| Objective The aim of this study was to explore more regulatory effect of let-7a at post-transcriptional level through bioinformatic approaches for let-7a target prediction and analysis.Methods Let-7a target site prediction was performed by using miRGen software. Access entry by using the hsa-let-7a and the intersection of the three algorithms: miRanda (microrna.org), PicTar (4-way), TargetScans, were chosen. Then, biological function of these targets was analyzed using the GO (gene ontology) annotation on the website of gene ontology through data linking provided by miRGen software. Genes with high conversation, low free energy and close correlation to cell proliferation, were selected as candidate targets.Results As a result, we obtained 82 possible targets for let-7a. Eight genes were screened as candidate targets, including NRAS,CDC34,TUSC2,NIRF,GAS7,DUSP1,RB1 and PDGFB. Given an important role of NIRF in cell proliferation, NIRF was chosen for further investigation. The NIRF gene is located in 9p23-24.1. Protein encoded by NIRF is 802 amino-acids and possesses four structural domains, including a NIRF_N domain, a PHD finger domain, a SRA domain, and a RING finger domain. By bioinformatics analysis, we found that NIRF 3′UTR (untranslation region) contains a binding site of let-7a. A good 9-nt match (nucleotides1-9) to the seed region at the 5′end of mature let-7a is present in the NIRF 3′UTR. The free energy of dimmer structure formed by let-7a and NIRF 3′UTR is -16.89kcal/mol. In addition, we found that the putative let-7a target site in NIRF 3′UTR is highly conserved in all four genomes, including human, mouse, rat, and canis.Conclusion let-7a has a large regulatory potential and NIRF may be a target of let-7a. Objective The purpose of this study was to verify whether NIRF was targeted by let-7a through experimental approaches.Methods A luciferase gene expression vector containing full-length 3′untranslated region (3′UTR) of NIRF gene was constructed. The resulting luciferase expression plasmids were named pRL-TK-NIRF 3′UTR. The firefly luciferase expression plasmid pGL3-control and the renilla luciferase construct pRL-TK or pRL-TK-NIRF 3′UTR with either let-7a mimics or mimics control were cotransfected into A549 cells, in which let-7a is known to be poorly expressed. The firefly luciferase expression plasmid pGL3-control and the renilla luciferase construct pRL-TK or pRL-TK-NIRF 3′UTR with either let-7a inhibitor or inhibitor control were cotransfected into HeLa cells, which were reported to express native let-7a. Then, the firefly and renilla luciferase activity were detected by using the dual glo luciferase assay system. After transfection of let-7a mimics or control mimics in A549 cells and transfection of let-7a inhibitor or inhibitor control in HeLa cells, expression of NIRF protein was determined by Western blot.Results The recombinant vector pRL-TK-NIRF 3′UTR was verified by sequencing. Luciferase activity was significantly reduced to 49.29% in A549 cells transfected with pRL-TK-NIRF 3′UTR and pGL3-control in the presence of let-7a mimics, compared with A549 cells transfected with pRL-TK and pGL3-control (P<0.01). Luciferase activity was significantly enhanced by 46.77% in HeLa cells transfected with pRL-TK-NIRF 3′UTR and pGL3-control in the presence of let-7a inhibitor, compared with HeLa cells transfected with pRL-TK and pGL3-control (P<0.01). Western blot detection demonstrated that a significant decrease of NIRF protein level in cells transfected with let-7a mimics (P<0.01), but not in cells transfected with mimics control (P>0.05), when compared to A549 cells; and a significant increase of NIRF protein level in cells transfected with let-7a inhibitor (P<0.01), but not in cells transfected with inhibitor control (P>0.05), when compared to HeLa cells.Conclusion These data strongly indicated that NIRF 3′UTR contained regulatory information, allowing let-7a to negatively regulate the expression of NIRF. Thus, NIRF is indeed a target of let-7a. Objective The object of this research was to access the effect of let-7a on the growth of A549 lung cancer cells and to explore the possible underlying mechanisms.Methods The let-7a expression construct and a control plasmid, named pGeneil-let-7a and pGeneil-control, were constructed. siRNA expression vectors specific to NIRF were also generated and were named si-1, si-2, si-3 and si-nc, respectively. Then pGeneil-let-7a and pGeneil- control were transfected into A549 cells, which were selected by G418 and verified by Real time RT-PCR to establish lung cancer cell lines stablely expressing pGeneil-let-7a and pGeneil-control, named A549-let-7a cell line and A549-control cell line. The living cells of A549, A549-control, and A549-let-7a cells were counted by MTT assay and cell growth curves were drew to analyze the cell proliferation. Cell cycle distribution of A549, A549-control, and A549-let-7a cells were determined by flow cytometry. The expression of NIRF, p21WAF1 and p27kip1 protein of A549, A549-control, and A549-let-7a cells were determined by Western blot. The expression of NIRF and p21WAF1 of A549, A549-control, and A549-let-7a cells were detected again by immunocytochemistry analysis. siRNA expression vectors specific to NIRF were transfected into A549 cells, and the pIRES2-EGFP empty vector and pIRES2-EGFP-NIRF construct were transfected respectively into A549-let-7a cells. After transient transfection, the cells were harvested to determine the expression of NIRF and p21WAF1 by Western blot.Results The recombinant vectors, including pGenesil-let-7a, pGenesil-control, si-1, si-2, si-3 and si-nc, were verified by sequencing. Real time RT-PCR displayed that let-7a was significantly enhanced in A549-let-7a cells compared with untreated A549 cells (P<0.01), and the expression of let-7a in A549-let-7a cells was 5.34 times as much as that in A549 cells, suggesting that stable transfected cell lines over-expressing let-7a and the control miRNA, A549-let-7a and A549-control cells, were established. The cell growth curves showed the proliferation of A549-let-7a cells were inhibited significantly compared with that of A549 cells and A549-control cells. Flow cytometry assay showed a lower distribution in S phase in A549-let-7a cells, compared with A549 cells and A549-control cells. Western blot indicted that a decrease in NIRF and a coordinated increase in p21WAF1 were observed in A549-let-7a cells, compared with untreated A549 cells (P<0.01). However, no significant change in the levels of p27kip1was found (P>0.05). Immunofluorescence assay revealed that NIRF was significantly reduced, whereas p21WAF1 was markedly enhanced in A549-let-7a cells, when compared with A549 cells (P<0.01). RNAi efficiency on the expression NIRF and p21WAF1 were detected by Western blot. Si-1, si-2 and si-3 significantly inhibited the expression of NIRF (P<0.01), and the inhibitory rate were 70.27%, 42.85%, 65.49%, respectively. The marked increase of p21WAF1 protein level were observed in the corresponding groups (P<0.01), and the expression of p21WAF1 enhanced by 64.21%, 25.73%, 34.54%, respectively. These results suggested that expression level of p21WAF1 inversely correlated well with NIRF. Western blot also showed that an enhancement in NIRF and a coordinated reduction in p21WAF1 were observed in A549-let-7a cells transfected with by pIRES2-EGFP-NIRF, compared with untreated A549-let-7a cells (P<0.01).Conclusion Our results strongly indicated that the growth inhibition effect of let-7a could be explained in part through le-7a-mediated elevation of p21WAF1 by targeting of NIRF. Objective The aim of this study was to determine the effect of let-7a on the growth of A549 lung cancer cells transplanted subcutaneously in nude mice and to investigate the possible underlying mechanisms.Methods A549, A549-control, and A549-let-7a cells were injected subcutaneously in the backs of nude mice, respectively. The growth of transplanted tumors was observed and the tumor inhibitory rate was calculated. After injection thirty days, the mice were sacrificed; the xenografts were excised and weighed. The expression of let-7a in tumor xenografts was assessed by Real-time RT-PCR. The expression of NIRF and p21WAF1 in xenografts were determined by Western blot and immunohistochemistry detection.Results A significant depress in tumor weight was observed in the xenografts of A549-let-7a cells-injected group, compared to A549 cells-injected group (P<0.01). The inhibitory rate of A549-let-7a cells-injected group on tumor growth was 27.51%. No significant difference in tumor weight was found between the A549-control cells-injected group and A549 cells-injected group (P>0.05). Real time RT-PCR indicted the expression of let-7a was significantly increased in the tumor xenografts of A549-let-7a cells-injected group compared to A549 cells-injected group (P<0.01). The expression of let-7a in the tumor xenografts of A549-let-7a cells-injected group was 4.25 times as much as that in the tumor xenografts of A549 cells-injected group. HE staining showed marked decrease in volume of cancer cells, in the ratio of nucleus to cytoplasm and in mitosis figures in the tumor xenografts of A549-let-7a cells-injected group, compared to the A549-control cells-injected group and A549 cells-injected group. Decreased NIRF and increased p21WAF1 in xenografts of A549-let-7a cells-injected group were detected by Western blot (P<0.01) and immunohistochemistry assay (P<0.01), compared with the xenografts of A549 cells-injected group. However, no significant change in the levels of NIRF and p21WAF1were found between the A549-control cells-injected group and A549 cells-injected group (P>0.05).Conclusion Overexpression of let-7a can inhibit the growth of A549 lung cancer cells in vivo through le-7a-mediated elevation of p21WAF1 by targeting of NIRF.
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