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. 2020 Dec 26;13(1):49. doi: 10.3390/cancers13010049

Figure 5.

Figure 5

circZNF609 promotes colorectal cancer development. (A) shRNA design for knocking down of circZNF609 based on pri-miR-331 cassette driven by Poll-II promoter. shRNA targeting circZNF609 was designed by replacing miR-331 sequence. Using this construct, circZNF609 is efficiently knocked down in HEK293T-sh-circZNF609 upon doxycycline induction. (B) Relative expression of circZNF609 and ZNF609 (using two different primer pairs P01 and P02) in HT29-sh-circZNF609 and HCT116-sh-circZNF609 upon doxycycline induction validated by qPCR. (C) Knockdown of circZNF609 decreases cell proliferation in both HT29-sh-circZNF609 and HCT116-sh-circZNF609 cell lines. (D,E) HT29-sh-circZNF609 cell lines were injected into spleen of the SCID mice. Spleens containing primary tumors (PT) and livers with metastases (LM) were imaged for macro analysis. The according tumors were resected and measured by tumor weight (n = 8). Relative expression of circZNF609 of RNA isolated from PT and LM by qPCR, which was normalized to ZNF609. (F) Relative expression of circZfp609 in CMT93-sh-circZfp609 upon doxycycline induction validated by qPCR. (G) In vivo data of tumor resected from the mice subcutaneously injected with CMT93-sh-circZfp609 upon doxycycline induction. The tumor development was calculated by measuring the tumor weight (n = 11). (H) CMT93-circZfp609 cell was induced by doxycycline and the cells were harvested for northern blot analysis. (I) In vivo data from the mice subcutaneously injected of CMT93-circZfp609 cells upon doxycycline induction (n = 12). The tumor development was calculated by measuring the tumor weight. Dox: Doxycycline. ns: not significant, p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.