Micro RNAs in colorectal cancer

王锡山

Introduction

In 2008 over 1．2 million new cases along with 608 700 estimated CRC-associated deaths have occurred［1］．In China，where it is now the third most common malignancy and the fifth leading cause of cancer-related death，the incidence of CRC is still increasing even with the improvements in the standard of living and changes in lifestyle．Despite improvements in diagnosis and treatment，＞52 000 patients were estimated to have died of CRC at the end of 2007，and approximately 50%of all patients diagnosed with CRC would die of metastatic disease［2］．Almost 90%CRC related deaths are due to metastasis，however the process of metastasis is still not fully understood［3］．CRC metastasis is a multistep and complex process including various stages like invasion of the surrounding tissues，vasculature intravasation，circulation translocation，distant sites parenchymal extravasation and formation of micrometastais followed by macroscopic secondary tumors［4］．Retrospective studies have investigated the genes and genes products involved in the metastasis［5］．MicroRNAs are small，endogenous，regulatory RNA molecules that modulate expression of their target genes posttranscriptionaly．Recently it has been investigated and proved the role of miRNAs in CRCmetastasis in various studies．During the process of tumor progression，the fragile chromosomal regions alter，where more than 50%of miRNA genes are situated［6］．They are evolutionary conserved molecules and may perform various physiological and pathological processes including CRC metastasis by influencing cancer stem-cell biology，angiogenesis，the epithelial-mesenchymal and mesenchymal-epithelial transition or drug resistance［7］．Aberration in the expression of miRNAs leads the progression of CRC［8］．This effect may be underlined by deletions，amplifications or point mutations of miRNA loci，epigenetic silencing，deregulation of transcription factors-modifiers of miRNA expression or inhibition of processing of primary miRNA to its mature form［9］．In addition，expression profiling analysis has revealed characteristic miRNA signatures that can predict the clinical outcomes of CRC［10］．The recent discovery of miRNAs in secreted membrane vesicles(exosomes)［11-12］，as well as in the blood serum［13-14］and other body fluids［15］suggests that miRNAs play a role in intracellular communication in a both paracrine and endocrine manner and some miRNAs have been identified as oncogenes or tumor suppressor genes in CRC［16］．It has also opened a new exciting direction for study of miRNAs as biomarkers for diseases，and cancer diagnostics by miRNA profile in blood serum has become a quickly growing field［17］．

CRC metastatic cascade and miRNAs

Metastatic cascade consists of multiple sequential steps starting from cancerous cells escaped from the primary site，followed by local invasion of surround tissues，into the circulatory or lymphatic system，and after extravasation and survival of tumor cells，proliferation and colonization into the secondary site．Chang et al［18］and others have identified the miRNAs that are associated with colorectal cancer metastasis［19］．As complex metastatic process can be broadly divided into two main stages，the first being the migration of tumor cells from their primary tumor environment to various distant tissues and the second being the colonization of these tumor cells in their new location［20］．Underlying these two main stages are a number of cellular hallmarks taking place during the development and metastasis of human tumors［21］．The various molecular，genetic，and epigenetic changes that occur define the multistep dissemination process of the tumor，also known as the“metastatic cascade”Cells．In this review we will describe the steps involved in the cascade schematically focusing particularly on miRNAs．

i)Angiogenesis:Angiogenesis，the generation of new blood vessels，is an essential physiological process that can be dysregulated in various pathological conditions，including cancer［22］．A key component of both primary and secondary(metastatic)tumor growth at any site is angiogenesis［23］．

Regulated by pro-and anti-angiogenic factors，angiogenesis，the formation of a network of blood vessels near the tumor，is essential for its growth and contact of the cancer with the bloodstream［24］．The results from recent studies indicate that miRNA may regulate angiogenesis by exerting pro-angiogenic or anti-angiogenic effects［25-26］．It seems that miRNA may exert distinct roles in CRCangiogenesis through regulation of the level of mRNA encoding inhibitors or activators of angiogenesis．Various miRNAs are involved with angiogenesis like miR-194，miR-17-92，miR-126， miR-210 and miR-424 enhancing angiogenesis while miR-221 and miR-222 inhibiting angiogenesis［27-33］．

ii)invasion:Prior to the development of frank metastatic lesions，cancer cells exhibit properties consistent with a propensity to migrate and invade into surrounding tissues and distal organs［34-35］．Cancer cell invasion from the tumor mass to adjacent tissue is an early step in tumor progression．Various cellular events are known to be associated with this increased potential for malignant cells to spread to local and distant sites including directional activation of proteolytic enzymes，including metalloproteases，degradation of extracellular matrix，transition of the cancer phenotype from epithelial to mesenchymal(EMT)，a process by which epithelial cells lose their cell to cell contacts and subsequently attain characteristics of mesenchymal phenotype，and translocation of cancer cells．The role of miRNAs in the process of cancer cell invasion has also been evaluated．Various miRNAs contributing to the process of invasion like miR-31，miR-122，miR-200 family，miR-328，miR-143，miR-145， miR-103， miR-107， miR-29a， miR-21， miR-17and miR-19a［36-44］．

iii)Intravasation，circulation and extravasation:a)Even though the exact mechanism，how a cancer cell intravastes blood/lymph vessels by crossing the basement membrane，is still elusive．Studies published recently have illustrated the connection of miRNAs with tumor growth，invasion and intravasation，even though these complex mechanisms are still not fully understood．b)Shear stress and immune system attack may result in degradation of cancer cells，as they enter the bloodstream［45］．A number of miRNAs have been discovered to play critical roles in modulation of T and B lymphocyte activation，innate and adaptive immune responses［46］．Even though the exact role played by miRNAs is still unknown but it seems that miRNAs help cancer cells to evade recognition by the immune system in the blood/lymph vessels．The escape of cancer cells from capillaries to invade the parenchyma is a step before creating cancer cell colonies in the site far from the primary tumor．Only two studies have illustrated these miRNAs regulated processes and it is hypothesized that miRNAs may influence cancer cell extravasation［47-48］．Many miRNAs regulate the processes of intrasation，circulation and extravasa-tion．They are miR-21，miR-126，miR-155，miR-17-92，miR-328，miR-26b，and miR-103/107［49-55］．

iv)colonization:Colonization to the distant secondary sites，being the final step in cancer metastasis，and circulating tumor cells or cancer cells in the bloodstream showing the affinity for the particular sites［56］is explained by the “seed and soil”hypothesis．The cancer cells is the“seed”and the specific organ microenvironment is the“soil”．Metastatic colonization of this microenvironment may be dependent on the ability of cancer cells to proliferate and to adapt to new conditions．Cancer stem cells are the subpopulation of cancer cells characterized by the ability of self-renewal and multipotency，which may aid in the establishment of distant metastases［57］．MiRNAs may regulate the pathways that are required for the phenotype of stem cells．The abnormal level of miRNA was observed in cancer stem cells compared to their non-stem counterpart．Since these miRNAs may regulate the stem-cell properties of cancer cells，they may play a role in CRC metastasis．Also miRNAs enable the colonization of CRC cells at a metastatic site，but the exact mechanism of that action is unknown［58］．

Other factors promoting metastatic cascade

i)Macrophage migration inhibitory factor(MIF)is an innate cytokine which plays a critical role in the host control of inflammation and immunity．MIF could inhibit p53 tumor suppressor activity［59］and also plays an important role in the colorectal carcinogenesis and hypoxia-induced apoptosis［60-61］．It has been analyzed through bioinformatic analysis that MIF is a potential target of miR-451．Reduction of cell proliferation and enhanced susceptibility to radiotherapy，down-regulated expression of MIF at both mRNA and protein level is related with over-expression of miR-451 in gastric cancer and CRC．The role of miR-451 as a tumor suppressor has been suggested，when biopsies of gastric tumors were observed，creating an inverse connection between miR-451 and MIF expression［62］．The role of miRNAs linking inflammation and tumorigenesis，and their close relationship with DNA methylation has also been elucidated．Methylation of CpG islands in the miR-34b/c gene was frequently observed in CRC cell lines(9 of 9，100%)and in primary CRC tumors(101 of 111，90%)instead of in normal colonic mucosa［63］．Five other miRNAs whose genes are located around/on a CpG island，were down-regulated in CRC samples．Expression of 3 of them(miR-9，miR-129 and miR-137)was restored after treating with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor in 3 CRC cell lines．Methylation of their genes was frequently observed in CRC cell lines and in primary CRCtumors instead of normal colonic mucosa［64］．These cells detach from the primary tumor site and enter into circulation which is believed to be responsible for tumor cell metastasis［65-66］．Studies have shown that detection of circulating miRNAs can be associated with clinical parameters such as relapse with metastatic disease［67］．It was reported that the expression of KRAS was inversely regulated by miR-143 in vivo［68］．It was frequently down-regulated in 87．5%(35 of 40)of CRC compared with their surrounding normal tissues and was inversely correlated with mRNA and the protein expression of DNMT3A in CRC．The tumor cell growth and soft-agar colony formation were inhibited，and the DNMT3A expression in mRNA and protein levels was down-regulated after the restoration of miR-143 expression［69］．MiRNAs have an impact on the expression of oncogenes［70］．It has been suggested that c-Myc could promote the development of cancers via miR-17-92 cluster［71］．The siRNA-mediated down-regulation of microtubuleassociated kinase(DCAMKL-1)resulted in a growth arrest of HCT116 cells，increased the level of pri-let-7a miRNA and decreased expression of Myc in tumor cell lines．It suggested that DCAMKL-1 may play a role in both stem cell differentiation and tumor growth［72］．An inverse correlation between the level of APC mRNA and miR-135 was observed in colorectal adenomas and carcinomas［73］．Human let-7a miRNA expression was significantly reduced in two of eight human CRC samples based on stem-loop qRT-PCR［74］．A low level of let-7a could activate the signal pathway including c-Myc and KRAS and lead to the formation and development of CRC．Meanwhile，a high level of let-7a could inhibit the drug-induced apoptosis of CRC cells．Cyclooxygenase-2(COX-2)，which has an important effect on the growth and invasiveness of CRC cells，was inversely correlated with miR-101 expression in colon cancer cell lines and translation of COX-2 mRNA was directly inhibited by miR-101 in vitro．

Conclusion

Although miRNAS have crucial role throughout the metastatic cascade and an important role the diagnosis and treatment of CRC，further large-scale evaluation in multiple independent cohorts is indispensable for determining their realistic expectation．And there is still need to find more specific bio-markers to diagnose CRC in the early stages and to limit metastases．

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