [47,105] (although p53-independent regulation of miR-145 exists in the mutant p53 background [52]). The p53 pathway is involved in both tumor growth and metastatis. In one metastatic CRC model, miR145 triggers the mesenchymal-like phenotype by targeting G1/S cell cycle checkpoint (represented by myc and CCND2) and neuregulin (represented by myc and ADAM17) via p53 pathways [112,113]. miR145 regulates apoptosis (Fig. 4) in colorectal mucosa by targeting DNA fragmentation factor-45 (DFF45) [114,115]. Additionally, miR145 inhibits angiogenesis (to be discussed later in this review). miR-126, consistently expressed in normal colon epithelium, may impede neoplastic growth by targeting the 3′-UTR of mRNA of p85β, a regulator subunit involved in stabilizing and propagating the phosphatidylinositol 3-kinase (PI3K) signal. Impaired PI3K signaling results in a threefold reduction of p85-β protein level, as well as a marked decrease of phosphorylated Akt, resulting in colon tumor inhibition. In contrast, in colon cancer cell lines, miR-126 expression is absent [116]. miR-365 acts as either a tumor suppressor or oncogene in different cancer types. In CRC, miR-365 functions as a tumor suppressor, inhibiting cell cycle progression and promoting CRC apoptosis (Fig. 4). It is down-regulated in human colon cancer tissues, resulting in cancer progression and poor survival. Augmentation of miR-365 in NIH3T3 cells via UVB irradiation effectively inhibits cell cycle progression, promoting 5-FU-induced apoptosis and repressing carcinogenesis in colon cancer cell lines by targeting the mRNAs of CD1 and Bcl-2 [14]. Myb-related protein B (Mybl2) is a target of miR-365, and exhibits significant suppressive capacity in colon epithelial cells during maturation along the cryptluminal axis [117]. Similarly, miR-345 is involved in cell proliferation (Fig. 3) and apoptosis (Fig. 4) in human CRC by targeting BCL2-

associated athanogene 3 (BAG3), resulting in decreased translation of an anti-apoptotic protein. However, because miR-345 is significantly down-regulated in 51.6% of CRC tissues, it serves as a good indicator for lymph node metastasis (LNM) and detrimental histological type [118]. The robust tumor suppressor miR-30a-5p contributes to cell cycle arrest at the G1 phase, inducing apoptosis (Fig. 4) by targeting denticleless protein homolog (DTL, which is overexpressed in CRC and modulates the cell cycle via the DTL–TP53– CDKN1A regulatory circuit [119]). DHFR (an S-phase-specific enzyme that converts dihydrofolate to tetrahydrofolate, which is essential for the synthesis of purine and thymidylate) is the direct target of miR-24, miR-192, and miR-215 [120]. miR-24 and p21 induce intra-S and G2 cell cycle arrest [121]. miR-24, which up-regulates tumor suppressor gene p53, is involved in cell cycle checkpoints, apoptosis (Fig. 4), and cellular senescence [122,123] While the antimitogenic action of p21 is independent of p53 function [121], both miR-192 and miR-215 (induced by p53) induce G1 and G2 cell cycle arrest [39,99,124,125]. Moreover, it has been confirmed that restoring the expression level of frequently down-regulated miR-342 in CRC cells can down-regulate DNMT1, inducing G0/G1 cell cycle arrest and the subsequent inhibition of cell proliferation (Fig. 3), which is implicated in an up-regulation of p21 and down-regulation of cyclin E and CDK2 [126]. Additionally, the tumor suppressor miR195 promotes cell apoptosis (Fig. 4) by targeting Bcl-2 in CRC cell lines HT29 and LoVo [127]. Bcl-xL, another anti-apoptotic protein, frequently over-expressed in CRC [128,129], is targeted by miR491 [128]. miR-133, miR-26, miR-107, miR-210, and miR-342 are all involved in the regulation of apoptosis in colorectal mucosa [114,115,130]. miRNAs as oncogenes Many miRNAs are considered oncogenes. Elevated expression of certain miRNAs are seen not only in CRC patients, but also in healthy volunteers with elevated CRC risk [84]. Subsequent genomic instability (like DNA mismatch repair failure) and chromosomal instability might ensue (Table 2). Among the oncogenes in CRC, the miR-17-92 cluster (including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-921) is expressed to greater degree in CRC tissues compared to normal tissues. Combinational gain of 8q24 (in which c-myc located) and 13q31 (in which miR-17-92 located) may be responsible for progression from colorectal adenoma-to-adenocarcinoma. The miR17-92 cluster, namely oncomir-1, is located within the third intron of the ORF 13 (C13orf25), and spans nearly 800 bases. Activated by c-myc, its transcription increases the DNA copy number of miR1792 upon 13q31, except for miR-18a. The miR-17-92 cluster is related to the E2F family of transcription factors, vital regulators of the cell cycle and apoptosis (Fig. 4). E2F1 and E2F3 directly activate transcription of the miR-17-92 family, which in turn regulates expression of E2F1 and E2F3, thus forming a negative feedback loop [172]. Overexpression of miR-17-92 on an elevated c-myc background promotes CRC growth by targeting E2F1. Expectedly, E2F1, E2F3, and c-myc participate in a complex regulatory network of apoptotic and proliferative signals. Moreover, the miR-17-92 cluster and its target E2F1 exhibit similar regulation of embryonic colonic mucosal cell proliferation and stage I CRC. However, the cell proliferation of stage II CRC is more similar to embryonic colonic mucosa, with a more aggressive phenotype and a greater proliferative capacity (Fig. 3). In addition, cyclin-dependent kinase inhibitor (CDKN1A, which controls cell cycle progression) and the BCL2-like 11 gene (BCL2L11, which controls cell death) are the putative targets of the miR-1792 cluster, with albeit tenuous confirmation [173]. Recently, Luo et al. detected three members of the miR-17-92 cluster (miR-17, miR18a, and miR-18b) and its homolog miR-106a were expressed more in CRC tissues compared to control. This overexpression enhanced

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Table 2 miRNAs as oncogenes in CRC. miRNA

Targets

Biological functions

Expression level

References

miR-17-92 family miR-17

TSP1, CTGF, E2F1 RND3

Promotion of angiogenesis Promotion of tumor cells proliferation, suppression of cell cycle arrest Promotion of lymph node metastasis, inhibition of apoptosis Inhibition of apoptosis Induction of chemotherapy resistance, suppression of apoptosis Suppression of apoptosis, promotion of lymph node metastases, involvement in cytokine interaction, cellular adhesion, promotion of invasion Blocking constitutive phosphorylation of MAPK Promotion of cell proliferation and malignant transformation, regulation of cell cycle Promotion of CRC progression, enhancement of migration and invasion, involvement in stem cell-like properties inhibition and inflammation, induction of chemotherapy resistance Promotion of migrasion, invasion, metastasis

↑ ↑

[23,71,81,172–174] [71,151,175]

↑

[18,19]

↑ ↑

[18,19] [21,158]

↑

[20,22,57,145,158,187]

↑↓ ↑

[16,17,71,91,175] [56]

↑

[20,25–31,34,48–50,53,72,73, 79,80,91,145,150,157,176–181]

↑

[20,29–31,53,100]

↑ ↑ ↑ ↑ ↑

[30,62,63] [24,122] [24,122] [20,56,71,81,174] [57–59]

↑ ↑

[60] [61,141,148]

↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑

[67,68] [182] [65,183] [40] [40,143] [75,97,185] [116,167] [82,83] [142]

miR-19a

WNT3

miR-19b-1 miR-20a

BNIP2

miR-92

BIM

miR-18a miR-675

RND3, ERK5, DNMT3A, K-ras RB

miR-21

PTEN, PDCD4, RECK, TIMP3, NFIB, SPRY2, TGFβR2, TIAM1

miR-31 miR-96 miR-135a miR-135b miR-106a miR-190

AXIN1, FOXC2, FOXP3, TIAM1, SATB2 CHES1 APC APC RND3 PHLPP

miR-95 miR-221

SNX1 CDKN1C/p57, TSP1

miR-155 miR-499-5p miR-103/107 miR-129* miR-137 miR-9 miR-126 miR-27b miR-10b

hMSH6, hMSH2, hMLH1 FOXO4, PDCD4 DAPK, KLF4

mRNA of E-cadherin SEMA6A,Dll4 and Spry2 HoxD10

Suppression of apoptosis Involvement in phenotype maintenance Involvement in differentiation Promotion of proliferation, malignant transformation and metastasis Promotion of cell growth Promotion of cell proliferation, suppress apoptosis, cell cycle arrest and differentiation, promote angiogenesis Suppression of apoptosis Promotion of migrasion, invasion, metastasis Promotion of invasion and metastasis Promotion of lymph node metastasis Promotion of lymph node metastasis Promotion of lymph node metastasis Promotion of angiogenesis Promotion of angiogenesis Promotion of angiogenesis

tumor cell proliferation, and suppressed G0/G1 and G1/S arrest by directly targeting RND3 in vivo [71]. RND3 is an atypical member of the Rho GTPase family, regulating cytoskeletal dynamics [81]. Down-regulation of RND3 induces hyper-proliferation of keratinocytes [174]. RND3 may act as a tumor suppressor gene, and has been implicated in the malignant transformation process from adenoma to carcinoma. In some studies, miR-17 suppresses expression of genes promoting and inhibiting proliferation, depending upon cell type and target mRNA functions [175]. miR-17-5p was found in high concentrations in the base layer of the colonic crypts. miR-17-5p levels gradually decrease toward the top of the colonic crypts, consistent with the presence of epithelial proliferative progenitor cells. As these progenitor cells migrate toward the intestinal lumen, a transient proliferation of undifferentiated cells occurs [151] (Fig. 3). One study cites miR-18a as an oncogene and its overexpression is related to poor CRC prognosis [16]. However, miR18a was also reported to function as a tumor suppressor by targeting the K-ras oncogene [17]. miR-18a targets ERK5 and DNMT3A, blocking constitutive MAPK phosphorylation [70,102]. Moreover, miR19 (along with miR-19a and miR-19b-1) has been demonstrated to decrease c-myc-induced apoptosis [18,19] (Fig. 4). Increased miR19a levels contribute to rectal carcinogenesis by targeting Wnt3. miR92, a key oncogenic component of the miR-17-92 cluster in colon cancer, is highly expressed in cancer tissues, and exhibits the most transcriptional activity of all six members of the miR-17-92 cluster. miR-92 is increased in intermediately differentiated tumors compared to advanced or poorly differentiated tumors. It is more abundant in rectal than colonic tumors. miR-92a directly targets the

anti-apoptotic molecule BCL-2-interacting mediator of cell death (BIM) to decrease apoptosis [20] (Fig. 4). Resistance to chemotherapeutic agents such as 5-FU, oxaliplatin (L-OHP), and teniposide (VM-26) leads to chemotherapy failure, cancer relapse, and poor prognosis. The responsible mechanisms include decreased uptake of water-soluble drugs by cancer cells, accelerated repair of DNA damage, altered drug metabolism and increased energy-dependent efflux of chemotherapeutic drugs. miRNA-mediated regulation of such processes remains unclear. In colorectal adenocarcinoma, over-expressed miR-20a reduces drug sensitivity, ultimately resulting in resistance to these chemotherapy agents. miR-20a knockdown results in restored chemotherapeutic sensitivity. miR-20a also interacts directly with the 3′UTR of proapoptotic factor BNIP2, down-regulating BNIP2 mRNA and protein, increasing tumor cell survival despite administration of proapoptotic drugs. BNIP2, a pro-apoptotic factor in estrogen-mediated neuroprotection, is a Bcl-2 homology domain 3 (BH3)-only member of the BCL-2 family. It plays a pivotal role in mitochondrionmediated apoptosis and constitutes the main mechanism of chemotherapeutic agent-induced apoptosis [21] (Fig. 4). miR-92a is significantly correlated with LNM, and carries great prognostic potential [22]. Furthermore, when activated by c-myc, the miR-1792 cluster targets anti-angiogenic factors thrombospondin-1 (TSP1) and connective tissue growth factor (CTGF), and therefore carries angiogenic impact [23]. miR-135 (including miR-135a and miR-135b), miR-21, and miR31 are up-regulated in colorectal neoplasms. miR-135a and miR135b are markedly up-regulated in CRC cells, contributing to APC

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mRNA suppression, liberating β-catenin and activating Wnt signaling; β-catenin translocates to the nucleus, activating target gene transcription [24,122]. Hypoxic challenge induces miR-21, which is associated with cellular outgrowth, migration, invasion, intravasation, extravasation, and metastasis, and is up-regulated in CRC tissues [25]. In CRC cells, the main targets of miR-21 include programmed cell death protein 4 (PDCD4), NFIB, and Sprouty2 (SPRY2). PDCD4, a tumor suppressor protein, inhibits PMA-induced neoplastic transformation, inhibiting invasion and intravasation. PDCD4 suppression is associated with poor CRC prognosis [25,176]. The relationship of NFIB, a versatile transcriptional repressor of many promoters, in malignant processes is unclear. SPRY2 is involved in cellular outgrowth, branching, and migration. The tumor suppressor gene PTEN is another target of miR-21 [177]. Expression of oncomir miR-21 may be induced by CD24 via Src within lipid rafts, while CD24 and Src are both post-transcriptionally down-regulated by the tumor suppressor miR-34a. CD24, a heat stable antigen, is a glycosylphosphatidylinositol (GPI)-anchored membrane protein, and regulates CXCR4 activity. CD24 also regulates proliferation, motility, integrin-mediated adhesion, tumorigenesis, metastasis, and portends a poor CRC prognosis. CD24 activates Src, which activates AP-1 family members c-Jun and c-Fos primarily via MAPK, increasing promoter activity and miR-21 expression, which in turn suppresses expression of PDCD4 and PTEN [178]. Yu et al. reported that high miR-21 expression was correlated with the clinical CRC stage (LNM and distant metastasis). Recently, miR-21 is confirmed to regulate stemness by modulating TGFβR2 signaling in CRC [179]. Given that the proportion of stem cells within tumors increases after conventional chemotherapy, and 30% of malignant colon tumors have TGFβR2 mutations (both of which are related to tumor recurrence [26]), further studies are warranted and carry great potential. After conventional chemotherapy, the percentage of undifferentiated cancer stem/stem-like cells (CSCs/ CSLCs) is relatively increased. CSCs/CSLCs are defined as a selfrenewing population of undifferentiated cells in a tumor. They grow slowly and are more prone to survive chemotherapy. They are responsible for populating the bulk of the tumor, tumor initiation, progression, metastasis, resistance to chemotherapy, and relapse [27]. Colon CSCs/CSLCs may be partly responsible for the age-related increase of CRC [73]. Recently, Yu et al. demonstrated that miR-21 was markedly increased in chemotherapy-resistant (CR) colon cancer cells. As increased miR-21 expression can modulate TGFβR2 signaling, this will downregulate Wnt signaling, maintaining stemness [180]. Via decreased Wnt signaling, the levels of β-catenin, c-myc, and cyclin-D, and T-cell factor/lymphoid enhancer factor (TCF/ LEF) activity are all augmented [179]. miR-21 down-regulation by antisense miR-21 could potently induce differentiation of colon cancer cells, rendering them susceptible to conventional or nonconventional chemotherapeutic regimens [181]. The involved targets include PTEN, PDCD4 [53,80] and TGFβR2 [48]. Suppression of PTEN by the up-regulated miR-21 in CRC leads to PI3K signaling activation and consequent CRC progression [49,50,122]. PDCD4 (introduced earlier in this review) can inhibit PMA-induced neoplastic transformation, tumor progression, invasion, and intravasation [25,176]. Furthermore, the elevated miR-21 in CRC could target RECK and TIMP3 genes, activating proteinases, especially urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs) [72], thereby markedly enhancing migratory and invasive CRC cell capacity [28]. miR-31, one of the most up-regulated miRNAs in colon tumors, is implicated in the tumor lymph node metastasis stage (in particular the pT stage and tumor invasive processes). Unexpectedly, its low expression is mainly observed in poorly differentiated tumors [29,30,53,100]. miR-21 and miR-31 can be up-regulated by TGF-β and its enhancer TNF-α, which in turn promote the biological effects of TGF-β [31]. TGF-β signaling exerts its tumor-suppressive effects

by strongly inhibiting cell proliferation (Fig. 3) and triggering apoptosis (Fig. 4). Smad4, a member of ligand-activated TGF-β, phosphorylates Smads, a pivotal cellular transducer of TGF-β signaling, resulting in the formation of an active nuclear transcriptional complex by certain cytoplasmic transducers. Mutation of Smads is associated with distant metastases in CRC, and is associated with poor prognosis via unknown mechanisms [54]. Since Smad4 activity and stability are regulated by post-translational modifications such as sumoylation, ubiquitination, and deubiquitination [55], investigating the post-transcriptional mechanism of Smad4 modulation in CRC may be of value. The target of miR-21 and miR-31 is T lymphoma invasion and metastasis gene 1 (TIAM1), a guanidine exchange factor of the Rac GTPase. TIAM1 also regulates migration and invasion [31]. Slaby et al. demonstrated that the targets of miR-31 include members of the Wnt signaling pathway (AXIN1) and forkhead family transcription factors (FOXC2 and FOXP3) [100]. In addition, H19, a maternally expressed oncofetal gene, highly expressed in embryogenesis (but nearly completely down-regulated postnatally), is the precursor of miR-675. H19 and miR-675 are upregulated in CRC tissue, promoting aggressive tumor cell growth and regulate the cell cycle by targeting tumor suppressor retinoblastoma (RB). RB exerts its regulation effect mainly via interaction with transcription factor E2F, which is known to activate H19. miR106a has been found to be consistently up-regulated in colon carcinoma, despite the fact that colon carcinoma does not express RB [56]. Associated with differentiation, miR-106a expression is markedly increased in intermediately-differentiated tumors compared to well- and poorly-differentiated ones, and higher in rectal tumors than in colonic ones [20]. miR-190 is a cell growth-regulating miRNA in CRC [57], and it enhances proliferation and malignant transformation. Elevated miR-190 down-regulates the translation of PH domain leucine-rich protein phosphatase (PHLPP), a negative regulator of Akt signaling. Increased miR-190 activates Akt, promotes the expression of VEGF, an Akt-regulated protein, and facilitates carcinogenesis and metastasis [58,59]. Ectopic expression of miR-95 in human CRC cell lines promotes cell growth in vitro and tumorigenesis in vivo by directly targeting sorting nexin 1 (SNX1). SNX1 is a putative tumor suppressor in CRC, plausibly acting in vesicle trafficking processes in oncogenesis and tumor suppression. However, SNX1, SNX2, SNX10 and SNX16 are implicated in mammalian tumorigenesis [60]. Moreover, miR-221 directly targets CDKN1C/p57, a member of the kinase inhibitor protein (CIP/KIP) family, and promotes CRC progression, particularly modulating cell proliferation (Fig. 3) and apoptosis (Fig. 4). The extent of miR-221 expression is positively correlated with CRC stage and aggressiveness. Down-regulation of CDKN1C/p57 in CRC cells results in reversal of G0/G1 phase arrest, enhanced aggressiveness, advanced tumor stage, poor differentiation, augmented tumor size, poor prognosis, and low disease-free survival after surgery [61]. miR-96 represses apoptosis partly by targeting transcription factor CHES1 [30,62,63] (Fig. 4). The marked down-regulation of five members of the miR200 family in CRC contributes to EMT, and augmented metastatic potential [64] (to be discussed in the next section). Moreover, miR103 and miR-107 possess oncogenic potential, particularly advancing metastasis [65]. As for angiogenesis, miR-107 serves as a potent angiogenic inhibitor by repressing hypoxia-inducible factor β (HIFβ)-mediated expression of VEGF [66]. miR-155 may maintain genetic integrity by targeting three enzymes involved in DNA repair, namely hMSH6, hMSH2, and hMLH1. The latter two proteins are the fundamental components of mismatch repair (MMR) in human cells. Specifically, over-expressed miR-155 may significantly downregulate core MMR proteins, which is implicated in replication errors and the pathogenesis of a number of sporadic microsatellite instability (MSI) tumors (including CRC). 12.7% patients are found to harbor MSI, a mutant phenotype [67]. Ovcharenko et al. observed that miR-155 is over-expressed in CRC, and suppresses apoptosis

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via a poorly understood mechanism [68]. miR-20b, which is significantly down-regulated in five CRC cell lines, exhibits a high functional consistency score with CRC, with an unknown oncogenic mechanism [69]. miR-29a (believed to act as a tumor suppressor in lung cancer) is significantly up-regulated in patients with advanced colorectal neoplasia. Brunet et al. reported that miR-29a was confirmed as significantly overexpressed in tumor samples when compared with normal samples. The serum levels of miR-29a was also significantly higher in CRC patients when compared to levels in the controls [32]. More importantly, Tang et al. further proved that miR-29a promotes colorectal cancer metastasis by modulating matrix metalloproteinase 2 and E-cadherin expression via KLF4 [33]. However, whether miR-29a plays an oncogenic or a tumor suppressor role needs further investigation [34]. miR-224, miR-335, miR-424, miR-301b, and miR-374a are all upregulated in CRC, via unclear mechanisms [35]. In conclusion, many onco-miRNAs have been discovered with largely unknown effects and mechanisms influencing CRC. Additional research is necessary and warranted to determine their future clinical applicability. miRNA and colorectal tumor migration, invasion, and metastasis The major cause of death from cancer stems from complications arising from migration, invasion, and metastasis [77]. Tumor migration and invasion are multi-step processes involving MMP activation, the breakdown of extracellular matrix (ECM), EMT onset (conversion of cellular phenotype from epithelium to mesenchyme via down-regulation of epithelial mRNAs and up-regulation of mesenchymal mRNAs by miRNAs, Fig. 2), microcirculation establishment, cell motility increase and invasion, and distant metastasis (dissemination via blood and lymph). Metastasis, a malignant hallmark of cancer, is related to epigenetic miRNA silencing with tumor suppressor capacities by CpG island hyper-methylation, a process largely unexplored [78] (Fig. 5).

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Many miRNAs play a metastasis-promoting role in CRC. As oncogenes, up-regulation of miR-21 and miR-31 in CRC are both associated with metastasis, where the former (miR-21) is associated with distant metastasis (particular liver) and the latter (miR31) is associated with local invasion. miR-21 is implicated in ECM breakdown by regulating RECK and TIMP3 genes [79]. miR-31 leads to metastasis and poor prognosis by targeting special AT-rich binding protein 2 (SATB2), which is involved in transcriptional regulation and chromatin remodeling. TIAM1, a guanidine exchange factor for Rac GTPase, is another target of miR-31. Their interaction promotes TGF-β- and TNF-α-dependent motility and invasion in CRC cell lines [20]. Although the function of miR-499-5p in cancer has not been fully elucidated, it has been demonstrated to be a prometastatic miRNA. miR-499-5p directly targets FOXO4 (a member of the Forkhead box O-class subfamily) and PDCD4, promoting CRC cell migration and invasion in vitro and pulmonary/hepatic metastasis in vivo. Specifically, FOXO4 is a tumor suppressor of metastasis during EMT in CRC, while PDCD4 is a tumor suppressor of migration and invasion [182]. Elevated expression of miR-103 and miR107 in CRC is responsible for its local invasion and liver metastasis. miR-103/107 targets the DAPK and KLF4 in CRC cells, leading to increased cell–matrix adhesion, aggressive propensity, decreased cell– cell adhesion via E-cadherin/claudin-3, and decreased epithelial marker expression. This process might be enhanced by EMTinducing signals transduced from tumor microenvironments. The concurrent repression of DAPK and KLF4 also potentiates the colonization of CRC cells at a metastatic site. DAPK, a metastasis suppressor, represses cell–matrix adhesion by inactivating integrin β1. Zinc finger transcriptional factor KLF4 is abundant in terminally differentiated epithelial cells near the luminal surface and decreases toward the base of crypts. Though hypoxia is a potent inducer of miR-103/107-mediated tumor metastasis, neither normoxia nor hypoxia affect the physiologic roles of miR-103 or 107. Recently, Chen et al. reported that increased miR-103/107, decreased DAPK, and decreased KLF4 expression may be prognostic

Fig. 2. CRC metastasis. Metastasis encompasses spread from primary CRC, intravasation, migration and survival in circulation, extravasation, growth and proliferation to form secondary tumors (indicated by numbers) with CRC cell participation, endothelial cells, host lymphocytes, and fibroblasts.

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Fig. 3. Network of miRNAs and protein-coding genes involved in proliferation. This figure demonstrates the interaction between miRNAs (brown boxes) and proteincoding genes (green boxes), and their respective functions. While the majority of miRNAs inhibit target mRNA(s), some can promote or induce target mRNA (i.e., miR-192 and miR-215 can induce p53, c-Myc can induce miR-17-92 cluster, miR-17-92 cluster may promote E2F1/3, E2F1/3 can induce miR-675, TGFβ2 can induce miR-21 and miR31). Promotion (red) or suppression (blue) of proliferative function is also indicated. Dashed lines indicate an interaction not well understood, and gray lines indicate unclear functions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

for increased metastatic recurrence and poor survival in CRC [65]. Additionally, miR-103 and 107 are oncogenic by inducing EMT, with concomitant Dicer suppression [183]. Paradoxically, increased Dicer expression is associated with high-grade tumor and poor prognosis [74,184].

When considering curative treatment for early colon cancer, preoperative assessment of LNM must be assessed. The methylation of the promoter region of hsa-miR-9 represses its expression. Additionally, the methylation of hsa-miR-9-1 is associated with LNM in CRC [75,97]. miR-9, targeting mRNA encoding E-cadherin (a key

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Fig. 4. Network of miRNAs and protein-coding genes involved in apoptosis. This figure demonstrates the interaction between miRNAs (orange boxes) and protein-coding genes (green boxes), and their respective functions. Promotion (red) or suppression (blue) of apoptosis is indicated. Dashed lines indicate an interaction not well understood, and gray lines indicate unclear functions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

protein in suppressing tumor metastasis), is up-regulated by myc and MYCN in various cancer cells, leading to β-catenin signaling activation, acting as a tumor-initiator miRNA [185]. A recent study confirms that hsa-miR-129* and hsa-miR-137 are LNM-associated miRNAs, up-regulated in lymph node-positive colon cancer specimens [40]. Another proportion of miRNAs are implicated in metastasis suppression. The miR-200 family (including miR-200a/b/c, miR-141, and miR-429) and miR-205 are pivotal regulators of EMT in various cancers, including CRC. Low levels of miR-200 family members may initiate EMT, initiating metastasis, and triggering emergence of epithelial cells in metastatic sites, forming a secondary tumor [7]. The zinc-finger E-box binding homeobox 1 (ZEB1) and TGFβ2 may trigger EMT via modulation of the miR-200 family. ZEB1, a pivotal inducer of EMT in CRC, is able to suppress expression of basement membrane components and cell polarity factors, directly restraining transcription of miR-141 and miR-200c by binding at least two highly conserved sites in their putative promoter. In turn, miR-141 and miR200c inhibit EMT by strongly activating epithelial differentiation, respectively targeting EMT activators TGFβ2 and ZEB1. ZEB1 triggers a miRNA-mediated feed-forward loop stabilizing EMT and potentiating CRC metastasis [122]. The miR-200 family, which also targets Sox2 and Klf2, also inhibits stem cells and inflammation [49,50]. Despite possessing their own separate targets, these coexpressed miRNAs are synergistic, possibly co-activated by a common promoter. ZEB1 is pivotal in EMT regulation, with a feed-forward effect in self-enhancing manner. Its aberrant expression reduces its own inhibitors miR-141 and miR-200c. Should initiating signaling (such as TGF-β) vanish, miR-141 and miR-200c may activate and re-induce an epithelial phenotype, thus explaining the strong phe-

notypic heterogeneity usually exhibited by invading cancer cells [41]. As described earlier, miR-34a (targeting CD44-encoded mRNAs [37]) in concert with miR-34 (targeting c-Met-encoded mRNAs) suppresses migration and invasion [7]. In addition to inhibiting tumor growth, mi-145 represses cell invasion and metastasis by directly targeting MUC1, a well-established metastasis gene. MUC1, frequently over-expressed in CRC patients, correlates with poor CRC survival. Suppression of MUC1 by miR-145 reduces β-catenin and oncogenic cadherin 11, both of which are co-expressed at the invasive border of CRC tissues, and are correlated with poor prognosis [38]. The let-7 miRNA family is well conserved across different species. During normal circumstances, let-7 family members are elevated, and carcinogenesis is inhibited. Absence or decreased levels of the let-7 family is implicated in cell proliferation (Fig. 3), differentiation, migration, and invasion via EMT. Moreover, low let-7 family levels have been observed in CSCs, contributing to unlimited selfrenewal CSC capability and subsequent cancer progression [42]. The downstream targets of the let-7 family include CDK6, vimentin, and E-cadherin, which are all correlated with the transition from epithelial to mesenchymal phenotype. Similarly, the miR-200 family functions as a fundamental EMT regulator by targeting the EMTdriving transcription factors ZEB1 and ZEB2. The underlying regulatory mechanism is unclear, and believed to be related to promoter hyper-methylation of miR-200c/141, reducing its expression in CRC tissues, and ultimate consequent acquisition of mesenchymal characteristics [43,44]. Over-expression of DNMT1 silences tumor suppressor gene by relevant DNA methylation, strongly contributive to carcinogenesis. The frequently down-regulated miRNA in CRC miR-342 directly targets DNMT1, inhibiting cell invasion [126]. Zhao et al. reported that miR-30 family over-expression facilitates

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Fig. 5. Network of miRNAs and protein-coding genes involved in migration, invasion, and metastasis. This figure demonstrates the interaction between miRNAs (brown boxes) and protein-coding genes (green boxes), and their respective functions. Promotion (red) and suppression (blue) of migration, invasion, and metastasis are indicated. Gray represents unclear functions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

epithelial phenotype maintenance, and suppresses tumor metastasis [7]. The down-regulation of tumor-suppressor miR-143 occurs in colon cancer and hepatic metastatic tissue [45]. This exposition of miRNAs promoting/suppressing migration, invasion, and metastasis is by no means exhaustive or complete, as many underlying responsible mechanisms remain largely unknown. Angiogenesis Angiogenesis is a critical process of both physiological and pathological conditions, in which miRNAs play an active role (Fig. 6). Diverse endothelium-associated miRNAs (miR-126, miR-130a, miR210, and miR-292) promote angiogenesis. miR-126, an endothelial cell-specific miRNA, promotes angiogenesis in the presence of various stimuli, and represses negative regulators of angiogenic signaling pathway. miR-27b also promotes angiogenesis, determining tip cell sprouting and venous specification by targeting endogenous angiogenesis inhibitor SEMA6A or controlling the expression of EfnB2, EfnB4, Flt1, and Flt4. Furthermore, miR-27b targets Dll4 and SPRY2, correlated with vascular guidance and tubular structure branching. Silencing miR-27B may therefore be a promising manner of

controlling tumor angiogenesis and growth, by utilizing agents such as pigment epithelial-derived factor (PEDF), an endogenous angiogenesis inhibitor [82,83]. c-myc activates the miR-17-92 cluster, promoting angiogenesis by targeting TSP1 and CTGF [173]. The let-7 family, miR-221, and miR-222 are all miRNAs capable of promoting angiogenesis by repressing endogenous angiogenic inhibitors such as TSP1. miR-10b, up-regulated by thrombin, binds the 3′-UTR of homeobox D10 (HoxD10), inducing HMEC-1 migration, tube formation, and angiogenesis. Heparin down-regulates miR-10b, inhibiting angiogenesis [142]. Therefore, the pro-angiogenic function of miRNA is frequently correlated to vascular-endothelium-related molecules, involving many signaling networks. Conversely, various miRNAs inhibit angiogenesis. miR-214, which directly targets Quaking (a protein critical for vascular development), is differentially expressed in compensatory arteriogenesis. miR-214 reduces the expression of pro-angiogenic growth factor, including vascular endothelial growth factor (VEGF), and inhibits endothelial cell sprouting [131]. miR-145, a tumor suppressor, inhibits potent angiogenic factors HIF-1α and VEGF by directly targeting p70S6K1 and the rapamycin (mTOR)/p70S6K1 signaling pathway. The mTOR/p70S6K1 signaling pathway is activated by the

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Fig. 6. Network of miRNAs and protein-coding genes involved in angiogenesis. This figure demonstrates the interaction between miRNAs (light blue boxes) and proteincoding genes (yellow boxes), and their respective functions. Promotion (red) or suppression (blue) of angiogenesis is indicated. The abundance of gray lines (indicating unclear functions) demonstrates the significant amount of work still needed in this field of study. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

dysfunction or genetic alterations of its upstream PI3K/PTEN/AKT signaling pathway or via p70S6K1 upregulation/amplification. The mTOR/p70S6K1 signaling pathway mediates tumor angiogenesis and tumor growth by altering HIF-1α and VEGF levels [105]. In endothelial cells, VEGF, VEGF receptor-2 (VEGFR2), and fibroblast growth factor receptor-1 (FGFR1) are all targets of miR-16 and miR-424. Overexpression of miR-16 or miR-424 contributes to reduced VEGFR2 and FGFR1, which respectively regulate VEGF and basic fibroblast growth factor (bFGF) signaling, decreasing endothelial cell proliferation, migration, in vitro cord formation, and in vivo blood vessel formation [132]. Therapeutic target for colorectal cancer Compared to single-gene targeting approaches, the most attractive aspect of miRNAs as therapeutic agents is their ability to target multiple molecules, usually in the context of a regulatory network. To target miRNA dys-regulated in cancer, direct and indirect strategies have been designed. Direct strategies involve anti-miRNA oligonucleotides (AMOs) or virus-based constructs blocking oncogenic miRNA expression or re-introduction of a tumor suppressor miRNA absent in cancer. Indirect strategies modulate miRNA expression [3]. Examples of viral vectors include retroviruses, lentiviruses, and adenoviruses [133]. The class of chemically engineered AMOs capable of silencing endogenous miRNAs is termed antagomiRs [134]. Recently, seminal technologies have been de-

veloped, such as anti-miRNA complex with interfering nanoparticles (iNOPs) [135] and liposome–polycation–hyaluronic acid (LPH) nanoparticle formulation. LPH is modified with tumor-targeting single chain antibody fragment (scFv) [186]. As the miR-200 and the miR-30 family are crucial regulators of EMT, they can be considered novel therapeutic CRC targets. As tumor suppressors, the let-7 and miR-200 families inhibit EMT and subsequent migration/invasion via targeting CDK6, vimentin, and E-cadherin. However, the expression of let-7 and miR-200 is low in CRC tissues. Acetyl-11-keto-β-boswellic acid (AKBA, one of the active components in boswellic acids), promisingly suppresses NFκB and STAT-3-related pathways, and may upregulate the let-7 and miR-200 families, leading to apoptosis and angiogenic inhibition in CRC, albeit via unknown precise mechanisms [43]. Additionally, miRNA could also manipulate the biological properties of CSCs, altering processes within tumor maintenance, tumor progression, therapy resistance, and distant metastasis [136]. miRNAbased therapeutics [137,146] may therefore be helpful in treating CRC. Employment of modified, locked-nucleic-acid antisense AMOs [147] may counter carcinogenesis-inducing miRNAs, usually with relatively low toxicity [138]. Other options include methods binding target miRNAs, such as synthetic mRNAs, lentiviral vectors, smallmolecule inhibitors, miRNA sponges, and miR-masking [166]. To amplify carcinogenesis-inhibiting miRNAs, viral/liposomal delivery systems [139] or double-stranded miRNA mimics [140] may increase expression of target miRNAs [7]. For example,

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miR-221-specific inhibitor potently suppresses CRC cellular growth, and may be a potential anti-tumor candidate in CRC treatment [141]. Epigenetic alterations such as DNA methylation regulate miRNA expression during carcinogenesis. Approximately 47% of human miRNA genes are related to CpG islands, which, when hypermethylated, silence tumor suppressor genes. Some miRNAs, like miR-345, are situated within or near a CpG island, and are methylation-sensitive. Such miRNAs may markedly increase after 5-aza-2′-deoxycitidine (5-aza-dC), a DNA methyltransferase inhibitor, treatment [118]. miR-137 (discussed earlier) inhibits cell proliferation, and is frequently silenced by promoter hypermethylation, in an early event of CRC progression. Thus, miR-137 has prognostic and therapeutic implications. Epigenetic drugs may have positive benefits via unexplored mechanisms [143]. miR-342 inhibits CRC proliferation and invasion by directly targeting DNMT1; a demethylating agent could therefore act as epigenetic cancer therapy [126]. miR-145 exhibits great pro-apoptotic and antiproliferation capacity. Recently, miR-33a has been demonstrated to down-regulate oncogenic kinase Pim-1 and inhibit cell-cycle progression. These tumor suppressing miRNAs have been the subject of nonviral cancer treatment strategies. Polyethylenimine (PEI)/ miRNA complexes have been employed to delivery intact unmodified miR-145 and miR-33a molecules to colorectal tissues, where they can exert local or systemic antitumor effects [144]. In conclusion, dys-regulated miRNAs as therapeutic CRC targets are promising, and warrant further investigation. miRNA as diagnostic and prognostic biomarkers for colorectal cancer Colonoscopy, the current gold standard for CRC diagnosis, is not widely adopted because it is an invasive and expensive procedure. The fecal occult blood test (FOBT), which detects blood in the stool leaked from disrupted vessels on the colorectal tumor has been widely used, despite of its relatively low sensitivity. The stool DNA test, detecting mutated DNA, cancer-related methylation analysis, and DNA integrity, is non-invasive, and highly sensitive (52%) and specific (94%). However, it is not widely applied for its laborintensive handling and high cost. The stool-based mRNA test is another frequently used CRC diagnostic tool. This analysis includes direct sequence analysis, single-strand conformational polymorphism analysis, and analysis of CRC-related gene expression based upon real-time reverse transcriptase-PCR. However, results may be unreliable if available mRNA concentration is too low or degraded. Testing for neoplasm-derived proteins, especially stool carcinoembryonic antigen, is also reported to be valuable with a sensitivity of 86% and a specificity of 93% for CRC. However, the test’s stability requires improvement [145]. As molecular biomarkers for early stage CRC, miRNAs are highly stable. Certainly, miRNAs can be reliably extracted and detected from frozen and paraffin-embedded tissues, blood (either total blood, plasma, or serum) [152,153], circulating exosomes [168], and other biologic fluids like urine [167], saliva [154,155] and even sputum [51,156]. It is a relatively efficient way to conduct miRNA expression analysis of exfoliated colonocytes isolated from feces, including three internal control miRNAs – U6snRNA, RNU6B, and miR-16. miRNAs in serum and plasma are remarkably stable among individuals of the same species. Even extracellular miRNA, purported by-products of dead cells, is astoundingly stable after 1 month, due to its binding of the Ago2 protein (a part of the RISC [30]). Hence, miRNAs are ideal noninvasive biomarkers for cancer detection. Two well-established oncogenic miRNAs miR-21 (the first serum miRNA biomarker) and miR-92a (a member of the miR-17-92 gene cluster) are increased in CRC tumor tissues. miR-92a is increased in the plasma of CRC patients compared to control. There is higher sensitivity of stool miR-92a, but not miR-21, for distal CRC (80.3%)

compared to proximal CRC (51.9%). Interestingly, low miR-92a levels are found in a substantial proportion of tumor tissues [20]. Stoolbased miRNA detection can be used alone or integrated into existing marker panels to augment sensitivity. miR-92a in stool samples is highly stable over 72 hours. During this period, a 30% reduction from the original levels is observed after 12 hours, without any further degradation in the remaining 60 hours. Wu et al. recently reported that the expression of miR-21 and miR-92a was significantly higher in CRC tissues compared with their adjacent normal tissues [145]. Both miRs are associated with poor survival and poor therapeutic outcome (miR-21 to significant extent) [29,30,157]. In addition, miR-15b, miR-21, miR-181b, miR-191, and miR-200c may also be linked to CRC development and progression, and may be prognostic CRC markers [34,150]. miR-92 and miR-17-3p are both encoded by the miR-17-92 gene cluster, promoting cell proliferation, suppressing cancer cell apoptosis, and inducing tumor angiogenesis. Compared to miR-17-3p, miR-92 is more likely related to CRC tumorigenesis. miR-92 alone represents an early predictive CRC marker, with 89% sensitivity and 70% specificity. However, no difference has been demonstrated between plasma miR-92 level and CRC stage, tumor volume, and metastatic status [57,187]. CRC is classified into 3 groups: Lynch syndrome, MSI, and microsatellite stable (MSS) tumors (based upon the presence or absence of MSI). The expression of a subset of nine miRNAs is markedly different between tumor and normal colonic mucosa tissues. Among these miRNAs, the differential expression of miR-622, miR1238, and miR-192 is the most pronounced between Lynch syndrome tumors and sporadic MSI tumors [142]. miRNA expression is verifiable, because they are quite stable dependent upon tissue type, and are protected against endogenous degradation [142]. In another classification scheme, Lynch syndrome is not included. CRC is divided into two major subgroups: MSS and MSI. MSI cancer carries a better prognosis. Any differences between normal colorectal, MSI, and MSS tissues may therefore hold diagnostic and therapeutic potential. The expression levels of both miR-320 and miR-498 are significantly lower in MSS compared with normal control and their expression levels are also associated with probability of recurrence-free survival, which demonstrate significant clinical value as predictive biomarkers [158]. In another study, miR20a and miR-92 are up-regulated in both MSI and MSS subtypes [158]. Moreover, circulating miRNAs could also serve as optimal early diagnostic biomarkers of disease because they are highly stable [140,152], RNase-resistant, capable of self-renewal [159], consistently expressed across the same population, and resistant to changes in temperature and pH. miR-485-5p, miR-361-3p, miR-326, and miR-487b are utilized as diagnostic CRC molecular targets [7]. Furthermore, plasma miR-221 is significantly greater in CRC patients than in healthy patients. A study of 79 solid tumor circulating miRNA biomarkers demonstrates 58% (46 of 79) are highly expressed in one or more blood cell types. Blood cells therefore contribute significantly to circulating miRNAs. Consequently, circulating miRNAs may reflect a blood cell-based phenomenon, rather than a cancerspecific origin [148]. Despite the high (75–80%) 5-year survival of patients with stage II (no lymph node or distant metastases) CRC after surgery, 20– 25% patients develop recurrent CRC and die from this disease, making clear the urgency in differentiating between positive and negative CRC patient prognosis. Some miRNAs (miR-31, miR-7, miR-99b, miR378, miR-133a and miR-125a) are expressed to different degrees dependent upon the CRC stage (mostly early stage II to late stage III) [30]. A panel of plasma miRNA markers may improve the sensitivity and specificity of this CRC screening assay. High accuracy is of course a prerequisite for any predictive clinical tool. However, the clinico-pathologic parameters (node involvement and tumor infiltration), microsatellite instability, and traditional DNA signatures such as CpG island hypermethylation are not yet sufficient. To

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categorize stage II colon cancer patients harboring elevated recurrence risk, possibly benefiting from adjuvant treatments if diagnosed early enough, a widespread test of epigenetic inactivation affecting ECM pathway genes in colon cancer was conducted [149]. The epigenetic inactivation starts from early tumor development and accumulates throughout cancer progression, and thereby predicts cancer cell growth, dissemination [160], metastasis, and risk of recurrence after surgical removal. A new DNA methylation signature involving 2 genes (the mismatch repair gene hMLH1 and the 06methylguanine-DNA methyl-transferase gene MGMT) may be promising. DNA hypermethylation of IGFBP3, EVL, FLNC, and CD109 are associated with increased mortality risk [161]. The currently used TNM system is less predictive of outcome for the intermediate CRC stage. Given the importance of precise prognostic prediction throughout cancer stages, novel biomarkers are under fervent investigation. Regardless of disease stage, all patients benefit from early diagnosis and treatment initiation. However, the pathophysiology of CRC is complex. Despite MSI being a generally positive prognostic indicator, early-stage K-ras-LCS6 wild-type patients have a poor prognosis if they harbor a sporadic MSI tumor [162]. A combined assessment of let-7a and SNP in LCS6 are more reliable clinically than single factor in the prediction of clinical outcome [87]. Given the difficulty in locating the tumor origin in the setting of ongoing metastases, miRNA dys-regulation patterns may prove helpful. One such pattern was utilized to accurately predict tumor tissue origin in 86% of cases in a blind test set, which included 77% metastatic cases [163]. Different subtypes of a particular cancer can be discriminated by gene expression profiling based upon microarray platforms [164]. Numerous methods detecting miRNAs have been developed, including quantitative real-time polymerase chain reaction (RT-qPCR), in situ hybridization [165,188], and high throughput sequencing [189]. As technology, creativity, and pathophysiology comprehension advances, so will our capability to diagnosis CRC earlier and more accurately [3]. miRNA in the prediction of treatment response in colorectal cancer patients No relationship has yet been established between CRC-modified miRNA transcriptome and the therapeutic response of individual CRC patients. However, because miRNA expression is closely correlated with therapy efficacy, prediction of the response of CRC patients to different treatment regimens is possible. Through comparing 667 miRNAs in 2 human CRC cell lines, Caco-2 (sensitive to cetuximab) and HCT-116 (resistant to cetuximab), Ragusa et al. found 21 and 22 miRNAs are differentially expressed in Caco-2 and HCT116, respectively [150]. Down-regulation of let-7b and let-7e and up-regulation of miR-17 can function as molecular markers predicting cetuximab resistance in CRC. After 24 hours of cetuximab treatment, 12 miRNAs (6 down-regulated and 6 up-regulated) were differentially expressed in Caco-2 cells, while 16 were differentially expressed (11 down-regulated and 5 up-regulated) in HCT116 cells. miR-330-5p is up-regulated and miR-610 is downregulated in both cell lines. After 48 hours, differential expression of 10 miRNAs (8 down-regulated and 2 up-regulated) was demonstrated in Caco-2 cells, 6 (5 down-regulated and 1 up-regulated) in HCT-116 cells. The authors cite that miRNA regulation begins after 24 hours of treatment. Additionally, as discussed earlier, 5-FU promotes stable miR-43 over-expression, inducing CRC apoptosis by incorporating into RNA and DNA, inhibiting thymidylate synthase. Though single use cetuximab has limited effect, combination use with novel chemotherapeutic agents has promising efficacy [70]. Likewise, miR-145, a tumor suppressor inhibiting angiogenesis, is markedly down-regulated in CRC cells; miR-145 rescue may be a potential therapeutic approach for CRC patients. Furthermore, miR145 controls cellular apoptosis by targeting DFF45 (Fig. 4), and

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inhibits colon cancer cell growth by targeting oncogene FLI1 via unclear mechanisms [105]. Work predicting the response of CRC patients to treatment regiments via miRNA expression remains ongoing. Potential therapeutic applications Of anti-tumor agents interfering with the EGFR pathway, cetuximab is a common CRC treatment, used alone or in combination with chemotherapy. Cetuximab blocks the EGFR signaling pathway, and can inhibit cell-cycle progression, angiogenesis, invasion and metastasis, and initiate apoptosis. Cetuximab has synergistic cytotoxicity with chemotherapy and radiotherapy, and is believed to alter the miRNA transcriptome in drug-sensitive and drug-resistant CRC cells. At steady state, the corresponding molecular networks might be prone to respond to EGFR inhibitors. When EGFR is blocked, K-ras fails to transduce proliferation signals, and as a result, replication of tumor cells is suppressed [150]. Additionally, cetuximab efficiently transfects precursor miR-145 into CRC cells, which will later be processed into mature miRNA, and abundant apoptotic morphology ensues [158]. miR-30a-5p is a potent tumor suppressor impairing CRC proliferation, and facilitates functional recovery of its target gene denticleless protein homolog (DTL) [119]. As a tumor suppressor, restoring of miR-491 into nude mice significantly decreases colorectal tumor formation in vivo. Its mechanism involves targeting Bcl-xl, which is associated with chemotherapy resistance [169,170], rendering it a therapeutic target of interest [128]. Proteins PI3K/PTEN/AKT enact signaling affecting apoptosis, cell growth and proliferation and inhibitory manipulation of their downstream targets mTOR/p70S6K1 show promise in animal experiments and clinical trials [171]. miRNA microtome analysis for anticipating patient response to treatment remains an immature but developing field of research. Conclusions As post-transcriptional regulators, miRNAs are differentially expressed in different types of cancers, including CRC. Some miRNAs are down-regulated in CRC tissues (tumor suppressors), while others are up-regulated (oncogenes). Both types of miRNAs are implicated in various intracellular signaling networks, and therefore exert different effects upon CRC carcinogenesis, including cell growth, differentiation, apoptosis, angiogenesis, migration, invasion, and metastasis. From a clinical perspective, miRNA dysregulation may serve as potential biomarkers for diagnosis and prognosis of CRC patients. Combination biomarker panels enhance sensitivity and specificity. Methods detecting plasma and fecal miRNAs continue to evolve rapidly with great future potential application. Certain miRNAs may serve as therapeutic targets against CRC. Significant future investigation on miRNAs with clinical applicability pertaining to CRC diagnosis, prognosis, and treatment is warranted. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81402396) and Yi Yao Foundation (Grant No. 14H0563). Conflict of interest The authors declare no conflicts of interest. References [1] N. Valeri, C.M. Croce, M. Fabbri, Pathogenetic and clinical relevance of microRNAs in colorectal cancer, Cancer Genomics Proteomics 6 (2009) 195–204.

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