Fig. 8

Therapeutic value of targeting TIMP1 alternative splicing in CRC. A The scheme depicts targeting the TIMP1 alternative splicing by CRISPR/dCasRx system. B RT-PCR results show that CRISPR/dCasRx targeting significantly decrease the ratio of TIMP1 transcripts with or without exon 4–5 in HCT-8 and SW480 cells transfecting with guides targeting intron–exon boundary compared with non-target guide. The ratio was quantified with ImageJ software. C, D Tumor cell migration (C) and invasion (D) assay of HCT-8 and SW480 cells with indicated treatments. P values were calculated by two-sided Student’s t test, *** P < 0.001. The migrated or invaded cells were quantified by counting in five fields. Scale bar, 100 μm. E MTT assay of HCT-8 and SW480 cells with indicated treatments. Data were shown as mean ± SD. P values were calculated by two-sided Student’s t test, ** P < 0.01, *** P < 0.001. F The injection schematic for in vivo anti-tumor effect analysis of targeting TIMP1 exon 4–5 skip by CRISPR/dCasRx system. G Xenograft mouse model established using SW480 cells in BALB/c nude mice with indicated treatment (n = 6 mice per group). In vivo generated tumors are depicted. H, I Analysis of tumor growth (H) and weight (I) in the xenograft mouse model. Data are presented as mean ± SEM of n = 6 mice per group. Two-way ANOVA and one-way ANOVA followed by Tukey test. J Representative H&E and IHC images of randomly selected tumors are shown. Scale bar, 100 µm