**2. The need for improved biomarkers**

The survival and prognosis of patients suffering from CRC depends on the stage of the tumour at time of detection. "Five year survival" significantly reduces from 93% for localized early cancerous lesions (Dukes A) to < 15% for advanced metastatic cancers (Dukes D). Unfortunately, approximately one third of patients with CRC have regional or distant spread of their disease at time of diagnosis (Ferlay, et al, 2008). Currently, bowel

MicroRNAs are Novel Biomarkers for Detection of Colorectal Cancer 3

target genes by directing specific messenger RNA cleavage or translational inhibition through the RNA induced silencing complex (RISC) (Bartel, et al, 2004 & 2009). So far around 1400 mature human miRNAs have been described in the Sanger miRBase version 17 (An international registry and database for miRNA nomenclature, targets, functions and their implications in different diseases). In the database, each mature miRNA in human and non-human species is assigned a unique identifier number for universal standardization. For example human microRNA 21 is designated as hsa-miR-21. Table 1 summarizes the

**Mechanism of Action Function** 

information from DNA to

RNA interference and RNA interference related

formation with piwi

(RNP) complexes to guide the enzymatic modification of target RNAs at sites determined by RNA:RNA antisense interactions

Transfers a specific active amino acid to a growing polypeptide chain at the ribosomal site of Protein

MiRNAs are mostly transcribed from intragenic or intergenic regions by RNA polymerase II into primary transcripts (pri-miRNAs) of variable length (1 kb- 3 kb). In the nucleus PrimiRNA transcript is further processed by the nuclear ribo-nuclease enzyme 'Drosha' thereby resulting in a hairpin intermediate of about 70–100 nucleotides, called pre-miRNA. The pre-miRNA is then transported out of the nucleus by a transporting protein exportin-5.

Translational Inhibition

Interference with gene

Transcriptional gene

retrotransposons and other genetic elements in germ

Chemical modifications of

Amino acid carriers and protein synthesis during

Protein synthesis in

Protein synthesis

expression

silencing of

line cells

other RNAs e,g methylation, pseudouridylation

translation.

ribosomes

different types of RNAs by size, mechanism of action and function in human cells.

17-23 RNA induced silencing complex (RISC)

the ribosomes

pathways

26-31 RNA-protein complex

proteins

70-200 Act as ribonucleoprotein

120-5050 Decode mRNA into amino

acids

900-1500 Conveys genetic

**Types of Non Coding RNA** 

MicroRNA (miRNA)

(mRNA)

Messanger RNA

Small interfering RNA (SiRNA)

Piwi-interacting RNA (piRNA)

Small Nucleolar RNA (SnoRNA)

Transfer RNA (tRNAs)

Ribosomal RNA

( rRNA)

Table 1.

**Size No of Nucleotides**

20-25 Double stranded

73 to 93 Clover Leaf

**5. MicroRNA biogenesis in human cells** 

cancer screening programmes in Europe use either flexible sigmoidoscopy (FS) or guaiacbased faecal occult blood testing (FOBT) as the primary screening tool, with the current gold standard colonic imaging modality of colonoscopy being reserved for patients testing positive. Both primary screening tests have proven to be of benefit in reducing the death rate from CRC in randomised controlled trials but are generally considered to lack the desired convenience or accuracy for use as a general screening test (Hewitson, et al, 2007). A comparative study of diagnostic sensitivities of FOBT, faecal immunochemical stool testing (FIT), flexible sigmoidoscopy (FS), colonoscopy and CT colonography (CTC) has revealed 20%, 32%, 83.3% 100% and 96.7% sensitivity, respectively for the detection of CRC and advanced adenomas (Graser, et al, 2009) . Endoscopic and radiological diagnostic modalities are expensive and are associated with risks such as bleeding, infection, bowel perforation and exposure to radiation. This explains why there is still a need for an improved, reliable, accurate and non-invasive biomarker for colorectal cancer detection.

### **3. Colorectal cancer development**

The development of CRC follows the sequential progression from adenoma to the carcinoma (Vogelstein, et al, 1988). Carcinogenesis pathways for colorectal neoplasia have become much clearer and precise in the past two decades. The common pathway for CRC development is dependent on Adenomatous Polyposis Coli (APC) & Tumour Protein-53 (TP53) gene mutations and is initiated through WNT signalling (Segditsas, et al, 2006). In this pathway colonic carcinoma originates from the colonic epithelium as a consequence of accumulation of genetic alterations in the tumour suppressor gene TP53 and oncogenic APC genes. The initial genetic alterations result in adenoma formation in which cells exhibit autonomous growth. During the further course of carcinogenesis, intestinal epithelial cells acquire the characteristics of invasion and the potential for metastasis. Another carcinogenesis pathway has recently gained acceptance and is commonly named as the serrated-neoplasia pathway. This pathway is for the most part APC and TP53 independent and shows distinct molecular features of somatic mutations such as BRAF mutation and concordance with high CpG islands methylation phenotype (CIMP-H), microsatellite instability (MSI+) and MutT homologue 1 (MLH1) methylation (Casey, et al, 2005 & Spring, et al, 2006,) . Sequential progression of colorectal neoplasia from adenoma to carcinoma highlights that opportunities exist to improve cancer specific survival by altering the natural course of disease development. Such interventions could potentially be chemo preventive for high risk individuals, the early detection of colorectal neoplasia, chemotherapy to down stage the cancer prior to surgical resection and therapy for palliation of symptoms in advanced stage cancer. Recent advances in proteomics and genomics provide a vast amount of information about the role of micro-molecules in several cancer related pathways. These advances have focused on the detection of micro molecules released from tumour cells and their utility as diagnostic biomarkers. The discovery of tumour specific microRNAs (miRNAs) has opened a new era of biomarker research that holds great potential for future cancer detection strategies.

#### **4. What are MicroRNAs**

MicroRNAs are single-stranded, evolutionarily conserved, small (17–25 ribonucleotides) noncoding (Lee, et al, 1993) RNA molecules. MiRNAs function as negative regulators of target genes by directing specific messenger RNA cleavage or translational inhibition through the RNA induced silencing complex (RISC) (Bartel, et al, 2004 & 2009). So far around 1400 mature human miRNAs have been described in the Sanger miRBase version 17 (An international registry and database for miRNA nomenclature, targets, functions and their implications in different diseases). In the database, each mature miRNA in human and non-human species is assigned a unique identifier number for universal standardization. For example human microRNA 21 is designated as hsa-miR-21. Table 1 summarizes the different types of RNAs by size, mechanism of action and function in human cells.


Table 1.

2 Biomarker

cancer screening programmes in Europe use either flexible sigmoidoscopy (FS) or guaiacbased faecal occult blood testing (FOBT) as the primary screening tool, with the current gold standard colonic imaging modality of colonoscopy being reserved for patients testing positive. Both primary screening tests have proven to be of benefit in reducing the death rate from CRC in randomised controlled trials but are generally considered to lack the desired convenience or accuracy for use as a general screening test (Hewitson, et al, 2007). A comparative study of diagnostic sensitivities of FOBT, faecal immunochemical stool testing (FIT), flexible sigmoidoscopy (FS), colonoscopy and CT colonography (CTC) has revealed 20%, 32%, 83.3% 100% and 96.7% sensitivity, respectively for the detection of CRC and advanced adenomas (Graser, et al, 2009) . Endoscopic and radiological diagnostic modalities are expensive and are associated with risks such as bleeding, infection, bowel perforation and exposure to radiation. This explains why there is still a need for an improved, reliable,

The development of CRC follows the sequential progression from adenoma to the carcinoma (Vogelstein, et al, 1988). Carcinogenesis pathways for colorectal neoplasia have become much clearer and precise in the past two decades. The common pathway for CRC development is dependent on Adenomatous Polyposis Coli (APC) & Tumour Protein-53 (TP53) gene mutations and is initiated through WNT signalling (Segditsas, et al, 2006). In this pathway colonic carcinoma originates from the colonic epithelium as a consequence of accumulation of genetic alterations in the tumour suppressor gene TP53 and oncogenic APC genes. The initial genetic alterations result in adenoma formation in which cells exhibit autonomous growth. During the further course of carcinogenesis, intestinal epithelial cells acquire the characteristics of invasion and the potential for metastasis. Another carcinogenesis pathway has recently gained acceptance and is commonly named as the serrated-neoplasia pathway. This pathway is for the most part APC and TP53 independent and shows distinct molecular features of somatic mutations such as BRAF mutation and concordance with high CpG islands methylation phenotype (CIMP-H), microsatellite instability (MSI+) and MutT homologue 1 (MLH1) methylation (Casey, et al, 2005 & Spring, et al, 2006,) . Sequential progression of colorectal neoplasia from adenoma to carcinoma highlights that opportunities exist to improve cancer specific survival by altering the natural course of disease development. Such interventions could potentially be chemo preventive for high risk individuals, the early detection of colorectal neoplasia, chemotherapy to down stage the cancer prior to surgical resection and therapy for palliation of symptoms in advanced stage cancer. Recent advances in proteomics and genomics provide a vast amount of information about the role of micro-molecules in several cancer related pathways. These advances have focused on the detection of micro molecules released from tumour cells and their utility as diagnostic biomarkers. The discovery of tumour specific microRNAs (miRNAs) has opened a new era of biomarker research that holds great potential for future

MicroRNAs are single-stranded, evolutionarily conserved, small (17–25 ribonucleotides) noncoding (Lee, et al, 1993) RNA molecules. MiRNAs function as negative regulators of

accurate and non-invasive biomarker for colorectal cancer detection.

**3. Colorectal cancer development** 

cancer detection strategies.

**4. What are MicroRNAs** 
