Identification Of Important Target Genes Of NF-κB

Nuclear factor-κB (NF-κB) is a ubiquitous DNA-binding transcription factor. In general, NF-κB proteins were sequestered in the cytoplasm as inactive forms by their inhibitors. However, upon the diverse stimulations such as TNFα and LPS, the proteins were activated and released into cell nuclear, where they bound to their cognate DNA-binding sequences, by which they regulated the expressions of their target genes, including protein- and microRNA-coding genes. Many studies indicated that NF-κB realized biological functions via controlling its target genes. In this way, NF-κB plays various key roles in some important physiological and pathological processes, such as immunity, inflammation, tumorigenesis and aging. NF-κB target gene thus becomes an important target for drug screening and disease therapy. Therefore, identification of NF-κB transcriptional targets induced by TNFα is beneficial for the deep understanding of its biological functions and the underlying molecular mechnisms. Fir this pupose, this thesis performed the following stuides and obtained some new findings.1. NF-κB regulates the expressions of miRNAsIn this study, we aimed to identify NF-κB target miRNAs. As a consequence, we identified 14 NF-κB target miRNAs in the TNFa-stimulated HeLa cells by using ChIP-Seq and miRNA-Seq techniques. The transcriptional regulations of these miRNAs by NF-κB were confirmed in the TNFα-stimulated HeLa and HepG2 cells by ChIP-qPCR and qPCR. Then, a total of 576 genes were predicted as targets of the identified miRNAs by three programs including miRanda, miRwalk and DIANA-microT-CDS. We next identified 16 target genes of 6 miRNAs including miR-125b-1-3p, miR-1286, miR-502-5p, miR-1276, miR-219-1-3p and miR-30b-3p, in the TNFa-stimulated HeLa cells. Target genes of miR-125b-1-3p and miR-1276 were validated in the HeLa and HepG2 cells by introducing their expressed plasmids and mimics. DAVID software revealed that two potential target genes of miR-1276, BMP2 and CASP9, were enriched in disease phenotype. The former is enriched in osteoarthritis, and the latter is enriched in Type 2 diabete and lung cancer, respectively. These findings suggest that this little known miRNA may play roles in these diseases via its two target genes of BMP2 and CASP9. The expression of miR-125b-1 regulated by NF-κB has been reported in diverse cell types under various stimuli, this study found that its expression was also significantly regulated by NF-κB in the TNFα-stimulated HeLa and HepG2 cells. Therefore, this miRNA was proposed as a central mediator of NF-κB pathway. These findings provided new insights into the functions of NF-κB in its target miRNA-related biological processes and the mechanisms underlying the regulations of these miRNAs.2. NF-κB regulates the expression of apoptosis gene CASP9NF-κB was reported to have pro-apoptotic function in some situations; however, the mechanism underlying this function remains unclear. In this study, we demonstrated that NF-κB directly bound and upregulated CASP9 expression at both mRNA and protein levels in the TNFa-treated HeLa and HepG2 cells. In addition, NF-κB also directly bound and downregulated miR1276 expression, whereas miR1276 repressed CASP9 expression at both mRNA and protein levels in these two cells. In this way, a typical coherent feed-forward loop was formed among NF-κB and its two direct target genes, CASP9 and miR1276, which controlled the upregulation of CASP9 in the detected cells. We found that NF-κB upregulating CASP9 by this loop contributed to the TNFa promoted apoptosis of HeLa and HepG2 cells induced by doxorubicin (DOX). TNFa stimulation increased the total and activated CASP9 proteins in these cells. Moreover, TNFa stimulation enhanced the apoptosis of the two cells induced by DOX. However, an inhibitor of CASP9 protein significantly repressed such TNFa-promoted apoptosis of DOX-induced cells. These findings indicate that CASP9 is a new mediator of NF-κB pro-apoptotic pathway in the detected cancer cells. This study therefore provides new insights into the NF-κB pro-apoptotic role and its underlying molecular mechanism. This study also shed new insights into the molecular mechanism underlying TNFa sensitizing of cancer cells to apoptosis induced by some anticancer drugs such as DOX.3. NF-κB regulates the expressions of cancer genesIdentification of target genes is beneficial for the deep understanding of the molecular functions of NF-κB in cancer process. Here we identified five new cancer-related NF-κB target genes. Firstly, it was found that 20 NF-κB potential target genes (PTGs) identified by ChIP-Seq and Genechip assay were enriched into the KEGG term of Pathways in cancer,16 of these genes were also enriched into the KEGG term of Small cell lung cancer, Chronic myeloid leukemia, Basal cell carcinoma, Pancreatic cancer, and Colorectal cancer. Therefore, these genes of 20 NF-κB PTGs were regarded as cancer-related potential target genes (crPTGs) of NF-κB. Apart from the known target genes, we also found some novel crPTGs of NF-κB, such as CYCS, M1TF, FZD1, FZD8, and PIAS1. Next, the five genes were identified as NF-κB novel target genes in TNFa-treated HeLa and HepG2 cells by several methods including ChIP-Seq, ChIP-qPCR, bioinformation assay, report genes, qPCR and Western blot. These findings provide more insights into the role of NF-kB in cancers.4. NF-κB regulates the expressions of TAC enzyme genesMetabolic reprogramming is one of the prominent characteristics of cancer tissues. NF-kB may promote tumor progression through metabolism alteration resulted by its target genes. Hence, identification of NF-kB metabolic target genes is also helpful for understanding NF-κB molecular roles in tumor development. Here we discovered four metabolic genes as NF-κB transcriptional targets. First, we found four genes IDH1, IDH3A, ACO2 and SUCLA2 that encoded tricarboxylic acid cycle (TCA) core enzymes were covered by many high-confidence NF-KB/RelA ChIP-Seq peaks in the TNFa-treated HeLa cells. Besides, a number of κB sites and the MEME motif which was statistically significantly similar to classical NF-κB motif were discovered in these peaks. The peaks were also confirmed by ChIP-qPCR assay in both TNFa-treated HeLa and HepG2 cells. Dual-luciferase reporter assay showed that NF-κB bound to the peak region for gene regulation. qPCR and Western blot assay were performed to check the regulation of the four TCA-related genes by NF-κB in the TNFa-treated HeLa and HepG2 cells. As results, it was found that NF-κB upregulated both mRNA and protein of IDH1, IDH3A, and ACO2, however, it downregulated SUCLA at mRNA level and exerted no significant effects on its protein. These findings provide new insights into the role of NF-κB in cellular metabolism that help for understanding tumorigenesis.5. NF-κB regulates the expression of neuroblastoma gene NBPFThe neuroblastoma breakpoint family (NBPF) has been reported to play potential roles in the development of neuroblastoma and human evolution. However, the exact regulation and function of this family is still unknown. In this study, the genes of NBPF family were found to be densely covered by many high-confidence ChIP-Seq peaks of NF-κB. The expressions of NBPF genes were thus deduced to be regulated by this transcription factor. It was therefore found that the expressions of NBPF genes were regulated by NF-κB in TNFa-treated HeLa and HepG2 cells. Therefore, the genes of NBPF family were identified as new bona fide direct target genes of NF-κB. In addition, NBPF was also identified as a nuclear protein by in silico prediction and immunolocalization. Finally, the bioinformatics analysis revealed that most of NBPF proteins contained classical nuclear localization signals (NLSs) and a conserved DNA-binding domain similar to the transcription factor stat3b/dna complex or stat-1/dna complex in their N-terminals. Therefore, this study concluded that NBPF was a nuclear protein that contained classical NLSs and a conserved known DNA-binding domain, and its expression was regulated by another important transcription factor, NF-κB. These findings suggest that NBPF may function as DNA-binding transcription factor in nucleus, which provides important new insight into the functions of NBPF genes in the human cells.In conclusion, these studies have demonstrated some important NF-κB transcriptional targets, such as miR-125b-1, miR-1276, CASP9, CYCS, MITF, FZD1, FZD8, PIAS1, IDH1, IDH3A, ACO2, SUCLA2 and NBPF family genes. Thus, these findings showed that the regulations of NF-κB to these genes or miRNAs may be underlying molecular mechanisms by which this factor play key roles in the tumor development. Therefore, these findings may provide new insights for cancer therapy.