| Colorectal carcinoma (CRC) is one of the leading causes of cancer-related death worldwide with an estimated one million new cases and a half million deaths each year. The CRC incidence and mortality in China increase rapidly in the past several decades. Screening for CRC allows early-stage diagnosis of the malignancy and potentially reduces mortality of the disease. New targeted therapies directed against molecules involved in the pathogenesis of CRC were recently reported as safe and effective. With the advent of new chemotherapeutic agents, such as angiogenesis inhibitor and TGF-a inhibitors, there is growing interest to identify new prognostic biomarkers and therapeutic targets for this disease.MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs (18-25nt), which regulate gene expression by translational inhibition or direct cleavage of target mRNAs. The miRNAs play important roles in the development, differentiation and function of various cell types, and in the pathogenesis of various human diseases, including cancer. Currently, more than800human miRNAs have been identified and registered in the miRNA database, miRBase. Strikingly, about30%of protein-coding human transcripts are predicted to be regulated by these miRNAs. However, in spite of the discovery of various miRNA targets implicated in human cancer, most mechanisms by which miRNAs are involved in tumorigenesis remain to be elucidated. Indeed, the identification of cancer-specific miRNA targets is critical for understanding the functional role of miRNAs in carcinogenesis and may be important for defining novel therapeutic molecules. The relationship between miRNAs and target mRNAs is not a "one to one" connection because the same mRNA can be regulated by more than one miRNA, and the choice of how many and which miRNAs target an individual3-untranslated region (UTR) is largely determined by the specific cellular environment. The development of reagents that can strongly suppress specific miRNAs has also generated much interest and will be important for both basic miRNA research and possible therapeutic strategies.MiR-338-3p was a recently discovered miRNA and was involved in cell differentiation. Although miR-338-3p is known to be specifically expressed in neuronal tissue, little is known about its abundance and function during carcinogenesis in certain cancers. We have found miR-338-3p was down-regulated in several CRC samples compared with adjacent non-tumorous tissues in our previous study, suggesting that miR-338-3p might act as tumor suppressor in CRC, however, the targets it regulated in CRC have not been reported. Smoothened (SMO), a protein that is related to G-protein-coupled receptors, is the key activator of Hedgehog (Hh) signaling pathway. Up-regulation of SMO in CRC was shown to correlate with a higher biological aggressiveness, advanced stage, poor differentiation, larger size, and high proliferative activity. Furthermore, it is also well known that SMO regulation, both in physiological and pathological conditions, is exerted mostly at a post-transcriptional level. Moreover, with the application of bio informatics predictions, we found that miR-338-3p and SMO mRNA3-UTR had complementary binding sites. Thus, we inferred that the non coding RNA miR-338-3p acts as a local regulator of SMO by binding to the3-UTR of its mRNA, thereby modulating CRC development.To confirm the molecular mechanism of miR-338-3p in CRC, it is necessary to observe the biological effects of the up-and down-regulation of miR-338-3p. We will construct a lentiviral vector, pLV-THM-miR-338-3p, and over-express this vector in the CRC-derived cell line, SW-620, in this study. Moreover, we designed a miR-338-3p inhibitor that is flanked by sequences that form8bp hairpin structures in conjunction with an miRNA binding site (MBS) with a3-nt linker on both sides. We will design an inhibitor with a combination of these two structures to establish a novel and potent miR-338-3p inhibitor based on a lentiviral vector. These provide the basis for further studies regarding the molecular function of miR-338-3p in CRC. We aimed to reveal a new regulating mechanism of miR-338-3p in the development of CRC, and provide a new miRNA and target gene for the clinical application.Part Ⅰ Construction of lentivirus-mediated microRNA-338-3p and its inhibitorAim:To construct a lentivirus-based hsa-microRNA-338-3p (miR-338-3p) and its inhibitor.Methods:The miR-338-3p and its inhibitor sequences were artificially synthesized and successfully inserted into the pLV-THM plasmid. Restriction endonuclease analysis and DNA sequencing confirmed the sequence of the recombinant plasmid pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor. HEK-293T cells were co-transfected with the lentiviral vectors pLV-THM-miR-338-3p or pLV-THM-miR-338-3p-inhibitor, psPAX2and pMD2.G. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and this supernatant was then used to infect SW-620cells. Flow cytometry was utilized for sorting the eGFP(+) cells. The expression of miR-338-3p was determined by real-time RT-PCR.Results:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were successfully constructed. The expression of miR-338-3p in SW-620cells infected with the lentivirus pLV-THM-miR-338-3p was significantly increased. Moreover, the expression of miR-338-3p in SW-620cells infected pLV-THM-miR-338-3p-inhibitor was significantly decreased.Conclusions:The successful construction of the lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor provide the basis for further studies regarding the molecular function of miR-338-3p in colorectal carcinoma. Part Ⅱ Suppression of cell migration by microRNA-338-3p in colorectal carcinomaAim:To investigate the suppressive effect of cell migration by microRNA-338-3p (miR-338-3p) in colorectal carcinoma (CRC).Methods:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully as described in part Ⅰ. The lentivirus particles were then used to infect CRC-derived cell line, SW-620. The expression of miR-338-3p was determined by real-time RT-PCR, and Western blot analysis was used to detect the expression of the smoothened (SMO) protein in SW-620cells. Additionally, the migration ability of the transfected SW-620cells was assessed by transwell assay.Results:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were successfully constructed. The expression of miR-338-3p in SW-620cells infected with the lentivirus pLV-THM-miR-338-3p was significantly increased. Furthermore, over-expression of miR-338-3p suppressed the expression of SMO protein in SW-620cells, which showed obviously suppressed invasive ability in transwell assay. The expression of miR-338-3p in SW-620cells infected with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased. Moreover, the down-regulated expression of miR-338-3p caused the up-regulated expression of the SMO protein in SW-620cells, which showed significantly enhanced migration in transwell assay.Conclusions:MiR-338-3p may suppress SMO gene expression and thereby inhibit CRC migration. |
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