MicroRNAs in Colorectal Cancer : from Diagnosis to Targeted Therapy

Colorectal cancer (CRC) is still one of the five most leading cancer-related death which have one million new cases every year (Pourhoseingholi, 2012; Siegel et al., 2012). Although since mid-1980’s the number of CRCrelated death has been decreasing due to the increased awareness and progresses in screening approaches, the prognosis of patients with metastatic CRC remains unknown (Terzic et al., 2010; Salimzadeh et al., 2012). Eventually, the study for early diagnosis and prognosis markers are still crucial and would allow selecting patients with early CRC stages for operative cancer management and developing novel targeted therapies. The involvements of non-coding RNAs in carcinogenesis and tumor progression have been confirmed by numerous functional studies in the past decades (Seton-Rogers, 2013). Among the all types of ncRNAs, microRNAs (miRNAs) received the greatest attention due to their frequent dys-regulations in CRC. miRNAs compromise a large subsequent of endogenous small ncRNAs that regulate gene expression posttranscriptionally and control various cellular mechanisms including tissue development (Johnston and Hobert, 2003; Zhao et al., 2005), cell proliferation (Cheng et al., 2005; 2006), cell division (Hatfield et al., 2005; Croce


Introduction
Colorectal cancer (CRC) is still one of the five most leading cancer-related death which have one million new cases every year (Pourhoseingholi, 2012;Siegel et al., 2012).Although since mid-1980's the number of CRCrelated death has been decreasing due to the increased awareness and progresses in screening approaches, the prognosis of patients with metastatic CRC remains unknown (Terzic et al., 2010;Salimzadeh et al., 2012).Eventually, the study for early diagnosis and prognosis markers are still crucial and would allow selecting patients with early CRC stages for operative cancer management and developing novel targeted therapies.
The involvements of non-coding RNAs in carcinogenesis and tumor progression have been confirmed by numerous functional studies in the past decades (Seton-Rogers, 2013).Among the all types of ncRNAs, microRNAs (miRNAs) received the greatest attention due to their frequent dys-regulations in CRC.miRNAs compromise a large subsequent of endogenous small ncRNAs that regulate gene expression posttranscriptionally and control various cellular mechanisms including tissue development (Johnston and Hobert, 2003;Zhao et al., 2005), cell proliferation (Cheng et al., 2005;2006), cell division (Hatfield et al., 2005;Croce
miRNAs have been reported to have pervasive effects on CRC tumorigenesis including, oncogenesis, progression, invasion, metastasis, and angiogenesis (Esquela-Kerscher and Slack, 2006;Huang et al., 2008;Zhang et al., 2012).Nevertheless, a comprehensive understanding of the functional role and thorough panel of target mRNAs for individual miRNAs are still in their infancy.Equally challenging or even more challenging is their applications in CRC diagnosis and prognosis, since clinical application of miRNAs have some drawbacks and some provocative reports directly contradict one or another outcomes of original assertions.
These discrepancies between studies could be explained by different factors.First, the heterogeneities observed in results arise from different tumor locations (Colon or Rectum, proximal or distal colon tumor) (Slattery et al., 2011).Secondly, different population sampling bearing various genomic pools could lead to different gene expressions and regulations.Therefore, miRNA expression pattern could differ among populations.On the other hand, the subgroup of tumor is an important defining factor since it has been reported that miRNA deregulation could vary between MSI and CIMP tumors (Schepeler et al., 2008;Earle et al., 2010;Haghighi et al., 2010;Shemirani et al., 2011).In addition, several other factors affect miRNA expression profiling and may hand in different data and results.These factors include the type of the sample (blood, tissue, serum, and faces), the timing between sampling and extraction, methods and kits used for profiling experiments and sample storage.
Despite all aforementioned disadvantages, accumulating proofs demonstrating the potential role of miRNAs in carcinogenesis reflect the worthy advantages of miRNA implications as tumor markers and their potential role for cancer therapies.These advantages include the need of a small amount of sample, the possibility of extracting miRNAs from body fluids, their stability in FFPE tissues, and non-invasive and poisonousness nature of their sampling methods.In the following, we provide a comprehensive review of scientific literature describing the aberrantly expressed miRNAs, and consequently dysregulation of targeted mRNAs along with the potential role of miRNAs in CRC diagnosis, prognosis, and response prediction.We also summarize the recent findings on miRNA-based manipulation methods with the aim to enhance knowledge of anti-CRC therapies.

Cancer diagnosis
CRC diagnosis based on a molecular signature implies that a specific expression level is found in tumour cells compared to non-tumour counterpart.Increasing profiling experiments have revealed that miRNA expression patterns are unique to specific cancers, unlike other markers currently available, offering capability of cancer diagnosis in early stages (Zhou et al., 2013a;Li et al., 2014c).The most extensively applied techniques of studying miRNA profiles are microarray, real-time PCR-based methods, as well as sequencing and in situ hybridization (ISH) (Weng et al., 2010;Xie et al., 2011).Next Generation Sequencing (NGS) added a new layer of accuracy as new method for miRNA expression measurement and normalization and provided new knowledge of changes in genome and relevant disease for translational studies (Yadav et al., 2014).
The first miRNAs found to be aberrantly expressed in CRC were miR-143 and miR-145 that were consistently downregulated at the adenomatous and cancer stages of CRC neoplasia compared to normal colon mucosa (Michael et al., 2003;Akao et al., 2006b).Since then the literature dedicated to dysregulation of miRNAs in three different series including tumour samples, both fresh frozen and formalin-fixed paraffin-embedded (FFPE), circulating miRNAs, both in serum and plasma, and faecal samples has grown considerably.

Dysregulated miRNAs in tissue samples and CRC cell lines
After discovering miR-143 and miR-145, several studies have been performed in order to investigate other CRC-related miRNAs.miR-143 was then found to be down-regulated in colon but not in rectum related cancer  Let-7a Down c-Myc, DLD1, KRAS (Cummins et al., 2006, Akao et al., 2006a, Fang et al., 2007, Earle et al., 2010) Let-7c Down MMP11, PBX3 (Han et al., 2012)

Dysregulated miRNAs in serum or plasma samples
Recent studies have revealed that dysregulated miRNAs are present in body fluids carried by small micelles and therefore, are protected from hostile ribonuclease activity (Chim et al., 2008;Lawrie et al., 2008;Mitchell et al., 2008).Many groups evaluated the feasibility of using circulating miRNAs as non-invasive biomarkers for CRC (Slattery et al., 2011).Chen et al. reported the existence of circulating miRNA in the serum of CRC patients for the first time (Chen et al., 2008).Since then, dysregulated miRNAs were found to be in plasma or serum of CRC patients at detectable levels and are capable of distinguishing normal samples from CRC samples (Huang et al., 2010;Wang and Zhang, 2012;Wang et al., 2012b;Kanaan et al., 2012).Furthermore, the discovery of miRNA transport, mediated by exosomes, opened a new research area of isolating tissue specific circulating exosome and their contained miRNAs to better analysis of their expression (Taylor and Gercel-Taylor, 2008).These dysregulated circulating miRNAs are summarized in Table 2.

Dysregulated miRNAs in stool samples
Stool-based test is a widely used non-invasive screening method of CRC patients (Lee et al., 2014).However, due to their poor sensitivity and specificity these tests were losing their significance.Nevertheless, reporting miRNAs as screening markers found in faecal specimens by Ahmad et al in 2009, elevated the practicability of using miRNAs as non-invasive diagnostic biomarkers (Ahmed et al., 2009).Surprisingly, analysed miRNAs in stool samples showed relatively high sensitivity and specificity (Link et al., 2010;Koga et al., 2010;Kalimutho et al., 2011;Li et al., 2012;Wu et al., 2012a).Studies were summarized in Table 3.These studies confirmed the hypothesis that miRNAs hold a potential of being used in a stool-based assay for early CRC detection.However, the capability of profiling such miRNAs for adenomatous polyposis is remained to be explored.

miRNA as Marker for Treatment Response Predictions
Aberrant expression of some miRNAs has been shown to be associated with treatment response and outcomes in patients with colorectal cancer.An increasing number of dysregulated miRNAs were revealed to have association with drug resistance or sensitivity which indicate their capability of predicting patients' responses to some anticancer agents (Hummel et al., 2010).For instance the upregulated miR-10b and miR-192/215 were reported to hold a potential to indicate chemosensitivity to the common 5-FU-based chemotherapy regimen (Boni et al., 2010;Nishida et al., 2012) as well as miR-19a which predicts resistance to FOLFOX chemotherapy in advanced CRC cases (Chen et al., 2013).Pichler et al. showed that miR-18a downregulation is associated with poor survival in patients with CRC and its expression could predict progression-free survival (PFS) in EGFR-targeted therapy (Pichler et al., 2014).Moreover, miR-203 was demonstrated to induce oxaliplatin, a common component DOI:http://dx.doi.org/10.7314/APJCP.2014.15.17.6989MicroRNAs in Colorectal Cancer: from Diagnosis to Targeted Therapies combination therapeutic regimen for use in patients with metastatic CRC, and reverse chemoresistance by negatively regulating ATM kinase and Akt, respectively (Li et al., 2011;Zhou et al., 2014).
Besides, experimental and clinical studies have cleared that hsa-let-7g and hsa-miR-181b are potential indicators for chemoresponse to S-1 based chemotherapy and miR-200a, miR-200c, miR-141, and miR-429 expression levels may identify CRC patients, including those with stage II disease, who are most likely to benefit from fluropyrimidine, an adjuvant chemotherapy agent (Nakajima et al., 2006;Diaz et al., 2014).

miRNA-Based Targeted CRC Therapy
The principle that miRNAs play important roles in CRC development and progression provide a rationale for CRC therapeutic investigations.Tumor suppressor and oncogenic miRNAs, are so called oncomiRs, hold a promising potential to be manipulated for clinical trials such as CRC treatment, blocking the progression of precursor lesions, prevention of distant metastasis, and improving responses to chemo-and radio-therapies (Tong and Nemunaitis, 2008).
miRNAs interact with different cancer signalling pathways and control cellular homeostasis.Hence, silencing overexpressed oncogenic miRNAs as well as restoring downregulated tumor suppressor miRNAs may eventually lead to the tumor growth and progression inhibition, apoptosis, and reduced cell viability (Du et al., 2014;Orang et al., 2014a).
Negative manipulation of oncogenic miRNAs'  expression has some similarities with siRNAs in principle and could be achieved through introducing silencing molecules into the cell.Antisense oligonucleotides along with some bearing anchored group, sponge and masking molecules, locked nucleic acid (LNA) are of important miRNA-based therapies.Elaboration of these methods could replace current anticancer therapies and find their ways into daily clinical practices.
On the other hand, the fact that miRNAs need partial sequence complementarity to target mRNA, and consequently could target plethora of mRNAs in a context of a network makes a hindrance to miRNA-based gene therapies, which may lead to toxic phenotype formations in unfavourable cells, off-target effects, and immune system responses.Moreover, rapid degradation of miRNAs or anti-miRNAs by cellular nucleases and poor cellular uptake are another drawback of theses clinical approaches.Therefore, using the lowest optimum concentration of miRNAs along with effective delivery systems such as viral and non-viral vectors and nanoparticles may minimize such side effects and provide dose-dependent accumulation of targeted vectors and nanoparticles in CRC cells.
Considering all together, the miRNA-based gene therapy depends mostly on both positive and negative miRNA expression manipulation.Although there are some administrative obstacles, these therapeutic tools should face a promising future.

Concluding Remarks and Future Challenges
miRNAs are attracting considerable interest and there are accumulating evidences that aberrant expression of miRNAs plays an important role in CRC development and progression by indirectly changing their plethora of cognate mRNAs, which act either as an oncogene via facilitating tumor initiation and proliferation, or in some cases as a tumor suppressor through inhibiting proliferation and invasion.Significant strengths of miRNA related research are the potential for an important complementary approach to assessing CRC risk, measurement of responses to traditional chemo-and radio-therapies and pharmacologic interventions, and as therapeutic targets for CRC risk and recurrence reduction.
Recently, miRNAs have gained substantial attention as therapeutic targets.Nevertheless, the complexity of gene networks that a single miRNA may control and the potential adverse effects of the miRNA and/or anti-miRNA in vivo deliveries remained to be deeply explored.
In summary, given the ever-expanding number of miRNAs, understanding their functional aspects represents a promising mission.Technologies that integrate RNA sequencing, proteomics, and system biology of gene network will allow a more comprehensive assessment and understanding of miRNA effects and provide exciting opportunities for new pathogenetic and treatment insights into colorectal cancer management.Novel therapeutic strategies will face the major challenge of developing standardized methods for miRNA inhibition that combine high transfection efficiency with targeted delivery.

Figure 1 .
Figure 1.Regulation of CRC-Related Pathways by miRNAs.According to Vogelstein's model Colorectal cancer pathogenesis includes sequence of changes and mutations in normal epithelium from dysplasia to carcinoma.Several miRNAs have been reported to regulate the Wnt, TGF-b, and MAPK signalling pathways, along with cell cycle and apoptosis through direct and indirect mechanisms

Table 1 . A Comprehensive List of Dysregulated miRNAs in Colorectal Cancer Tissues and Cell Lines
doi.org/10.7314/APJCP.2014.15.17.6989MicroRNAs in Colorectal Cancer: from Diagnosis to Targeted Therapies