Additional file 2 of circCUL2 regulates gastric cancer malignant transformation and cisplatin resistance by modulating autophagy activation via miR-142-3p/ROCK2

Additional file 2: Figure S1. A: Heatmap of circRNA sequencing (GSE100170); B: Volcano plot of GSE100170. C and D: GO (C) and KEGG (D) analyses of differentially expressed circRNA host genes. Figure S2. A-I: The associations of circCUL2 expression with clinical stage (A), lymph node status (B), histological type (C), gender (D), age (E), tumor size (F), tumor depth (G), Helicobacter pylori infection status (H) and Lauren classification (I) as determined through qRT-PCR. Figure S3. A: Schematic representation and target sequences of the siRNAs specific to the backsplice junction of circCUL2. B-C: The proliferation of SGC-7901 cells transfected with circCUL2-specific siRNA or an overexpression plasmid was assessed by EdU (B) and colony formation assays (C). D: Wound healing assay to assess the effect of circCUL2 on cell migration. E: Transwell assay to assess the migration and invasion of SGC-7901 cells. Figure S4. A: The mRNA expression of CUL2 was significantly increased in cells transfected with the pcDNA-3.1 CUL2 vector (pcDNA3.1-CUL2) and decreased in cells transfected with CUL2 siRNA. B and C: The proliferation of GC cells transfected with pcDNA3.1-CUL2 and CUL2 siRNA was assessed by CCK-8 (B) and EdU (C) assays. D: Overexpression and knockdown of CUL2 did not change the migration and invasion capacities of GC cells. Figure S5. A: The protein level of ROCK2 in GC tissues was evaluated by IHC. B-H: The association of circCUL2 expression with clinical stage (B), lymph node status (C), histological type (D), gender (E), age (F), tumor size (G) and tumor depth (H). I: The mRNA levels of circCUL2, miR-142-3p and ROCK2 in mouse tumor tissues. J: The ROCK2 protein level in mouse tumor tissues, as evaluated by IHC. Figure S6. A: circCUL2, miR-142-3p and ROCK2 expression in AGS and SGC-7901 cells with circCUL2 overexpression or knockdown. B: miR-142-3p and ROCK2 expression in AGS and SGC-7901 cells transfected with miR-142-3p mimics or an inhibitor. C: Luciferase reporter assay was used to detect the binding of miR-142-3p to circCUL2 and ROCK2 in SGC-7901 cell lines. D: qRT-PCR of circCUL2 and miR-142-3p expression pulled down from SGC-7901 cell lysates and enriched with a circCUL2-specific probe. E-F: Cotransfection of miR-142-3p mimics and circCUL2 overexpression plasmids or miR-142-3p inhibitors and circCUL2-specific siRNA to detect the mRNA (E) and protein (F) levels of ROCK2 in SGC-7901 cell lines. G-K: Cotransfection of miR-142-3p mimics and circCUL2 overexpression plasmids to investigate malignant transformation by CCK-8 (G), EdU (H), colony formation (I), wound healing (J) and Transwell (K) assays in the SGC-7901 cell line. Figure S7. A and B: The miR-142-3p (A) and ROCK2 (B) levels in GC patients from TCGA database. C: The negative correlation of miR-142-3p with ROCK2 based on TCGA data (P < 0.01). D-E: The association of miR-142-3p (D) and ROCK2 (E) expression with the clinical stage. F: The mRNA expression of ROCK2 in cells cotransfected with miR-142-3p inhibitors and ROCK2 siRNA. G and H: The proliferation, migration and invasion of cells cotransfected with miR-142-3p inhibitors and ROCK2 siRNA were measured by CCK-8 (G), EdU (H) and Transwell assays(I). Figure S8. A-C: Relative viability of circCUL2 overexpression plasmid- or siRNA-transfected cells exposed to MMC (A), DOX (B) and 5-FU (C) at the indicated concentrations for 48 h. Figure S9. A-D: Cell growth (A), IC50 values (B), and apoptosis (C and D) in SGC-7901/DDP cell lines cotransfected with miR-142-3p mimics and circCUL2 overexpression plasmids. E: mRNA levels of circCUL2, miR-142-3p and ROCK2 in mouse tumor tissues. F: ROCK2 protein levels in mouse tumor tissues, as evaluated by IHC.

附加文件2：图S1。A：环状RNA（circRNA）测序热图（GSE100170）；B：GSE100170数据集的火山图。C、D：差异表达环状RNA宿主基因的基因本体（GO）富集分析（C）与京都基因与基因组百科全书（KEGG）富集分析（D）。
图S2。A~I：通过实时荧光定量聚合酶链反应（qRT-PCR）检测环状RNA CUL2（circCUL2）的表达与临床分期（A）、淋巴结状态（B）、组织学类型（C）、性别（D）、年龄（E）、肿瘤大小（F）、浸润深度（G）、幽门螺杆菌感染状态（H）及Lauren分型（I）的相关性。
图S3。A：靶向circCUL2反向剪接连接位点的小干扰RNA（siRNA）的序列示意图与靶序列。B、C：分别采用5-乙炔基-2'-脱氧尿苷（EdU）染色实验（B）与集落形成实验（C）评估转染circCUL2特异性siRNA或过表达质粒的SGC-7901细胞的增殖能力。D：划痕愈合实验检测circCUL2对细胞迁移能力的影响。E：Transwell小室实验检测SGC-7901细胞的迁移与侵袭能力。
图S4。A：转染pcDNA3.1-CUL2载体的细胞中CUL2的mRNA表达水平显著升高，而转染CUL2 siRNA的细胞中CUL2的mRNA表达水平降低。B、C：分别采用细胞计数试剂盒-8（CCK-8）实验（B）与EdU染色实验（C）评估转染pcDNA3.1-CUL2及CUL2 siRNA的胃癌（GC）细胞的增殖能力。D：过表达与敲低CUL2均未改变胃癌细胞的迁移与侵袭能力。
图S5。A：采用免疫组化（IHC）检测胃癌组织中ROCK2的蛋白表达水平。B~H：circCUL2的表达与临床分期（B）、淋巴结状态（C）、组织学类型（D）、性别（E）、年龄（F）、肿瘤大小（G）及浸润深度（H）的相关性。I：小鼠肿瘤组织中circCUL2、miR-142-3p及ROCK2的mRNA表达水平。J：采用免疫组化检测小鼠肿瘤组织中ROCK2的蛋白表达水平。
图S6。A：过表达或敲低circCUL2的AGS与SGC-7901细胞中circCUL2、miR-142-3p及ROCK2的表达水平。B：转染miR-142-3p模拟物或抑制剂的AGS与SGC-7901细胞中miR-142-3p与ROCK2的表达水平。C：采用荧光素酶报告基因实验检测SGC-7901细胞系中miR-142-3p与circCUL2及ROCK2的结合情况。D：采用qRT-PCR检测从SGC-7901细胞裂解液中通过circCUL2特异性探针富集、下拉得到的样本中circCUL2与miR-142-3p的表达水平。E、F：共转染miR-142-3p模拟物与circCUL2过表达质粒，或共转染miR-142-3p抑制剂与circCUL2特异性siRNA，以检测SGC-7901细胞系中ROCK2的mRNA（E）与蛋白（F）表达水平。G~K：共转染miR-142-3p模拟物与circCUL2过表达质粒，采用CCK-8实验（G）、EdU染色实验（H）、集落形成实验（I）、划痕愈合实验（J）及Transwell小室实验（K）检测SGC-7901细胞系的恶性转化情况。
图S7。A、B：来自癌症基因组图谱（TCGA）数据库的胃癌患者样本中miR-142-3p（A）与ROCK2（B）的表达水平。C：基于TCGA数据的miR-142-3p与ROCK2的负相关关系（P<0.01）。D、E：miR-142-3p（D）与ROCK2（E）的表达与临床分期的相关性。F：共转染miR-142-3p抑制剂与ROCK2 siRNA的细胞中ROCK2的mRNA表达水平。G：采用CCK-8实验检测共转染组细胞的增殖、迁移与侵袭能力；H：采用EdU染色实验检测；I：采用Transwell小室实验检测。
图S8。A~C：转染circCUL2过表达质粒或siRNA的细胞在不同浓度丝裂霉素C（MMC）（A）、多柔比星（DOX）（B）及5-氟尿嘧啶（5-FU）（C）中孵育48小时后的相对存活率。
图S9。A~D：共转染miR-142-3p模拟物与circCUL2过表达质粒的SGC-7901/DDP细胞系的细胞生长情况（A）、半数抑制浓度（IC50）（B）及细胞凋亡情况（C、D）。E：小鼠肿瘤组织中circCUL2、miR-142-3p及ROCK2的mRNA表达水平。F：采用免疫组化检测小鼠肿瘤组织中ROCK2的蛋白表达水平。