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: Metastasis and drug resistance contribute substantially to the poor prognosis of colorectal cancer (CRC) patients. However, the epigenetic regulatory mechanisms by which CRC develops metastatic and drug-resistant characteristics remain unclear. This study aimed to investigate the role of miR-302a in the metastasis and molecular-targeted drug resistance of CRC and elucidate the underlying molecular mechanisms. : miR-302a expression in CRC cell lines and patient tissue microarrays was analyzed by qPCR and fluorescence hybridization. The roles of miR-302a in metastasis and cetuximab (CTX) resistance were evaluated both and . Bioinformatic prediction algorithms and luciferase reporter assays were performed to identify the miR-302a binding regions in the NFIB and CD44 3'-UTRs. A chromatin immunoprecipitation assay was performed to examine NFIB occupancy in the ITGA6 promoter region. Immunoblotting was performed to identify the EGFR-mediated pathways altered by miR-302a. : miR-302a expression was frequently reduced in CRC cells and tissues, especially in CTX-resistant cells and patient-derived xenografts. The decreased miR-302a levels correlated with poor overall CRC patient survival. miR-302a overexpression inhibited metastasis and restored CTX responsiveness in CRC cells, whereas miR-302a silencing exerted the opposite effects. NFIB and CD44 were identified as novel targets of miR-302a. miR-302a inhibited the metastasis-promoting effect of NFIB that physiologically activates ITGA6 transcription. miR-302a restored CTX responsiveness by suppressing CD44-induced cancer stem cell-like properties and EGFR-mediated MAPK and AKT signaling. These results are consistent with clinical observations indicating that miR-302a expression is inversely correlated with the expression of its targets in CRC specimens. : Our findings show that miR-302a acts as a multifaceted regulator of CRC metastasis and CTX resistance by targeting NFIB and CD44, respectively. Our study implicates miR-302a as a candidate prognostic predictor and a therapeutic agent in CRC.
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Linkage studies of complex genetic diseases have been largely replaced by genome-wide association studies, due in part to limited success in complex trait discovery. However, recent interest in rare and low-frequency variants motivates re-examination of family-based methods. In this study, we investigated the performance of two-point linkage analysis for over 1.6 million single-nucleotide polymorphisms (SNPs) combined with single variant association analysis to identify high impact variants, which are both strongly linked and associated with cardiometabolic traits in up to 1414 Hispanics from the Insulin Resistance Atherosclerosis Family Study (IRASFS). Evaluation of all 50 phenotypes yielded 83 557 000 LOD (logarithm of the odds) scores, with 9214 LOD scores ⩾3.0, 845 ⩾4.0 and 89 ⩾5.0, with a maximal LOD score of 6.49 (rs12956744 in the LAMA1 gene for tumor necrosis factor-α (TNFα) receptor 2). Twenty-seven variants were associated with P<0.005 as well as having an LOD score >4, including variants in the NFIB gene under a linkage peak with TNFα receptor 2 levels on chromosome 9. Linkage regions of interest included a broad peak (31 Mb) on chromosome 1q with acute insulin response (max LOD=5.37). This region was previously documented with type 2 diabetes in family-based studies, providing support for the validity of these results. Overall, we have demonstrated the utility of two-point linkage and association in comprehensive genome-wide array-based SNP genotypes.
A functional complex consisting of androgen receptor (AR) and forkhead box A1 (FOXA1) proteins supports prostatic development, differentiation, and disease. In addition, the interaction of FOXA1 with cofactors such as nuclear factor I (NFI) family members modulates AR target gene expression. However, the global role of specific NFI family members has yet to be described in the prostate. In these studies, chromatin immunoprecipitation followed by DNA sequencing in androgen-dependent LNCaP prostate cancer cells demonstrated that 64.3% of NFIB binding sites are associated with AR and FOXA1 binding sites. Interrogation of published data revealed that genes associated with NFIB binding sites are predominantly induced after dihydrotestosterone treatment of LNCaP cells, whereas NFIB knockdown studies demonstrated that loss of NFIB drives increased AR expression and superinduction of a subset of AR target genes. Notably, genes bound by NFIB only are associated with cell division and cell cycle. To define the role of NFIB in vivo, mouse Nfib knockout prostatic tissue was rescued via renal capsule engraftment. Loss of Nfib expression resulted in prostatic hyperplasia, which did not resolve in response to castration, and an expansion of an intermediate cell population in a small subset of grafts. In human benign prostatic hyperplasia, luminal NFIB loss correlated with more severe disease. Finally, some areas of intermediate cell expansion were also associated with NFIB loss. Taken together, these results show a fundamental role for NFIB as a coregulator of AR action in the prostate and in controlling prostatic hyperplasia.
Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression.
SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.
The genomic copy multiplicity of the CCAAT transcription complex component enhancer factor I subunit A (EFIA) has been examined. When a mammalian genomic Southern blot was hybridized to a rat EFIA cDNA, a complex pattern consisting of numerous related sequences was found in all the species examined, with Bos taurus being the least complex. An EFIA#1 cDNA from Bos taurus was isolated from a primary lung endothelial cell cDNA library by screening with the 1489-bp rat EFIA cDNA. The deduced bovine EFIA#1 amino acid (aa) sequence is 98% identical to rat EFIA and 100% identical to human EFIA/DbpB/YB-1 family member DNA-binding protein B (DbpB). In addition, a processed EFIA pseudogene from Bos taurus, designated bovine psi EFIA#1, was obtained from a genomic library by screening with a rat EFIA cDNA probe. The bovine psi EFIA#1 gene has an ORF which, if expressed, would encode a 140-aa sequence, with aa 31-140 having 84% identity to bovine EFIA#1. The genomic cloning data indicate that processed pseudogenes are partially responsible for the complexity of the EFIA genomic Southern blots. The phenomenon of 'repeat induced point mutation' (ripping) at bovine psi EFIA#1 gene CpG dinucleotides occurs at a 6.5-fold higher frequency than expected from random mutagenesis. Therefore, ripping is likely to be the mechanism by which the bovine EFIA#1 pseudogene's ectopic recombination potential was inactivated.
The peripherin gene, which encodes a neuronal-specific intermediate filament protein, is transcriptionally induced with a late time course when nerve growth factor stimulates PC12 cells to differentiate into neurons. We have defined a negative regulatory element (NRE) that has a functional role in repressing peripherin expression in undifferentiate and nonneuronal cells. Nerve growth factor-induced derepression of peripherin gene expression is associated with alterations in proteins binding to a GC-rich DNA sequence in the NRE as detected by the DNA electrophoretic mobility shift assay (EMSA). We have utilized DNA affinity chromatography to purify from rat liver a 33-kDa DNA-binding protein that specifically recognizes the NRE. Microsequencing reveals identity with NF1-L, a member of the CTF/NF-1 transcription factor family. This protein forms a single complex when incubated with the NRE probe using EMSA analysis. The more slowly migrating complexes characteristic of crude undifferentiated PC12 cell extract are reconstituted by mixing the purified protein with the flow-through from the DNA affinity column, thereby demonstrating that protein-protein interactions are involved in complex formation. Supershift experiments incubating anti-CTF-1 antibody with undifferentiated PC12 cell extract prior to EMSA analysis confirm that NF1-L, or a closely related family member, is the DNA-binding protein component of the multiprotein complex at the NRE.
The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor-beta 1 (TGF beta 1). TGF beta 1 plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGF beta 1 might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGF beta 1 increased ecNOS mRNA in a dose-dependent manner. TGF beta 1 also increased ecNOS protein content. The production of nitrogen oxides (NOx), assessed by chemiluminescence, and nitric oxide synthase activity, assessed by arginine/citrulline conversion were increased in TGF beta 1-treated cells. Transcriptional activity of the 5'-flanking promoter region of the ecNOS gene was increased by TGF beta 1, as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGF beta 1-response element was present between nucleotides -1269 and -935 from the first transcription start site, in which a putative nuclear factor-1 (NF-1) binding site existed. Gel shift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding site in a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGF beta 1. In conclusion, TGF beta 1 increases ecNOS expression associated with an increase in production of NO in BAEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter.
Enhancer factor I (EFI) is a trans-acting factor which binds to the Rous sarcoma virus long terminal repeat enhancer and promoter at two inverted CCAAT-box motifs. We demonstrate that two forms of EFI DNA binding activity exist in nuclear extracts of avian cells. One form requires two heterologous components (EFIA)(EFIB) for high affinity, specific DNA binding activity, whereas a second form is not dependent on EFIB for binding and may be composed solely of EFIA, perhaps as a multimer. Both forms give rise to the same mobility shift in gel retardation assays, but the two forms can be separated chromatographically under buffer conditions which stabilize the two DNA binding activities. A cDNA for EFIA has been isolated from a rat liver cDNA expression library. The 1489-base pair EFIA cDNA encodes a 322-amino acid protein which is nearly identical to two previously described human DNA binding proteins. These are dbpB, a DNA binding protein of unknown specificity which binds to the epidermal growth factor receptor enhancer and c-erbB-2 gene promoter (Sakura, H., Maekawa, T., Imamoto, F., Yasuda, K., and Ishii, S. (1988) Gene (Amst.) 73, 499-507), and YB-1, a protein which recognizes the Y-box (inverted CCAAT motif) of the HLA-DR alpha chain gene (Didier, D. K., Schiffenbauer, J., Woulfe, S. L., Zacheis, M., and Schwartz, B. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7322-7326). EFIA/dbpB/YB-1 share a highly conserved region of 100 amino acids with dbpA, another protein identified by Sakura et al. (1988) which binds to the epidermal growth factor receptor enhancer and c-erbB-2 gene promoter, and with two Xenopus CCAAT binding proteins, FRG Y1 and FRG Y2 (Tafuri, S. R., and Wolffe, A. P. (1990) Proc. Natl. Acad. Sci. U. S. A., in press). This highly conserved domain among all six proteins is presumed to represent or contain a DNA binding domain for the CCAAT motif. In addition, we note that the EFIA/dbpB/YB-1 polypeptide contains a novel arrangement of alternating clusters of positively and negatively charged amino acids not yet reported for any trans-acting factor. The functional significance of this novel structural motif, which is also conserved in dbpA, FRG Y1, and FRG Y2, will be discussed.