Study On The Mechanism Of Transcription Factor CTCF Transcriptional Regulation Of SFPQ Promoting The Malignant Biological Behavior Of Osteosarcoma

Background:Osteosarcoma(OS)is the most common malignancy of bone tissue,which occurs in adolescents,but its pathogenesis is still being explored.Studies have shown that SFPQ is a potential oncogenic factor in the pathogenesis of osteosarcoma,but the specific regulatory mechanism of SFPQ on osteosarcoma remains unclear.CTCF is a candidate tumor regulatory gene encoding multifunctional transcription factors,and its role in osteosarcoma and related mechanisms remains unclear.This study aims to search for potential upstream transcription factors of SFPQ and explore their roles in the proliferation,migration and invasion of osteosarcoma,providing new molecular targets for the clinical treatment of osteosarcoma.Objective:The objective of this study was to clarify the transcriptional regulation of CTCF on SFPQ in osteosarcoma,and the effect of CTCF regulation on the expression of SFPQ on the proliferation,migration,and invasion of osteosarcoma cells.Methods:(1)The carcinogenic effect of SFPQ in osteosarcoma was confirmed by real-time fluorescence quantitative PCR(qRT-PCR),survival curve and Depmap database analysis;Online transcription factor analysis websites(PROMO,Animal TFDB3.0,JASPAR)and UCSC database were used to screen the potential transcription factors of SFPQ as CTCF,and the correlation between the two genes was reversed-verified using hTFtarget and GEPIA2 database.Then,real-time quantitative PCR(qRT-PCR)and Western Blotting(WB)were used to detect the expression level of CTCF in osteosarcoma cell lines.(2)The expression level of CTCF in osteosarcoma cell lines was regulated by transfection of si-RNA or overexpressed plasmid.The effects of different expression levels of CTCF on the proliferation,migration and invasion of osteosarcoma cells were detected by cell plate cloning experiment,EdU experiment,CCK-8 experiment,cell scratch experiment and Transwell migration and invasion experiment.(3)Double luciferase reporting experiment was used to verify the targeting relationship between CTCF and SFPQ.(4)The rescue experiment was carried out by co-transfection of CTCF overexpression plasmid and si-SFPQ in osteosarcoma cells to detect the regulatory relationship between CTCF and SFPQ in osteosarcoma cell lines and their common effects on the biological behavior of osteosarcoma cells.Results:(1)SFPQ was identified as an oncogene in osteosarcoma by qRT-PCR,survival curve and Depmap database analysis.UCSC and GEPIA2 databases identified CTCF as a potential transcription factor of SFPQ,and there was a significant correlation between the two.qRT-PCR and WB assay showed that CTCF was highly expressed in osteosarcoma cell lines.(2)Cell function test results showed that the proliferation,migration and invasion of osteosarcoma cells were inhibited after CTCF knockdown in osteosarcoma cell lines.However,overexpression of CTCF in osteosarcoma cell lines enhanced the proliferation,migration and invasion of osteosarcoma cells.(3)The results of dual luciferase reporting experiment showed that compared with NC,the activity of dual luciferase was enhanced after CTCF was overexpressed in the reporter vector group.CTCF can bind to the promoter sequence of SFPQ and enhance its expression level.(4)Rescue experiments showed that overexpression of CTCF could reduce the inhibition of knockdown SFPQ on proliferation,migration,and invasion of osteosarcoma cell lines.Conclusion:CTCF plays a carcinogenic role in osteosarcoma.As a transcriptional activator of SFPQ,CTCF positively regulates and promotes the expression of SFPQ,and enhances the proliferation,migration and invasion ability of osteosarcoma cells,suggesting that targeting CTCF/SFPQ can provide research strategies for further exploration of clinical treatment of osteosarcoma.