Cloning And Preliminary Analysis Of MicroRNA Let-7a-1/7f-1Promoter In Human Lung Cancer Cells

BackgroundmicroRNAs(miRNAs)are a class of small non-coding RNAs of about22nueleotides,which are found in diverse organisms. By perfect or imperfect complementary binding to the untranslated regions(3’-UTRs) of the target mRNA, miRNAs can induce RNA degradation and/or interfere with translation, and thus play key roles in down-regulating gene expression at the post-transcription level.They have been demonstrated to play major roles in a wide range of biological processes, such as development,cell growth, differentiation and apoptosis,etc.And concordant with this, impaired miRNA expression are found to be implicated in various human diseases and cancers. Study showed that about50%of annotated human miRNAs are located in areas of the genome, known as fragile sites, that are associated with cancer. And some tissue-specific changes in RNA expression profiles can be found in breast cancers, colorecta cancers,lung cancers, pituitary tumors and glial cell tumors.These all indicate the crucial functions of microRNAs in tumorgenesis and tumor development. microRNAs can act as either oncogenes or tumor suppressors in tumorgenesis and tumor progression. Studies on miRNAs can contribute to elucidating the molecular mechanisms of human tumors and are expected to provide new targets for human cancer diagnosis and treatment.has-let-7is the first known miRNA in human and was found to be expressed in various human tissues, with the lung being the organ having the most abundant expression. Takamizawa etal found that the expression of has-let-7was frequently reduced in lung cancers both in vitro and in vivo. Furthermore, lung cancer patients with lower level of has-let-7expression were found to have significantly worse prognosis after potentially curative resection, and in vitro,however,the over expression of let-7inhibited the growth of lung cancer cells.These indicate the role of let-7as a tumor suppressor gene in human lung carcinogenesis and the study on it can help us find a new target for gene therapy and drug treatment of human lung cancer.However, the underlying molecular mechanisms of let-7reduction in lung cancer are not well understood. The qRT-PCR results from Takamizawa and Yanaihara etal. both have shown the significant down-regulation of pri-let-7in lung cancer tissues and cells, suggesting the inhibition on transcription level maybe one of the mechanisms for let-7expression reduction in lung cancer. But our understanding of let-7transcription pattern in lung cancer cells is still limited. Thus, cloning and characterization of the5’flanking region of let-7a-l and let-7f-l, which are the most abundant species of let-7family and clustered within a few hundred bases in the human genome may well be a start point for elucidating the transcription regulation mechanism of let-7and its function in human lung cancer.ObjectiveTo determine the transcripton start site of let-7a-1/7f-1gene cluster and clone its promoter and then to do the preliminary analysis of the promoter so as to help elucidate the transcriptional molecular basis of microRNA let-7a-1/7f-1gene cluster in human lung cancer.MethodsFirstly,5’RACE was carried out to identify the transcriptional start site of let-7a-1/7f-1gene cluster, and then a2.1kb fragment of its5’flanking region(-1999bp/+124bp) was cloned into PGL3-basic vector to construct the pGL3-2123recombinant and test the promoter activity in A549cells. A549cells were transfected by the constructed pGL3-2123vector and then treated for48h with ATRA,9cRA,1,25(OH)2D3or DEX, respectively. After the completion of the treatment procedure, these cells underwent the dual-luciferase reporter assay, following the protocol recommended by Promega. For co-transfection experiments, pGL3-2123was transfected into A549cells along with one of the eukaryotic expression plamids pCMV-p53,pcDNA3.1(+)-Sp1and pcDNA3.1(+)-NFκBp50, pcDNA3.1(+)-PPRγ2,pcDNA3.1(+)-c/EBPα, pBABEpuro-ras or pBABE hygro-myc.Then all the cells underwent the dual-luciferase reporter assay as well as above48h after the completion of the transfection procedure.ResultsThe transcriptional start site of let-7a-1/7f-1gene cluster was identified by5’RACE.The pGL3-2123recombinant formed by inserting2.1kb-promoter fragment upstream of let-7a-1/7f-1gene into PGL3-basic vector was proved to be correct through the restriction enzyme digestion and DNA sequencing. The M1/M2value of dual-luciferase reporter assay was5.24at48hours after pGL3-2123was transfected into A549lung cancer cells with pRL-TK and that value of pGL3-control and pGL3-basic was38.76and0.26,respectively. The promoter activity of let-7a-1/7f-1gene cluster could be increased by dexamethasone, whereas treatment with9-cis-retinoic acid, all-trans retinoic acid or1,25-(OH)2D3resulted in little induction of the2.1kb promoter. Ectopic expression of either c/EBPa or p53could enhance the transcription level of let-7a-1/7f-1, while none of the PPARy2, NF-KappaB, Spl, ras and myc had significant effect on the promoter activityConclusionThe transcriptional start site of let-7a-1/7f-1gene cluster was determined, and the let-7a-1/7f-1promoter-luciferase reporter construct (pGL3-2123) was successfully cloned.The2.1kb-promoter of let-7a-1/7f-1proved to have a lower activity in A549lung cell line, which was consistent with that let-7a-1/7f-1was downregulated in A549cells,and meanwhile the promoter activity could be significantly enhanced by ectopic expression of c/EBPa or p53, and treatment with dexamethasone.The finding of this study provides a foundation for further study on identifying the functional responsive cis-elements within the let-7a-1/7f-1promoter and elucidating regulatory mechanisms of them.