The Regulating Function Of MiR-143/miR-145 In Colorectal Cancer Development

Background: Recently,a highlighted research field is regarding a kind of non-coding RNAs,the microRNAs(miRNAs).They are small RNAs of 20? 2 nucleotides,typically functioning as post-transcriptional gene regulators,miRNA genes are transcribed by RNA poly-merase(?),forming long? up to several kb? primary miRNA(pri-miRNAs). These pri-miRNAs contain one or more stem(?)oop or hairpin structures(～70 nucleotides) that are recognized and cleaved by a ds-RNA specific RNaseⅢendonuclease Drosha.The resulting precursor miRNA(pre-miRNA) is then shuttled to the cytoplasm by the nuclear export factor Exportin-5.In the cytoplasm,a second endonucleolytic cleavage occurs,mediated a second RNaseⅢendonuclease Dicer, yielding a 20? 5 nucleotide imperfect miRNA duplex,which in association with its RNA binding partner TRBP.generating a double-stranded RNA that is recruited to the RNA-induced silencing complex(RISC).After degradation into single strand,mature miRNA exhibit a partial complementarity in binding the 3(?)ntranslated region (3(?)TR) of target mRNAs,leading to translational repression and/or degradation of target mRNA.In this way miRNA takes part in individual development and metabolism by playing an important role in cells proliferation,differentiation and apoptosis.Cancer is a complex genetic disease involving structural and expression abnormalities of both coding and non-coding genes.Initially identified in B-cell chronic lymphocytic leukaemia(CLL),changes in the expression level of miRNAs have subsequently been detected by different groups in many types of human tumours, including breast cancer,glioma,lung cancer,colorectal cancer,gastric cancer,liver cancer and pancreatic cancer,miRNAs have been proposed to contribute to oncogenesis because they can function either as tumor suppressors or oncogenes.The genomic abnormalities found to influence the activity of miRNAs are the same as those previously described for protein-coding genes,such as chromosomal rearrangements,genomic amplifications or deletions,mutations and epigenetic changes.Colorectal cancer accounts for the 3rd most common cancer and one of the most fatal diseases.Colorectal cancer is associated with altered expression of miRNAs,which play a very important role in oncogenesis and cancer development.The expression of miR-143,miR-145,miR-34a and let-7 was reported to be consistently reduced in colorectal cancer.Later,they were proved to be the regulating molecules of this cancer by targeting mRNA of ERK5,Ras,c-myc and E2F respectively,functioning as tumor suppressors.Thus,Looking for more colorectal cancer-related miRNAs and elucidating their function mechanisms will be great help to molecular research of colorectai cancer.These altered expression of related miRNAs may be further used to as indices for early diagnosis,therapy evaluations and new drugs development. Our research is based on former preliminary research results,with further study on miRNAs tightly related with oncogenesis and development of colorectal cancers.We successfully identified miR-143/miR-145 as very important regulators of colorectal cancer growth,by suppressing the expression of unreported targets ErbB3.Nras. And the results were further certification by several molecular and cell biology methods,our conclusions will be a great help to further research of this kind of cancer.Objectives:①Retrieval and certification of miRNAs that are tightly related to the development of colorectal cancer.②Elucidation the biological effects on colorectal cancer cells exerted by these miRNAs.③Predicting the targets of these miRNAs with bioinformatics and knowledge of colorectal cancer molecular biology.④Verification of the predicting results with tests.And demonstration the function mechanisms,by which these miRNAs influence colorectal cancer biology.⑤Enrichment the theory of colorectal cancer etiology and molecular pathology;Providing new ways to colorectal cancer diagnosis and treatments.Results:①According to previous reports,we found the expression changes of several miRNAs were related to the development of colorectal cancers tightly,expression of some was enhanced in cell lines and tissues constantly and obviously,such as miR-21; others were greatly reduced,such as miR-143 and miR-145.②With Northern blot and miRNA sequencing to detect the expression of miR-143 and miR-145 in tumor tissues and cell lines,simultaneous reduced expression of them was found,compared to normal colorectal mucosal tissues.③Pre-miR-143 and Pre-miR-145 were synthesized and transfected into colorectal cells.Analysis with Flow cytometry indicated that miR-143 can reduce cancer cells proliferation:miR-145 can promote cancer cell apoptosis.④With the help of KeyTar,a targets predicting software of miRNAs,and the knowledge of colorectal molecular biology,we found that the targets for miR-143 are ErbB3 and K-ras in colorectal cancer,and N-ras for miR-145.⑤Results of luciferase assay and mutant tests verified the predicting results.⑥Western blot indicated that miR-143 functions as gene repressor of ErbB3 and K-ras;miR-145 as repressor of N-ras.Conclusions:①Expression of miR-143 and miR-145 in clinical colorectal cancer tissues and cell lines was simultanously reduced,which is the same as reported before.②miR-143 and miR-145 act as colorectal cancer cells proliferation inhibitor and apoptosis promoter respectively,which indicating that these two miRNAs work as tumor suppressors in colorectal cancer development.③miR-143 exerts its biological function on colorectal cancer cells through inhibiting the expression of ErbB3 and Kras genes;miR-145 through Nras gene.④The gene locations of miR-143 and miR-145 on chromosome are very near to each other,constituting a cluster,which accounts for their simultaneous expression change in tumor tissues and cell lines.⑤Their targets of ErbB3,K-ras,N-ras are very important components of growth factor signaling pathway,thus miR-143/miR-145 cluster functions as tumor repressor through inhibiting this signaling pathway.