Fig. 7

LINC01852 is transcriptionally regulated by HOXD8. (A) The flow chart outlines a general approach for screening transcription factors (TFs) that potentially regulate the expression of LINC01852. (B) qRT-PCR was used to verify the expression of HOXD8, LINC01852, SRSF5, and PKM2 in HOXD8-overexpressing CRC cells. (C) A dual-luciferase reporter assay was used to identify the specific binding site of HOXD8 in the LINC01852 promoter. The left panel shows the HOXD8 binding sites in the LINC01852 promoter; the middle panel shows a schematic representation of the truncated (FL, F1, F2, and F3) and mutated (FM) promoters of LINC01852 in the PGL3-Basic-LINC01852-luc reporter plasmid; the right panel shows the relative luciferase reporter activities of the indicated reporter plasmids. (D) ChIP assays were performed to investigate the binding site of HOXD8 in the LINC01852 promoter. (E) qRT-PCR was used to measure the expression level of HOXD8 in CRC tissues (n = 23), and Pearson correlation analysis was performed to analyze the correlation between HOXD8 mRNA expression and LINC01852 expression. (F, G) The protein levels of HOXD8 in CRC tissues (n = 146) and NCTs (n = 131) were measured via IHC. Representative images of HOXD8 IHC staining (left) and scores (right) in CRC and NCT tissues (F). The pie chart indicates the alteration in HOXD8 expression in paired samples (n = 118) (G). (H) Kaplan-Meier survival analyses based on the expression of HOXD8 in CRC tissues. (I) Violin plots were generated to visualize the distribution of LINC01852 in each group stratified based on the protein expression level of HOXD8. (J) Kaplan-Meier survival curves generated based on the expression of LINC01852 and HOXD8 in CRC tissues