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

Abstract Background Prostate cancer threatens the health of men over sixty years old, and its incidence ranks first among all urinary tumors among men. Enzalutamide remains the first-line drug for castration-resistant prostate cancer, however, tumors inevitably become resistant to enzalutamide. Hence, it is of great importance to investigate the mechanisms that induce enzalutamide resistance in prostate cancer cells. Methods Bioinformatic analyzing approaches were used to identified the over-expressed genes in prostate cancer tumor tissues from three GEO datasets. qRT-PCR, western blotting and immunochemistry/In situ hybridization staining assays were performed to assess the expression of SNHG4, RRM2, TK1, AURKA, EZH2 and RREB1. Cell cycle was measured by flow cytometry. CCK-8, plate colony formation and EdU assays were performed to assess the cell proliferation. Senescence-associated β-Gal assay was used to detect the cell senescence level. γ-H2AX staining assay was performed to assess the DNA damages of PCa cells. Luciferase reporter assay and RNA immunoprecipitation assay were performed to verify the RNA-RNA interactions. Chromatin immunoprecipitation assay was performed to assess the bindings between protein and genomic DNA. Results We found that RRM2 and NUSAP1 are highly expressed in PCa tumors and significantly correlated with poor clinical outcomes in PCa patients. Bioinformatic analysis as well as experimental validation suggested that SNHG4 regulates RRM2 expression via a let-7 miRNA-mediated ceRNA network. In addition, SNHG4 or RRM2 knockdown significantly induced cell cycle arrest and cell senescence, and inhibited DNA damage repair and cell proliferation, and the effects can be partially reversed by let-7a knockdown or RRM2 reoverexpression. In vitro and in vivo experiments showed that SNHG4 overexpression markedly enhanced cell resistance to enzalutamide. RREB1 was demonstrated to transcriptionally regulate SNHG4, and RREB1 was also validated to be a target of let-7a and thereby regulated by the SNHG4/let-7a feedback loop. Conclusion Our study uncovered a novel molecular mechanism of lncRNA SNHG4 in driving prostate cancer progression and enzalutamide resistance, revealing the critical roles and therapeutic potential of RREB1, SNHG4, RRM2 and let-7 miRNAs in anticancer therapy

RUNX2 interacts with SCD1 and activates Wnt/β‐catenin signaling pathway to promote the progression of clear cell renal cell carcinoma

Abstract Background Previous studies have demonstrated that Runt‐associated transcription factor 2 (RUNX2) serves as the main transcription factor for osteoblast differentiation and chondrocyte maturation. RUNX2 is related to a variety of tumors, particularly tumor invasion and metastasis, while the expression and molecular mechanisms of RUNX2 in clear cell renal cell carcinoma (ccRCC) keep to be determined. Stearyl CoA desaturase 1 (SCD1), an endoplasmic reticulum fatty acid desaturase, transfers saturated fatty acids to monounsaturated fatty acids, is expressed highly in numerous malignancies. Methods The Cancer Genome Atlas (TCGA) datebase and Western blot was used to analyzed the mRNA and protein levels of the target gene in ccRCC tissues and adjacent tissues. The proliferation ability of ccRCC cells was tested by colony forming and EdU assay. The migration ability of cells was detected by transwell assay. Immunoprecipitation was utilized to detect protein–protein interaction. Cycloheximide chase assay was used to measure the half‐life of SCD1 protein. Results In this study, the expressions of RUNX2 and SCD1 are increased in ccRCC tissues as well as ccRCC cell lines. Both RUNX2 and SCD1 could promote proliferation and migration in ccRCC cells. Furthermore, RUNX2 could physically interact with SCD1. In addition, the functional degradation and the inactivation of Wnt/β‐catenin signaling pathway triggered by the downregulation of RUNX2 could be partly offset by the overexpression of SCD1. Conclusion The findings indicate that the RUNX2/SCD1 axis may act as a potential therapeutic target via the Wnt/β‐catenin signaling pathway of ccRCC

Additional file 4 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 4: Table S3. Primer sequences for qRT-PCR

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

Additional file 2 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 2: Table S1. Associations between expression of SNHG4/RRM2 and clinicopathological characteristics of 60 PCa patients

Additional file 3 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 3: Table S2. Clinical information of prostate cancer patients included in this study