Professor Fan Xianqun, affiliated to the Ninth People's Hospital of Shanghai Jiaotong University School of Medicine, specializes in ocular tumors and orbital surgery, and has made outstanding achievements in basic research and clinical treatment. The eye tumor team led by ChIRP-MS technology (a technique for studying RNA-protein interactions) combined with RNA-seq, ChIP and other in vitro and in vivo experiments on the new mechanism of action of retinoblastoma. The study found that Ch12p 13.32 chromatin is open in tumor cells and activates a novel lncRNA GAU1 expression. GAU1 can be enriched in the promoter region of the oncogene GALNT8, recruiting the transcription factor TCEA1 to the promoter region, promoting its expression, thereby affecting tumorigenesis. This study proposes a new carcinogenic mechanism of chromatin dynamics, and the clinical data of the study also found that high expression of GAU1 predicts lower patient survival and provides important detection for early diagnosis and prognosis of patients with retinoblastoma. Indicators and new targets for treatment. The research results were recently published in the internationally renowned academic journal Nucleic Acids Research ( IF:11.561 ). ( ChIRP-MS experiments are provided by technical services )
Research Background
Changes in chromatin structure can regulate the ability of genes to bind to regulatory factors during transcription, replication, recombination, and damage repair. Open chromatin exposes the binding surface of many free transcription factors, while closed chromatin exhibits increased density and blurred protein binding sites. The destruction of chromatin dynamics leads to abnormal gene expression, chromosomal translocation and tumorigenesis. Therefore, chromatin dynamics has received more and more attention in the pathogenesis of human diseases.
Abnormal transformation of dynamic chromatin can dysregulate the expression of protein-coding genes, which may be an important inducer of tumorigenesis. Chromatin dynamics can also regulate the expression of long-chain non-coding RNA (lncRNA) to affect tumor formation. However, in tumorigenesis, whether dynamic changes in chromosome structure can simultaneously regulate long-chain non-coding RNA and protein-coding genes remains uncertain. In our study, we identified for the first time a new dynamic chromosome-driven mechanism that can co-activate the gene cluster in chr12p13.32, a new lncRNA GAU1 and a protein-coding gene GALNT8, via RNA-seq, A series of in vitro and in vivo experiments such as ChIRP-MS found that GAU1 can be enriched in the promoter region of the oncogene GALNT8, recruiting the transcription factor TCEA1 to the target gene promoter region, promoting its expression, thereby affecting tumorigenesis.
Research ideas
First, the authors studied the chromatin dynamics of retinoblastoma. RNA-seq and FAIRE found that chr12 p13.32 is open in tumor cells and can activate LOC101929549 and the protein-coding gene GALNT8. qPCR and WB found that GALNT8 and LOC101929549 were significantly overexpressed in tumor cells. And the open chromatin histone signal was significantly increased in the LOC101929549 / GALNT8 locus. Subsequently, the author determined that LOC101929549 has no protein coding ability through bioinformatics analysis, vector construction, immunofluorescence analysis and WB experiments. It belongs to a novel lncRNA and is named GAU1.
Next, the authors studied the function of GAU1/GALNT8. In vitro knockout/overexpression of GAU1/GALNT8 revealed that knockdown resulted in decreased colony formation and inhibition of cell cycle, while overexpression promoted these effects. In vivo orthotopic transplantation experiments showed the same expected results as in vitro experiments. It shows that GAU1/GALNT8 can play a role in cancer-promoting genes and regulate tumorigenesis. qPCR and immunohistochemical staining showed that GAU1/GALNT8 was up-regulated in retinoblastoma tissues. TCGA, R2, Dorsman and other database analysis and KM curve analysis found that high expression of GAU1/GALNT8 corresponds to poor prognosis and can be used as a potential indicator tumor. The biomarker that occurred.
Finally, the authors studied the mechanism of action of GAU1/GALNT8. Nuclear isolation and RNA-FISH revealed that GAU1 was distributed in the nucleus. Vector construction, qPCR, WB and other experiments found that GAU1 can cis-regulate the expression of downstream target gene GALNT8. The authors then studied lncRNA GAU1 by ChIRP. ChIRP-PCR, ChIRP-MS , ChIP, WB, qPCR and other experiments found that GAU1 can be significantly enriched in the GALNT8 promoter region, and TCEA1 can interact with the GAU1/GALNT8 promoter region. The above results indicate that in retinoblastoma, open chromatin promotes co-activation of GAU1/GALNT8, and GAU1 can recruit the transcription factor TCEA1 to the promoter region of the oncogene GALNT8 to promote its expression and thereby promote tumorigenesis.
Technical route
Result display
Figure 1. Chromatin kinetics study. Chr12p 13.32 region chromatin is open in tumor tissues, resulting in co-activation of GAU1 and GALNT8, and GAU1 belongs to the novel lncRNA.
Figure 2 Functional study results, GAU1/GALNT8 promotes tumorigenesis, high expression corresponds to poor prognosis
Figure 3 Mechanism study results, GAU1 is a nuclear lncRNA that regulates GALNT8 expression
Figure 4. In-depth mechanism study, GAU1 is enriched in the GALNT8 promoter region, and the transcription factor TCEA1 is recruited to the promoter region to activate its expression and promote tumorigenesis.
Significance
In this study, the researchers used ChIRP-MS technology combined with RNA-seq, ChIP and other in vitro and in vivo experiments to study the new mechanism of action of retinoblastoma. The chromatin of chromosome 13.32 in tumor cells is open, which can activate the expression of a novel lncRNA GAU1. GAU1 can be enriched in the promoter region of oncogene GALNT8, and the transcription factor TCEA1 is recruited to the target gene promoter region to promote its expression. Thus affecting the occurrence of tumors, this study presents a new dynamic mechanism of chromatin regulation. At the same time, it provides important detection indicators and new therapeutic targets for early diagnosis and prognosis of patients with retinoblastoma.
Original source
Dynamic chromosomal tuning of a novel GAU1 lncing driver at chr12p13.32 accelerates tumorigenesis. Nucleic Acids Research, 2018
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