Fig. 2

circRHOBTB3 is secreted outside of tumor cells and suppresses CRC cells in vitro and in vivo. (A) Expression of circRHOBTB3 in CRC or normal colonic epithelial cell lines and (B) exosomes derived from them. (C) Nucleoplasmic RNA isolation and RT-qPCR for the localization of circRHOBTB3. ACTB and GAPDH were used as cytoplasmic positive controls, and MALTAL1 was used as a nuclear positive control. (D) Overexpression of circRHOBTB3 by lentivirus vector in RKO and HCT116 cells. The quantification analysis results of (E) cell proliferation, (F) migration and invasion assays of circRHOBTB3-OE RKO and HCT116 cells. (G) Schematic of gRNA design and construction for circRHOBTB3. (H) Specific KD of circRHOBTB3 by RfxCas13d-BSJ-gRNA in SW480 cells. The quantification analysis results of (I) Cell proliferation, (J) migration and invasion assays of circRHOBTB3 KD SW480 cells. (K) Images of the subcutaneous xenograft tumors. (L) Tumor volume and (M) tumor weight of xenograft tumors. (N) Ki67 staining of xenograft tumors. The right column diagram shows the quantification analysis results. (O) The quantification of the liver distant metastatic foci of liver distant metastasis model established by splenic injection of control (empty vector) and circRHOBTB3-OE HCT116 cells in nude mice. (P) HE staining in liver metastatic foci. (Q) Expression of circRHOBTB3 in the plasma and (R) urine of mice inoculated with control or circRHOBTB3-OE HCT116 cells. Data were shown as mean ± SD (A, B, C, D, E, H, I, L, M, N, O, Q, X) or mean ± SEM (F, J), * P < 0.05, ** P < 0.01, *** P < 0.001, NS P > 0.05, in Student’s test (A, B, C, D, E, H, I, L, M, N, O, Q, R) or paired Student’s test (F, J)