| Background:Cancer metastasis remains a leading cause of treatment failure for cancer patients despite advancements in our understanding of this complex process. Exploration and characterization of genes involved in the initial steps of metastasis, including cell migration and invasion, could lead to novel targets for retarding cancer dissemination.MicroRNAs (miRNAs) are small, highly conserved non-coding RNAs that have been reported to participate in the metastatic process by negatively or positively regulating the expression of metastasis associated genes through posttranscriptional repression, mRNA degradation, or promoter activation. MiR-744 is identified as a tumor related gene recently, and its dysregulation was found in the serum or specimens from patients with head and neck cancers, multiple myeloma, gastric cancer, hepatocellular carcinoma, or breast cancer. We previously also reported that miR-744 expression was upregulated in NPC specimens and miR-744 upregulation was significantly associated with NPC progression. Furthermore, we confirmed for the first time that miR-744 involves in the complex process of cancer metastasis as a proto-oncogene. However, it has never been reported about the regulatory mechanism of miR-744 expression dysregulation in tumors. Only Sanchez-Jimenez C et al. observed the increase of miR-744 expression in HeLa cells with transient depletion of T-cell intracellular antigen (TIA)-proteins which function as regulators of cell homeostasis.Several mechanisms can control miRNAs expression (10), such as structural genetic alterations, defects in the miRNAs biogenesis machinery, epigenetic changes and so on. An altered transcription factor activity is also one of the important regulatory mechanisms for the deregulation of miRNAs expression. For instance, myogenic transcription factor MyoD could directly bind the miR-182 promoter to increase miR-182 expression hence contribute to cancer progression Oncoprotein MYC is able to both positively regulate transcription of oncogenic miR-17-92 cluster and directly repress tumor suppressors let-7 and miR-29 family members transcription.Objective::By using bioinformatics prediction technology,we predicated that there existed six transcription factor binding sites in the upstream promoter regions of miR-744 to c-Jun.Using qPCR we validated the transcription binding sites and the correlation of mir-744 expression and c-Jun, With immune coprecipitation of chromatin (CHIP) technology we determined the direct binding sites of the transcription factors and mir-744, and we used dual luciferase report experiment for further verification. Finally, in nasopharyngeal carcinoma cell line 5-8 F,HONE1 and non-samll-cell lung cancer cell line SPC-A-1, A549,we explorated the common mechanism of c-Jun and miR-744 in tumor cell migration and invasion.Then finally we determined the regulation of transcription factors to miR-744 in promoting cancer gene expression and their mechanism Since the-500 to-1 bp region was validated to be the key promoter region of miR-744 and be responsive to c-Jun, furthermore, the interaction of c-jun with the putative binding motif in the-343 to-349 bp region (Site 4) upstream of miR-744 gene was demonstrated by a ChIP analysis of c-Jun-treated A549 cells, this binding site was chosen for further experiment to validate promoter activation of miR-744 by c-Jun through directly binding to its promoter.The identified TF-binding site,the key region of miR-744 promoter was converted to the complementary sequence to eliminate potential TF binding by using a site-direct mutagenesis approach. The luciferase activity of firefly reporter gene fused with wild-type TF-binding site and the mutation of the site completely abrogated in 5-8F, HONE1, SPC-A-1 and A549 cells was then tested.We used bioinformatics prediction software PROMO2.0 to predict possible binding sites in mir-744 upstream promoter regions 2 kb of possible transcription factors. Then We designed the transcription factor overexpression plasmid and small interfering RNA, chosen nasopharyngeal carcinoma cell line 5-8F, HONE1 and lung cancer cell line SPC-A-1, A549 for transient transfection, After cultivating for 48 hours, TRIZOL method was used to extract the RNA in the cells, with poiA tail method forr reverse transcription into cDNA and qPCR to detecti mir744 expression in cells, compared with blank control group to explore the correlation between transcription factors and mir-744 expression.Then, we used chromatin immune coprecipitation technology (CHIP) to find the the direct binding sites of specific transcription factor and mir-744 promoter region. We chosen the lung cancer cell line A549 for transfection of overexpression plasmid of transcription factor..After 48 hours,we performed the CHIP assay,and finally used the designed specific primers of the binding sites for qPCR amplificationanalysis. Thus found transcription factors could directly bind to mir-744 promoter region.To determine the most potential promoter region of miR-744, we cloned -2000 to-1bp of 5’-flanking region upstream of miR-744 gene into pGL3-reporter vector upstream of the luciferase gene and conducted a dual luciferase assay in 5-8F, HONE1, SPC-A-1 and A549 cells. To further screen for the most potential promoter like sequence, we created another two sequence deletion versions of the promoter constructs ranging from-1000 to-1 bp of miR-744 gene. Then we observe the luciferrasy of the cells after transfection.To validate the existence of the TF response element in this key promoter region of miR-744, we performed co-transfection studies. The luciferase activity were detected in 5-8F, HONE1, SPC-A-1 and A549 cells transfected with both miR-744 key promoter or empty pGL3-basic vector and TF plasmid or its corresponding empty vector control for 48h.The identified TF-binding site, was converted to the complementary sequence to eliminate potential TF binding by using a site-direct mutagenesis approach. The luciferase activity of firefly reporter gene fused with wild-type c-Jun-bindingsie site and the mutatant one in 5-8F, HONE1, SPC-A-1 and A549 cells was observedWe then examined whether TF caused promotion of cancer cell migration and invasion could be counteracted by miR-744 inhibitor. In scratch assay, transwell migration and invasion assay, we used the overexpression plasmid of TF for transfection 5-8F, HONE1, SPC-A-1 and A549 and observe cells migration and invasion.whereas we also observed whether the positive effect of TF treatment on migration and invasion would be almost completely attenuated by miR-744 inhibitor.Results:PROMO2.0 bioinformatics prediction showed that transcription factor c-Jun have six possible binding sites in the upstream 2kb promoter regions of mir-744,:-1482～*1482bp,-1311～-1317 bp,-890～-896 bp,-1261～-1267 bp,-349～-343 and-1582 to-1588bp,. After transfection of c-Jun overexpression plasmid, we used qPCR technology to detect the expression of mir-744, and found that in nasopharyngeal carcinoma cell line 5-8F HONE1 and lung cancer cell line SPC-A-1,A549, mir-744 expression obviously increased compared with blank control groups, and transfection of c-Jun siRNA made the expression of miR-744 in four cell lines decreased obviously compared with control groups.These results showed that the c-Jun can regulate the expression of miR-744, and probably by binding to miR-744 promoter regions to regulate the transcription of mir-744.In CHIP asssy, we designed four specific primers for-1482 to-1488bp and-1582 to-1582bp(sitel),-1311 to-1317 bp and-1261 to-1261bp(site2),-890～-896 bp(site3),-343～-349 bp (site4) for qPCR amplification, the results of qPCR showed thet all the four bingding sites could directly bind to mir-744 promoter region.That meaned c-Jun could regulate miR-744 transcription through directly binding to the binding sites in the promoter region of mir-744. Dual luciferase report was then performed,and found that miR-744 key promoter regions was-1～-500 bp, with-343～-349 bp in this area, thus indicated the c-Jun transcriptional regulation of mir-744 is most probably by binding to-343～-349bp. Dual luciferase asssay further confirmed. We used-500 bp～-bp fragment of fluorescence activity of miR-744 with c-Jun ovefexpression plasmid or without for transfection in nasopharyngeal carcinoma cell line 5-8F, HONE1 and lung cancer cell line SPC-A-1,A549, and found increased luciferase activity in four cell lines after transfection of-1bp～- 500 bp fragment of fluorescence activity of miR-744 with c-Jun ovefexpression plasmid compared with control group,which meaned c-Jun can binding to miR-744 ey promoter region-1bp～-500 bp to influence the transcription of miR-744. At the same time, transfection of c-Jun overexpression plasmid and the wild type miR-744 key promoter region or the mutant type with specifc mutated in-389～-383bp in nasopharyngeal carcinoma cell line 5-8F, HONE1 and lung cancer cell line SPC-A-1, A549 was performed and the luciferase activity in four cell lines all showed significantly increasing in the group with transfection of c-Jun overexpression plasmid and the wild type miR-744 key promoter region while there wasn’t any changes with the mutant type compared with blank group, thus strongly confirmed the c-Jun could regulate the transcription of mir-744 by directly binding to-343 to-349 bp in promoter region of mir -744.In cell function experiments, we used the c-Jun overexpression plasmid to transfect nasopharyngeal carcinoma cell line 5-8F, HONE1 and lung cancer cell line SPC-A-1, A549, after incubation for 36 hours, scratches assay was performed, compared with blank group, the healing percentage in control group significantly increased, while transfection of c-Jun overexpression plasmid and miR-744 inhibitor: anti-mir-744 in four cell line shwed no differece compared with blank control groups,. In transwell experiments, we used the c-Jun overexpression plasmid to transfect nasopharyngeal carcinoma cell line 5-8 F, HONE1 and lung cancer cell line SPC-A-1, A549,and after incubation for 24 hours the migration and invasion of cells significantly increased compared with blank group, while transfection of c-Jun overexpression plasmid and mir-744 inhibitor:anti- mir-744 in four cell lines showed no differece compared with blank control groups These results suggested that miR-744 mediated the promotion of cancer cells migration and invasion by c-Jun Conclusion:In this study, we found that:1.MiR-744 transcription is potentially up-regulated by c-Jun.2.C-Jun enhances the promoter activity of miR-744 by directly binding to its promoter.3. MiR-744 mediates the promotion of cancer cells migration and invasion by c-Jun.4. Our data first proved regulatory mechanism of c-Jun acted on the proto-oncogene miR-744 transcription, and revealed the underlying mechanism of the oversxpression of miR-744 in tumosr, provided a sight for further study. |
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