The Transcription Factor SP2 Regulates The Carcinogenic Behavior Of Salivary Gland Mucoepidermoid Carcinoma By Inhibiting CLIC3’s Transcription

Objective: Chloride intracellular channel protein 3(CLIC3)is significantly expressed in salivary gland mucoepidermoid carcinoma(MEC),but its function and possible mechanism in MEC need to be further studied.In this study,CLIC3 was evaluated in the process of tumor proliferation,migration and invasion by downregulating the expression of CLIC3 in MEC.The upstream transcription factor SP2 was predicted and screened by bioinformatics to verify its relationship with the upstream and downstream regulation of CLIC3 expression and its possible role in MEC,so as to find new targets for the treatment of salivary gland mucinous epidermoid carcinoma.Methods: The effect of CLIC3 knockdown on the biological behavior of salivary gland mucoepidermoid cancer cells was verified by EdU experiment,cell scratch experiment and Transwell experiment.Step-down and identification of the core promoter region of the CLIC3 gene using a double luciferase reporter experiment,narrowing thresholds in JASPAR and UCSC databases,predicting and screening transcription factors bound upstream of the CLIC3 gene.The double luciferase experiment and chromatin immunoprecipitation experiment were used to further verify whether the transcription factor SP2 binds to the promoter region of the CLIC3 gene to regulate its transcriptional activity.Subsequently,in order to confirm the effect of SP2 on the expression of CLIC3,the level of SP2 in salivary gland mucus epidermoid cancer cells was overexpressed or knocked out,and the expression level of CLIC3 was detected by RT-q PCR and Western Blot experiment,and the expression correlation between the two in cancer cells was analyzed.Finally,the EdU experiment,cell scratch experiment and Transwell experiment were further used to evaluate the role of SP2 in the transcriptional regulation of CLIC3,thereby affecting the proliferation,migration and invasion ability of salivary gland mucinous epidermoid cancer cells.Results: EdU experiment,Transwell experiment and cell scratch experiment confirmed that after knocking down CLIC3,the proliferation,migration and invasion ability of salivary gland mucus epidermoid cancer cells decreased significantly.Dual luciferase reporter experiments showed that the core promoter region of CLIC3 was located 262 bp upstream of the transcription initiation site.Based on database grafting,the transcription factor SP2 upstream of the CLIC3 gene was predicted,and subsequent endogenous and exogenous CHIP experiments confirmed that SP2 could bind to the core promoter region of CLIC3.By binding to a specific sequence on the CLIC3 promoter,SP2 targets the regulation of CLIC3 expression,and after overexpression of SP2,firefly activity decreases,and CLIC3 m RNA and protein levels also decrease.After knocking down SP2,the activity of fireflies was enhanced,and the expression level of CLIC3 was also locally regulated.In the salivary gland mucus epidermoid carcinoma M3SP4 cells,overexpression of SP2 can significantly reduce the proliferation,migration and invasion ability of tumors with down-regulated CLIC3.Knocking down the expression of SP2 can improve the decrease in tumor biological function caused by downregulation of CLIC3 gene.Moreover,overexpression SP2 also reversed the promotion effect of overexpressed CLIC3 on salivary gland mucus epidermoid cancer cells,and changed the biological behavior of tumor growth,migration and invasion.Conclusions: The high expression of CLIC3 can promote the proliferation,migration and invasion of salivary gland mucus epidermoid cancer cells.In this study,the core promoter region of the CLIC3 gene was identified for the first time,predicting and confirming that the transcription factor SP2 binds directly to the CLIC3 gene,thereby affecting the biological behavior of salivary gland mucus epidermoid cancer cells.SP2 can lead to transcriptional inhibition of CLIC3,and overexpression or downregulation of SP2 can reverse the effects of overexpressed or knocked down CLIC3 on the growth,migration and invasion of salivary gland mucoepidermoid cancer cells,suggesting that the SP2/CLIC3 axis is a potential therapeutic target for MEC.