Long Noncoding RNA DLGAP1-AS2 Promotes Tumorigenesis and Metastasis by Regulating the Trim21/ELOA/LHPP Signaling Axis in Colorectal Cancer
Xue WangJ. ZhaoHan ChengJiuming LiYing ChenKaisa CuiTian LuJia ZhangChaoqun LiShengbai SunYuyang FengSurui YaoZehua BianShenglin HuangBojian FeiZhaohui Huang
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Abstract Background: Long noncoding RNAs (lncRNAs) have driven research focused on their effects as oncogenes or tumor suppressors involved in carcinogenesis. However, the functions and mechanisms of most lncRNAs in colorectal cancer (CRC) remain unclear. Methods: The expression of DLGAP1-AS2 was assessed by quantitative RT-PCR in multiple CRC cohorts. The impacts of DLGAP1-AS2 on CRC growth and metastasis were evaluated by a series of in vitro and in vivo assays. Furthermore, the underlying mechanism of DLGAP1-AS2 in CRC was revealed by RNA pull down, RNA immunoprecipitation, RNA sequencing, luciferase assays, chromatin immunoprecipitation, and rescue experiments. Results: We discovered that DLGAP1-AS2promoted CRC tumorigenesis and metastasis by physically interacting with Elongin A (ELOA) and inhibiting its protein stability by promoting tripartite motif containing 21(Trim21)-mediated ubiquitination modification and degradation of ELOA. In particular, we revealed that DLGAP1-AS2decreases phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) expression by inhibiting ELOA-mediated transcriptional activating of LHPP and thus blocking LHPP-dependent suppression of the AKT signaling pathway. In addition, we also demonstrated that DLGAP1-AS2 was bound and stabilized by cleavage and polyadenylation specificity factor (CPSF2) and cleavage stimulation factor (CSTF3). Conclusions: The discovery of DLGAP1-AS2, a promising prognostic biomarker, reveals a new dimension into the molecular pathogenesis of CRC and provides a prospective treatment target for this disease. Keywords: colorectal cancer, DLGAP1-AS2, ELOA, Trim21, LHPPKeywords:
Chromatin immunoprecipitation
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Chromatin immunoprecipitation (ChIP) experiments with differentiated adipocytes are challenging because lipid droplets interfere with immunoprecipitation efficiency. Here, the author describes optimized procedures to minimize the burden of lipid droplets by using hypotonic buffer to enrich nuclear fraction before formaldehyde crosslinking, thus increasing the sensitivity and specificity of ChIP experiments with differentiated adipocytes. The author also describes steps after fixation, including sonication, immunoprecipitation, washing, reverse-crosslinking, and purification. This protocol is compatible with ChIP-qPCR and ChIP-seq of various transcription factors and histone modifications. For complete details on the use and execution of this protocol, please refer to Hiraike et al. (2022).1.
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Abstract Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein‐DNA complexes are cross‐linked with formaldehyde ( UNIT ), and are purified via two successive immunoprecipitations, with each immunoprecipitation targeting a different protein. Protein‐DNA cross‐links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co‐occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co‐occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae . Cells from a wide variety of organisms can be used.
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Abstract In the past few decades, numerous approaches have been developed to investigate protein‐protein and protein‐nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co‐immunoprecipitation (co‐IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two‐step co‐immunoprecipitation (TIP) technique. As compared to standard co‐IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein‐protein complex from Burkitt lymphoma cells and from primary human CD4 + T cells. In addition, this unit describes an application of TIP for the isolation of transcription‐factor‐bound chromatin. © 2018 by John Wiley & Sons, Inc.
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This protocol can be ued for chromatin immunoprecipitation of RNAPII and associated factors, as well as histones. The settings are given for HeLa cells and should be adapted for other cell types.
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To verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo.Chromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein.The p53 binding sequences on the promoters of p21WAF1/CIP1 and hsp90 beta gene were found in the p53 antibody immunoprecipitated DNA fragments and p53 was detected in the immunoprecipitated samples.p53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.
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Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simultaneously, and is more sensitive than the standard ChIP assay.
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DNA-protein interactions are a major challenge in the research of gene transcription regulation.Chromatin immunoprecipitation assay(CHIP)provides a powerful tool to analyze this interaction in vivo.This article summarizes the methods of ChIP assay,and highlights the application and recent progress in the application of this technique.
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