Additional file 1 of LncRNA SNHG4 promotes prostate cancer cell survival and resistance to enzalutamide through a let-7a/RREB1 positive feedback loop and a ceRNA network

Additional file 1: Figure S1. Flow chart of the study. Figure S2. a. The staining intensity of RRM2 was significantly stronger in PCa tumors (n=20) than in adjacent normal prostate tissues (n=20) and BPH tissues (n=10) by IHC staining. b. qRT-PCR analysis suggested that RRM2 was highly expressed in PCa cell lines (DU145, PC3, 22Rv1 and LNCaP) compared to normal prostate epithelial cell line RWPE-1. c. RRM2 levels were significantly decreased or increased in response to RRM2 knockdown or overexpression in 22Rv1 and LNCaP cells by qRT‒PCR and western blotting. d. Knockdown of RRM2 notably induced cell cycle arrest in the G1 stage in 22Rv1 and LNCaP cells. The image for each experiment is shown in Fig. 2h. e. The correlation between the expression levels of NEAT1 and RRM2 in PCa tumor samples (n=499) was not significant. The data were obtained from the TCGA_PRAD dataset. f. Knockdown of NEAT1 had no effect on RRM2 levels in RV-a and LNCaP cells, as determined by western blotting. g. Let-7a-5p levels were significantly decreased or increased in response to transfection of let-7a-5p inhibitor or mimics in 22Rv1 and LNCaP cells by qRT‒PCR. h. High SNHG4 levels indicate poor progression free interval in PCa patients, data from the TCGA_PRAD dataset. i. qRT-PCR analysis suggested that SNHG4 was highly expressed in PCa cell lines (DU145, PC3, 22Rv1 and LNCaP) compared to normal prostate epithelial cell line RWPE-1. j. SNH4 coexpressed genes were enriched in the biological term “Cell Cycle”, indicating a potential role of SNHG4 in regulating the cell cycle of PCa cells. k. Representative ISH/IHC staining images of the indicated gene/protein expression in a series of clinical pathological sections from 30 PCa patients. The staining intensity of each gene/protein was scored as 0 to 5 (0: no staining, 1: very weak staining, 2: weak staining, 3: medium staining, 4: strong staining, 5: very strong staining), and 1-3 were classified as low expression, whereas 4-5 were defined as high expression. l. SNHG4 level is positively correlated with each indicated protein in PCa tumors (p<0.05, Fisher’s exact test). Figure S3. a. The knockdown efficiency of siRNAs against each indicated gene was measured by qRT‒PCR and western blotting. b. Knockdown of EZH2, AURKA or TK1 reduced the proliferation of PCa cells. Representative images of EdU staining of Fig. 6c and d. Magnification: 100X. Figure S4. a. Knockdown of each indicated gene significantly induced cell senescence in LNCaP cells, and senescent cell numbers were counted and compared. Magnification: 200X. b and c. qRT-PCR analysis showed that SNHG4 levels in 22Rv1 and LNCaP cells were significantly decreased in response to SNHG4 knockdown, whereas let-7a knockdown or RRM2 overexpression rescued SNHG4 expression. d. Western blot analysis showed that SNHG4 knockdown significantly decreased RRM2 expression, whereases let-7a knockdown or RRM2 overexpression rescued RRM2 expression in 22Rv1 and LNCaP cells. e. Knockdown of SNHG4 reduced the proliferation of PCa cells, whereas let-7a knockdown or RRM2 overexpression rescued cell proliferation of 22Rv1 and LNCaP cells. Representative images of EdU staining of Fig. 7b. Magnification: 100X. Figure S5. a. γ-H2AX foci were detected in PCa cells treated with negative control, SNHG4 knockdown, double knockdown of SNHG4 and let-7a, or SNHG4 knockdown with RRM2 overexpression by immunofluorescence staining. The indicated cells were treated with Docetaxel (10 nM) for 24 hours. Magnification: 200X. b. SNHG4 knockdown significantly induced cell cycle arrest in G1 phase, whereas knockdown of let-7a or RRM2 overexpression rescued the arrested cell cycle. The cell cycle was measured by FACS in pretreated 22Rv1 and LNCaP cells. Supplemental Methods.