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LncRNA ZNF649-AS1 Induces Trastuzumab Resistance by Promoting ATG5 Expression and Autophagy.

ABSTRACT: The regulatory mechanism of long non-coding RNAs (lncRNAs) in trastuzumab resistance is not well established to date. In this research, we identified differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer. HiSeq sequencing and quantitative real-time PCR were performed to identify the dysregulated lncRNAs. Mass spectrometry, RNA fluorescence in situ hybridization (RNA-FISH), and immunoprecipitation assays were performed to identify the direct interactions between ZNF649-AS1 and other associated targets, such as polypyrimidine tract binding protein 1 (PTBP1) and autophagy related 5 (ATG5). Our results showed that ZNF649-AS1 was more highly expressed in trastuzumab-resistant cells compared to sensitive cells. Increased expression of ZNF649-AS1 was associated with a poorer response and shorter survival time of breast cancer patients. ZNF649-AS1 was upregulated by H3K27ac modification at the presence of trastuzumab treatment, and knockdown of ZNF649-AS1 reversed trastuzumab resistance via modulating ATG5 expression and autophagy. Mechanically, ZNF649-AS1 was associated with PTBP1 protein, which further promoted the transcription activity of the ATG5 gene. In conclusion, we demonstrated that H3K27ac modification-induced upregulation of ZNF649-AS1 could cause autophagy and trastuzumab resistance through associating with PTBP1 and promoting ATG5 transcription.

SUBMITTER: Han M

PROVIDER: S-EPMC7647669 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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lncRNA ZNF649-AS1 Induces Trastuzumab Resistance by Promoting ATG5 Expression and Autophagy.

Han Mingli M Qian Xueke X Cao Hui H Wang Fang F Li Xiangke X Han Na N Yang Xue X Yang Yunqing Y Dou Dongwei D Hu Jianguo J Wang Wei W Han Jing J Zhang Fan F Dong Huaying H

Molecular therapy : the journal of the American Society of Gene Therapy 20200721 11

The regulatory mechanism of long non-coding RNAs (lncRNAs) in trastuzumab resistance is not well established to date. In this research, we identified differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer. HiSeq sequencing and quantitative real-time PCR were performed to identify the dysregulated lncRNAs. Mass spectrometry, RNA fluorescence in situ hybridization (RNA-FISH), and immunoprecipitation assays were performed to identify the dire ...[more]

PMID: 32735773

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Project description:BackgroundAlthough trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Recent evidence suggests that long noncoding RNAs (lncRNAs) play important roles in tumorigenesis and chemoresistance. However, the regulatory mechanism of lncRNAs in trastuzumab resistance is not well established to date. In this research, we identified the differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer.MethodsLncRNA microarray and qRT-PCR were performed to identify the dysregulated lncRNAs. Transmission electron microscopy, differential ultracentrifugation and qRT-PCR were used to verify the existence of exosomal AFAP1-AS1 (actin filament associated protein 1 antisense RNA 1). Bioinformatics prediction, RNA fluorescence in situ hybridization (RNA-FISH) and immunoprecipitation assays were performed to identify the direct interactions between AFAP1-AS1 and other associated targets, such as AU-binding factor 1 (AUF1) and ERBB2. Finally, a series gain- or loss-functional assays were done to prove the precise role of AFAP1-AS1 in trastuzumab resistance.ResultsAFAP1-AS1 was screened out due to its higher expression in trastuzumab-resistant cells compared to sensitive cells. Increased expression of AFAP1-AS1was associate with poorer response and shorter survival time of breast cancer patients. AFAP1-AS1 was upregulated by H3K27ac modification at promoter region, and knockdown of AFAP1-AS1 reversed trastuzumab resistance. Moreover, extracellular AFAP1-AS1 secreted from trastuzumab resistant cells was packaged into exosomes and then disseminated trastuzumab resistance of receipt cells. Mechanically, AFAP1-AS1 was associated with AUF1 protein, which further promoted the translation of ERBB2 without influencing the mRNA level.ConclusionExosomal AFAP1-AS1 could induce trastuzumab resistance through associating with AUF1 and promoting ERBB2 translation. Therefore, AFAP1-AS1 level may be useful for prediction of trastuzumab resistance and breast cancer treatment.

Project description:The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10- 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10- 25). The inflammatory "TNFA signaling via NFKB" gene set was downregulated the most (Padj=1 × 10- 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10- 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10- 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.

Dataset Information

LncRNA ZNF649-AS1 Induces Trastuzumab Resistance by Promoting ATG5 Expression and Autophagy.

Publications

lncRNA ZNF649-AS1 Induces Trastuzumab Resistance by Promoting ATG5 Expression and Autophagy.

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