Abstract
BackgroundMicroRNAs are the endogenous small non-coding RNA molecules capable of silencing protein coding genes at the posttranscriptional level. Based on computer-aided predictions, a single microRNA could have over a hundred of targets. On the other hand, a single protein-coding gene could be targeted by many potential microRNAs. However, only a relatively small number of these predicted microRNA/mRNA interactions are experimentally validated, and no systematic validation has been carried out using a reporter system.MethodsIn this study, we used luciferease reporter assays to validate microRNAs that can silence cyclin D1 (CCND1) because CCND1 is a well known proto-oncogene implicated in a variety of types of cancers. We chose miRanda http://www.microRNA.org as a primary prediction method. We then cloned 51 of 58 predicted microRNA precursors into pCDH-CMV-MCS-EF1-copGFP and tested for their effect on the luciferase reporter carrying the 3'-untranslated region (UTR) of CCND1 gene.ResultsReal-time PCR revealed the 45 of 51 cloned microRNA precursors expressed a relatively high level of the exogenous microRNAs which were used in our validation experiments. By an arbitrary cutoff of 35% reduction, we identified 7 microRNAs that were able to suppress Luc-CCND1-UTR activity. Among them, 4 of them were previously validated targets and the rest 3 microRNAs were validated to be positive in this study. Of interest, we found that miR-503 not only suppressed the luciferase activity, but also suppressed the endogenous CCND1 both at protein and mRNA levels. Furthermore, we showed that miR-503 was able to reduce S phase cell populations and caused cell growth inhibition, suggesting that miR-503 may be a putative tumor suppressor.ConclusionThis study provides a more comprehensive picture of microRNA/CCND1 interactions and it further demonstrates the importance of experimental target validation.
Highlights
MicroRNAs are the endogenous small non-coding RNA molecules capable of silencing protein coding genes at the posttranscriptional level
Having demonstrated ectopic expression of these microRNAs and construction of cyclin D1 (CCND1)-3'-untranslated region (UTR) luciferease reporters, we introduced each of microRNAs listed in Table 2 into 293T cells along with Luc-CCND1-3'-untranslated region (3'-UTR)-1 or Luc-CCND1-3'UTR-2
Our study suggests that the ratio of true target/predicted target is relatively low in our system, and further suggest the complexity of the microRNA/mRNA interactions
Summary
MicroRNAs are the endogenous small non-coding RNA molecules capable of silencing protein coding genes at the posttranscriptional level. Based on computer-aided predictions, a single microRNA could have over a hundred of targets. A single protein-coding gene could be targeted by many potential microRNAs. only a relatively small number of these predicted microRNA/mRNA interactions are experimentally validated, and no systematic validation has been carried out using a reporter system. The overall microRNA expression profile of normal tissues is distinct from that of tumor tissues [7] and ,. MicroRNAs can function as tumor suppressors or oncogenes depending on whether they target oncogenes or tumor suppressor genes [9,10,11]. Tumor suppressive microRNAs such as let-7, miR-15 and miR-16 are downregulated or deleted in lung cancer and leukemia [11,12,13]. Oncogenic microRNAs, such as miR-21 and miR-155, are overexpressed in tumors or tumor cell lines [7,14,15]
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