Fig. 7

CDK13-circCDK13-miR-212-5p/miR-449a-E2F5 regulatory axis participates in prostate tumorigenesis. a and b, PC3 cells engineered to stably overexpress GFP-shcircCDK13 (LV-shcircCDK13), GFP-shE2F5 (LV-shE2F5) or negative control (LV-GFP) were injected subcutaneously in 200 μl PBS/Matrigel (50:50) into the mouse forelimb (left: LV-GFP; right: LV-shcircCDK13) (left panel), or GFP-shE2F5 (left: LV-shE2F5) or both (right: LV-shcircCDK13 + LV-shE2F5) (right panel). At the final time point (21 days after injection), the tumor volumes in each group were measured both in situ by fluorescence imaging (A) and after resection of tumors (B) (n = 12 in each group). c, Tumor volume was determined by direct measurement with calipers and calculated by the formula: volume = [(length × width2) / 2]. *P < 0.05, **P < 0.01 vs. LV-GFP (n = 12 in each group), #p<0.05, ##p<0.01 vs. LV-shcircCDK13 or LV-shE2F5 (n = 10 in each group). d, Western blotting detected the expression of E2F5, CDK13 and p21 in xenograft tumors prepared as in (A). e, The xenograft models of nude mice were prepared as in (A), the TUNEL staining detected cell apoptosis in xenograft tumors. Blue staining represents the nucleus, and red staining indicates TUNEL-positive cells. Bar = 25 μm. f, PC3 and 22RV1 cells were treated with indicated pharmacological inhibitors for different signaling pathways, and Western blot analysis detected the expression of CDK13 and E2F5 protein. g-i, RT-qPCR detected the E2F5 mRNA (G), CDK13 mRNA (H), and circCDK13 (I) expression in PC3 and 22RV1 cells treated with 1-Azak or DMSO. *P < 0.05 vs. the vehicle. j, PC3 and 22RV1 cells were transfected with shE2F5 or empty vector pLKO and treated with 1-Azak for 24 h, and then cell viability was measured by MTS assay. *P < 0.05 vs. the vehicle, #p<0.05 vs. shE2F5 or 1-Azak. k, PC3 and 22RV1 cells were transfected with shcircCDK13 alone or combined with 1-Azak treatment. Cell proliferation was measured by colony formation assay. Right panel shows the quantitative analysis of colony numbers from three independent experiments. *P < 0.05 vs. the vehicle, #p<0.05 vs. shcircCDK13 or 1-Azak. l, PC3 and 22RV1 cells were treated as in (K), cell apoptosis was detected by AnnexinV/PI flow cytometry. Right panel shows the apoptosis rate of three independent experiments. *P < 0.05 vs. the vehicle, #p<0.05 vs. shcircCDK13 or 1-Azak