Supplementary Material for: circZMYM2 Competed Endogenously with miR-335-5p to Regulate JMJD2C in Pancreatic Cancer

Background/Aims: We aimed to study the involvement of circZMYM2 (hsa_circ_0099999) in pancreatic cancer (PC) cell proliferation, apoptosis and invasion and to figured out the underlying mechanism of circZMYM2 regulating miR-335-5p and JMJD2C. Methods: CircRNA differential expressions in twenty PC samples and paired normal tissue samples were analyzed using Arraystar Human CircRNA microarray V1. CircZMYM2 expression level was determined via qRT-PCR. The effects of circZMYM2 inhibition and overexpression on cell proliferation, cell apoptosis and cell invasion were investigated by CCK-8 assays, Flow cytometry assays and Transwell assays. An animal experiment on nude mice was put forward to test the influence of circZMYM2 knockdown on tumor growth. The relationship between circZMYM2, miR-335 and JMJD2C was verified by RNA pull down, dual-luciferase reporter assays and rescue experiment. The effect of circZMYM2 and miR-335-5p on the expression of JMJD2C protein was detected by western blot. Results: CircZMYM2 overexpression was observed in both PC tissues and cells. Knockdown of circZMYM2 inhibited proliferation, induced apoptosis, and weakened invasion ability of cancer cells. Tumor growth was restrained in vivo. CircZMYM2 repressed the expression of its target miR-335-5p. MiR-335-5p attenuated pancreatic cancer development via inhibition of JMJD2C. Conclusion: Our study demonstrated that circZMYM2 promoted PC progression. CircZMYM2 had a sponge effect on miR-335-5p and modulated the downstream oncogene JMJD2C.

研究背景与目的：本研究旨在探究环状RNA circZMYM2（hsa_circ_0099999）在胰腺癌（pancreatic cancer, PC）细胞增殖、凋亡及侵袭过程中的作用，并阐明circZMYM2调控miR-335-5p与JMJD2C的潜在分子机制。
方法：采用Arraystar人类环状RNA微阵列V1对20例胰腺癌组织样本及其配对的正常组织样本中的环状RNA差异表达情况进行分析。通过实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测circZMYM2的表达水平。分别采用细胞计数试剂盒-8（cell counting kit-8, CCK-8）实验、流式细胞术及Transwell实验，探究circZMYM2抑制与过表达对细胞增殖、凋亡及侵袭能力的影响。构建裸鼠动物实验模型，以验证circZMYM2敲低对肿瘤生长的影响。通过RNA下拉实验、双荧光素酶报告基因实验及挽救实验，验证circZMYM2、miR-335与JMJD2C三者间的调控关系。采用蛋白质印迹法（western blot）检测circZMYM2与miR-335-5p对JMJD2C蛋白表达的影响。
结果：在胰腺癌组织及细胞中均检测到circZMYM2的高表达。敲低circZMYM2可抑制癌细胞增殖、诱导细胞凋亡并削弱其侵袭能力。体内实验显示肿瘤生长受到抑制。circZMYM2可靶向抑制其靶分子miR-335-5p的表达。miR-335-5p通过抑制JMJD2C的表达，进而减缓胰腺癌的恶性进展。
结论：本研究证实circZMYM2可促进胰腺癌的恶性进展。circZMYM2对miR-335-5p具有海绵吸附作用，并通过调控下游癌基因JMJD2C的表达发挥生物学功能。