Alteration Of Exosome-carried MicroRNA In OSCC By BRD4 Inhibitor JQ1

[Background]Oral squamous cell carcinoma(OSCC),an oral and maxillofacial malignant tumor,has a high morbidity and mortality,the 5-year survival rate is less than 50%,and the average onset age decreases year by year.As a member of the bromine domain and extra terminal family,bromine domain protein 4(BRD4)regulates cell cycle by regulating cell transcription,so its abnormal expression is often related to the occurrence of many diseases,especially malignant tumors.Exosomes are heterogeneous nano-sized vesicles released by various cells for intercellular communication.Exosomes can specifically package and transport various proteins,lipids and nucleic acids,and mediate the interaction between tumor cells and tumor microenvironment.Its production and cargo loading are regulated by a variety of Rab family proteins.Small molecule BRD4 inhibitor JQ1 inhibits the transcription of oncogenes and has a bright future in the treatment of malignant tumors.[Objective]To find out whether JQ1 can affect the function of tumor cells and other cells in tumor microenvironment by affecting the types and relative content of miRNA in exosomes of OSCC,in order to explore the potential of miRNA as a biomolecular marker in early screening,diagnosis,prevention and treatment of OSCC.[Method]1.Culture,passage and JQ1 treatment of CAL27 and SCC25 cell lines;2.Determination of cell proliferation rate;3.Western blotting analysis;4.Real-time fluorescence quantitative reverse transcriptase polymerase chain reaction(qRT-PCR);5.Construction and sequencing of miRNA library of exosomes in cell supernatant;6.Bioinformatics analysis of miRNA sequencing in exosomes;7.Extraction and sequencing of mRNA from tongue squamous cell lines;8.Bioinformatics analysis of intracellular mRNA sequencing;9.Comparison and validation of sequencing results;10.Data processing and analysis[Results]1.After treatment with JQ1,the proliferation rate of tongue squamous cell carcinoma cell lines CAL27 and SCC25 decreased in varying degrees with the increase of JQ1 concentration,and the working concentrations of JQ1 for CAL27 and SCC25 cell lines were 4μM and 2μM,respectively.2.The species and relative content of miRNA in exosomes of tongue squamous cell carcinoma cell lines CAL27 and SCC25 treated with working concentration of JQ1 changed significantly3.The expression of some genes in tongue squamous cell carcinoma cell lines CAL27 and SCC25 also changed significantly after treatment with working concentration of JQ1.4.The changes of the types and relative contents of some miRNA in exosomes of tongue squamous cell carcinoma cell lines CAL27 and SCC25 are closely related to the expression of some genes in the cells,which participate in a variety of physiological activities including tumor immunity.5.The expression of Rab5,Rab7,Rab11,Rab27A and Rab35 in tongue squamous cell carcinoma cell lines CAL27 and SCC25 decreased at nucleotide and protein levels after treatment with working concentration of JQ1.[Conclusions]JQ1 treatment can inhibit the expression of Rab family proteins in tongue squamous cell carcinoma cells,and then change the types and relative content of miRNA carried by exosomes.After that,exosomes will be transferred to the tumor microenvironment and act on immature dendritic cells,affecting their maturation and function.