Mechanism Of BRD4 Inhibitor JQ1 Inhibiting The Proliferation Of Human Oral Squamous Cell Carcinoma Cells

Research BackgroundOral squamous cell carcinoma is one of the invasive solid malignancies with high incidence,easy lymph node metastasis,and poor prognosis.Therefore,it is very important to develop effective drugs to inhibit the proliferation of OSCC.BRD4 is an important class of epigenetic regulatory proteins,and it has the role of "reader" and transcription co-factors.On the one hand,the N-terminal bromine domain of BRD4 recognizes and binds to acetylated histones;on the other hand,the CTM and ET domains of the C-terminal of BRD4 recruit transcriptional regulators such as RNA polymerase Ⅱ to form transcription complexes to regulate the transcription of downstream target genes and participate in the regulation of important life activities such as the cell cycle.BRD4 inhibitor(JQ1)can significantly inhibit some blood tumors and solid tumors.However,whether the BRD4 inhibitor JQ1 can inhibit OSCC cell proliferation and the molecular mechanism is not fully understood.Research PurposesWe aim to clarify the role and molecular mechanism of BRD4 inhibitor JQ1 in the proliferation of OSCC cells,and lay the foundation for investigating the mechanism of OSCC development and developing new targeted drugs.Research contents1.This chapter explore the effect of JQ1 on the proliferation of SCC15 and SCC251)TCGA database analysis of mutation rate and expression of BRD4 and cell cycle related genes in HNSCC;2)The effect of JQ1 on the proliferation of SCC15 and SCC25 cells was analyzed by MTT experiment,EdU experiment and colony formation cell assay;3)Flow cytometry was used to detect the effect of JQ1 on apoptosis and cell cycle of SCC15 and SCC25 cells.2.This chapter explore the effect of JQ1 on the senescence of SCC15 and SCC25.1)Analysis of the proportion of positive cells with senescence-associated beta-galactosidase(SA-β-gal)and senescence-associated heterochromatin foci(SAHF)after JQ1 acting on SCC15 and SCC25 cells for 48h.2)Analysis of mRNA level of senescence-associated secretory phenotype(SASP)by qRT-PCR after JQ1 acting on SCC15 and SCC25 cells for 48h.3)Immunofluorescence was used to detect the expression of p16 and p21 protein in situ after JQ1 acting on SCC15 and SCC25 cells for 48h.4)Western Blot was used to detect the senescence marker p16,p21 and Lamin B1 protein levels.3.This chapter explore the mechanism of BRD4 inhibitor JQ1 inducing the senescence of SCC15 and SCC25.1)Analysis of the effect of JQ1 on the transcription level in SCC 15 and SCC25 cells by whole transcriptome sequencing.Analysis of the change of genes related to cell.cycle,cell senescence and cytokine-cytokine receptor interaction pathways by clustering heat map and GSEA;2)SCC 15 and SCC25 cell lines that knockdown BRD4 were constructed,and we analyzed the change of cell cycle,SA-β-gal activity and SASP factors;3)Clustering heat map analysis of E2F1 target genes.Western Blot detection of related pathway proteins involved in up-regulation of p21 expression on SCC 15 cells by JQ1 after 48h;4)EZH2 over-expression SCC15 cell was constructed,and we analyzed the change of cell cycle,SA-β-gal activity and SASP factors.Research Results1.JQ1 inhibits the proliferation of SCC15 and SCC25.1)TCGA database showed high frequency mutation and high expression of BRD4 and cell cycle related genes in HNSCC;2)JQ1 significantly inhibited the proliferation of SCC15 and SCC25 cells,and promoted cell arrest in G1 phase.2.JQ1 induces senescence of SCC15 and SCC25.1)Both types of cells exhibited a senescence phenotype,including increase of the proportion of SA-β-gal and SAHF positive cells.2)The SASP of SCC15 and SCC25 cells were significantly different.IL-8 and MMP3 were significantly upregulated in SCC15 cells,while IL-6 was significantly upregulated in SCC25 cells.3)It’s significant upregulation of p21 but not p16 protein in the two cell lines.3.Mechanism of BRD4 inhibitor JQ1 inducing the senescence of SCC15 and SCC251).The gene expression profile of OSCC cells induced by JQ1 was significantly changed.In the KEGG pathway analysis and GSEA,down-regulated genes were enriched in cell cycle,cellular senescence and cytokine-cytokine receptor interaction;2)OSCC cell lines with knock-down BRD4 were induced cellular senescence,promoted cell arrest in G1 phase,increased the activity of SA-β-gal and upregulated the mRNA level of SASP factors;3)JQ1 can significantly inhibit the expression of E2F1 and EZH2 in SCC15,which in turn causes DDR,and activates the ATM-p53-p21 signaling pathway;Overexpression of EZH2 can antagonize JQ1-induced senescence of SCC15 cells;meanwhile,JQ1 can also down-regulate c-Myc through a p53-independent pathway and directly up-regulate p21 expression.Research Conclusion1.In vitro experiments preliminarily showed that BRD4 inhibitor JQ1 induced senescence of SCC15 and SCC25 cells,significantly down-regulated OSCC-related oncogenes such as CCND1,CDK4,CDK6,E2F1 and c-Myc;2.Compared to the up-regulation of SASP factors in the classic cell senescence model,most proinflammatory cytokines were down-regulated after JQ1 on SCC15 and SCC25 cells;3.Mechanistically,through the E2F1-EZH2-p53-p21 axis and down-regulating c-Myc,JQ1 up-regulated the expression of p21 and promoted SCC15 cell arrest in G1 phase,then induced senescence.