Screening And Identification Of CCAT2-associated Transcription Factors In KM12SM Cells

Objectives To investigate the transcriptional regulation mechanism of long-chain noncoding gene CCAT2,by detecting the difference of G / T-allele transcription activity and analyzing the associated transcription factors in KM12SMMethods To construct of double luciferase reporter gene plasmid based on G-allele / Tallele site,which were asigned p GL3-153Gs-LUC,p GL3-153Ts-LUC and p GL3-837 GsLUC and p GL3-837Ts-LUC.The transcriptional activity of CCAT2 promoter was detected by double luciferase reporter gene analysis in low metastatic colorectal cancer cell line KM12 C and high metastatic colon cancer cell line KM12 SM.Four reporter gene plasmids were constructed driven by different promoter truncations,including G-type 153 bp,T-type153 bp and G-type 837 bp,T-type 837 bp.Based on the bait of the responsible promoter segment,Pull-Down combined protein mass spectrometry was used to screen the candidate transcription factors binding to the promoter of CCAT2.The m RNA and protein expression of transcription factors were verified by q PCR and Western-blot.Ch IP combined with q PCR and dd PCR was used to detect the binding activity of transcription factors in the CCAT2 promoter.Results In KM12 C cells,there was no relative activity difference between the pGL3-GsLUC group and p GL3-Ts-LUC group,while in KM12 SM cells,the difference between the two groups,p GL3-153Gs-LUC and p GL3-153Ts-LUC,was statistically significant(P<0.05).Pull-Down combined with mass spectrometry analysis showed that the differential proteins were Probable ATP-dependent RNA Helicase DDX17(DDX17)、Vimentin、Recombining Binding Protein Suppressor of Hairless(RBPJ)、Probable ATPdependent RNA Helicase DDX5(DDX5).The m RNA and protein expression of transcription factors were verified by q PCR and Western-blot.The results showed that the expression of RBPJ and Vimentin were low in KM12 cells,while DDX5 was highly expressed,and the expression of DDX5 in KM12 SM cells was higher than that in KM12 C cells.Western-Blot results showed that the expression level of DDX5 protein in KM12 SM is higher than that in KM12 C cells.Ch IP results showed the DDX5 binding in CCAT2 promoter.The results of dd PCR showed that the G-type allele-binding activity was higher than the T-type allele.Conclusions 1 DDX5 is one of transcription factors of CCAT2.2 data showed that DDX5 could mainly bind to the G allele promoter in KM12 SM cells.