| microRNAs (miRNAs) are a class of small non-coding RNAs of 21 to 25 nucleotides. They bind to the 3'- untranslated regions (3'-UTRs) of target genes and then the binding event causes translational repression of the target gene or stimulates rapid degradation of the target transcript, so they play key roles in regulation of gene expression. miRNAs were found both in animals and plants, and they correlated with various cancers closely. Most of known miRNA located at genomic regions involved in cancers. Some tissue-specific changes in miRNA expression profiles can be found in colorectal cancer, lung cancer, breast cancer, glial cell tumors, pituitary tumors.Lung cancer is the common cancer in the world. Takamizawa et al found that the expression of let-7 was frequently reduced in lung cancers both in vitro and in vivo. Furthermore, lung cancer patients with reduced let-7 expression were found to have significantly worse prognosis after potentially curative resection, but the overexpression of let-7 inhibited growth of lung cancer cells in vitro. let-7 may play a major role in human lung carcinogenesis as a tumor suppressor gene. let-7α2 is a member of let-7 family, and located on chromosome 11q24.1.1,25-dihydroxyvitamin D3(1,25-(OH)2D3) is the active metabolite of vitamin D. It regulates cell differentiation and proliferation. The actions of 1,25-(OH)2 D3 are mediated by the nuclear vitamin D receptor (VDR). Human VDR gene maps to chromosome 12q13.11 and the full length of human VDR gene is about 4669bp. VDR is the ligand-dependent transcriptional factor.1,25-(OH)2D3 activates VDR to form complex of VDR/RXR/accessory transcription factor, then the complex binds to VDR responsive element(VDRE) of target gene to affect the cell growth, differentiation, apoptosis mediated by RNA polymeraseⅡtranscription of certain genes.1,25-(OH)2 D3 could reduce the growth of lung cancer cell lines and induce cell apoptosis. The mRNA expression of VDR was decreased in lung cancer. The change of expression and function of VDR will influence the celluar responses to 1,25-(OH)2D3, and this action will influence regulation of cell growth, differentiation and apoptosis by 1,25-(OH)2D3.So far, little is known about the role and the regulatory mechanisms of let-7α2 expression in lung cancer. In the present study, we found that 1,25-(OH)2D3 could upregulate let-7α2 gene expression in transcriptional level in A549 cancer cells. Our work is focused on the regulatory mechanisms of let-7α2 expression by 1,25-(OH)2 D3 at transcriptional level in lung cancer. (1) Firstly,5'RACE was carried out to identify the transcriptional start site of let-7α2 gene, then a 2.8 kb fragment of its 5'flanking region(+336 bp to-2523 bp) was cloned into pGL3-basic vector to construct pGL3-p7a2 recombinant and test its promoter activity in A549 cells. (2) A series of transfection and luciferase reporter assay were carried out to test its promoter activity, as well the RT-PCR and transfection of let-7α2 target sequence-reporter plasmid to detect transcriptional level of let-7α2 gene in A549 treated with 9-cis-RA, AT-RA, LiCl, Dex or 1,25-(OH)2 D3. (3) The VDR responsive element (VDRE)-green fluorescence protein(GFP) reporter plasmid was constructed and transfected into A549 cells treated with 1,25-(OH)2 D3, then the expression of GFP was detected. 1,25-(OH)2 D3 activated VDR to bind VDRE in let-7α2 promoter region and then enhanced the expression of reporter gene. (4) EMSA and ChIP were carried out to investigate the binding activities of VDRE with transcription factor VDR. (5) The correlation between let-7α2 and VDR expression was detected in 18 lung cancer specimens by RT-PCR. (6) The recombinant eukaryotic expression plasmid of let-7α2 was constructed and expressed in lung cancer A549 cells. The effect of let-7α2 on A549 cell proliferation and apoptosis was tested by MTT and flow cytometry analysis.The results showed that:(1) The transcriptional start site of let-7α2 was identified by 5'RACE. A 2.8 kb fragment of its 5'-flanking region was cloned into pGL3-basic vector to construct pGL3-p7a2 recombinant plasid which could represent a promoter activity. (2) The promoter activity could be enchanced by 9-cis-RA, AT-RA or 1,25-(OH)2D3 treatment and down-regulated by LiCl treatment or CEBPαtransfection. (3) The expression of GFP in recombinant plasmid of VDRE increased with the treatment of 1,25-(OH)2 D3 than that in control cells. These results indicated that 1,25-(OH)2 D3 could enhance the binding of VDRE in let-7α2 promoter region with VDR. (4) The functional VDRE upstream of let-7α2 gene (-1730 bp～-1706 bp) were identified by EMSA and ChIP, which were involved in the positive regulation of let-7α2 gene expression by 1,25-(OH)2D3. (5) The expression of let-7α2 with VDR was closely correlated in lung cancer specimens, the expression of let-7α2 and VDR mRNA were simultaneously down-expressed acocounting for about 44%(8 out of 18) in lung cancer specimens and normal lung tissue, and positive correlation of VDR and let-7α2 expression was about 67%(12 out of 18). (6) The eukaryotic expression plasmid of let-7α2 is successfully constructed and effectively expressed in A549 cells. let-7α2 could inhibit the proliferation of A549 cells and induce their apoptosis..In summary, our research suggested that let-7α2 were related to lung cancer closely. The expression of let-7α2 was possibly regulated by retinoid acid,1,25-(OH)2 D3, lithium choride and transcriptional factor CEBPαin lung cancer cell A549. The positive regulation of let-7α2 gene by 1,25-(OH)2D3 was mediated by the binding of VDR and VDRE upstream of let-7α2 promoter. Our findings will be the foundation for further study in the action of let-7α2 and 1,25-(OH)2 D3 on lung cacer.
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