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.

附加文件1：图S1 本研究的流程图。

图S2 a. 免疫组化（Immunohistochemistry, IHC）染色结果显示，前列腺癌（Prostate Cancer, PCa）组织样本（n=20）中RRM2的染色强度显著强于配对正常前列腺组织（n=20）及良性前列腺增生（Benign Prostatic Hyperplasia, BPH）组织（n=10）。
b. 实时定量荧光PCR（Quantitative Real-time Polymerase Chain Reaction, qRT-PCR）分析表明，相较于正常前列腺上皮细胞系RWPE-1，RRM2在PCa细胞系（DU145、PC3、22Rv1及LNCaP）中呈高表达。
c. 通过qRT-PCR与蛋白质印迹法（Western Blotting）检测发现，在22Rv1与LNCaP细胞中，敲低RRM2可显著降低其表达水平，而过表达RRM2则可显著上调其表达。
d. 敲低RRM2可显著诱导22Rv1与LNCaP细胞发生G1期细胞周期阻滞，相关实验结果见图2h。
e. 来自癌症基因组图谱前列腺癌数据集（TCGA_PRAD）的499例PCa组织样本分析显示，NEAT1与RRM2的表达水平无显著相关性。
f. 蛋白质印迹法检测结果表明，敲低NEAT1对RV-a与LNCaP细胞中RRM2的表达水平无显著影响。
g. qRT-PCR检测显示，在22Rv1与LNCaP细胞中转染let-7a-5p抑制剂可显著下调let-7a-5p表达，而转染let-7a-5p模拟物则可显著上调其表达水平。
h. 来自TCGA_PRAD数据集的分析结果显示，SNHG4高表达的PCa患者无进展生存期更短。
i. qRT-PCR分析表明，相较于正常前列腺上皮细胞系RWPE-1，SNHG4在PCa细胞系（DU145、PC3、22Rv1及LNCaP）中呈高表达。
j. SNH4的共表达基因显著富集于“细胞周期”生物学过程，提示SNHG4可能参与调控PCa细胞的细胞周期进程。
k. 纳入30例PCa患者的系列临床病理切片中，针对指定基因/蛋白的代表性原位杂交（In Situ Hybridization, ISH）/免疫组化染色图像。各基因/蛋白的染色强度评分为0~5分（0：无染色；1：极弱染色；2：弱染色；3：中等染色；4：强染色；5：极强染色），其中1~3分归类为低表达，4~5分归类为高表达。
l. PCa组织中SNHG4的表达水平与各指定蛋白的表达呈正相关（p<0.05，Fisher精确检验）。

图S3 a. 通过qRT-PCR与蛋白质印迹法检测了针对各指定基因的小干扰RNA（Small interfering RNA, siRNA）的敲低效率。
b. 敲低EZH2、AURKA或TK1可抑制PCa细胞的增殖。图6c与6d的5-乙炔基-2'-脱氧尿苷（EdU）染色代表性图像，放大倍数：100×。

图S4 a. 敲低各指定基因可显著诱导LNCaP细胞发生细胞衰老，并对衰老细胞数量进行了计数与比较，放大倍数：200×。
b、c. qRT-PCR分析显示，在22Rv1与LNCaP细胞中，敲低SNHG4可显著下调SNHG4的表达水平；而敲低let-7a或过表达RRM2可逆转该抑制效应，恢复SNHG4的表达。
d. 蛋白质印迹法检测显示，敲低SNHG4可显著降低RRM2的表达水平；而敲低let-7a或过表达RRM2可逆转该抑制效应，恢复22Rv1与LNCaP细胞中RRM2的表达。
e. 敲低SNHG4可抑制PCa细胞的增殖，而敲低let-7a或过表达RRM2可逆转这一增殖抑制效应，恢复22Rv1与LNCaP细胞的增殖能力。图7b的EdU染色代表性图像，放大倍数：100×。

图S5 a. 通过免疫荧光染色检测了经以下处理的PCa细胞中的γ-H2AX焦点：阴性对照处理、敲低SNHG4、同时敲低SNHG4与let-7a、或敲低SNHG4同时过表达RRM2。所有受试细胞均经多西他赛（10 nM）处理24小时，放大倍数：200×。
b. 荧光激活细胞分选术（Fluorescence Activated Cell Sorting, FACS）检测显示，敲低SNHG4可显著诱导22Rv1与LNCaP细胞发生G1期细胞周期阻滞，而敲低let-7a或过表达RRM2可逆转该细胞周期阻滞效应。

补充方法。