METHODS: The RNA levels of circUCK2, miR-149-5p and uridine-cytidine kinase 2 (UCK2) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). EdU incorporation assay and colony formation assay were respectively performed to analyze cell proliferation and colony formation. Wound healing assay and transwell assay were conducted for cell migration and invasion. Flow cytometry was used for cell apoptosis analysis. Western blot assay was conducted to determine the protein levels of E-cadherin, N-cadherin, matrix metallopeptidase 9 (MMP-9) and UCK2. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were conducted to confirm the interaction between miR-149-5p and circUCK2 or UCK2. The xenograft model was established to explore the role of circUCK2 in tumor growth in vivo.
RESULTS: CircUCK2 level was elevated in HCC, and circUCK2 depletion suppressed HCC cell proliferation, colony formation, migration and invasion and accelerated cell apoptosis. Mechanistically, circUCK2 could positively modulate UCK2 expression by interacting with miR-149-5p. Furthermore, the repressive effects of circUCK2 knockdown on the malignant behaviors of HCC cells were alleviated by UCK2 overexpression or miR-149-5p inhibition. The promoting effects of circUCK2 overexpression on HCC cell malignancy were alleviated by UCK2 silencing or miR-149-5p introduction. Additionally, circUCK2 knockdown hampered tumor growth in vivo.
CONCLUSIONS: CircUCK2 contributed to HCC malignant progression in vitro and in vivo via targeting miR-149-5p/UCK2 axis, demonstrating that circUCK2 might be a novel therapeutic target for HCC.
方法:通过定量实时聚合酶链反应(qRT-PCR)检测circUCK2,miR-149-5p和尿苷-胞苷激酶2(UCK2)的RNA水平。分别进行EdU掺入测定和集落形成测定以分析细胞增殖和集落形成。对细胞迁移和侵袭进行伤口愈合试验和transwell试验。流式细胞术用于细胞凋亡分析。Westernblot检测E-cadherin蛋白水平,N-钙黏着蛋白,基质金属肽酶9(MMP-9)和UCK2。双荧光素酶报告基因测定,进行RNA免疫沉淀(RIP)测定和RNA下拉测定以确认miR-149-5p与circUCK2或UCK2之间的相互作用。建立异种移植模型以探索circUCK2在体内肿瘤生长中的作用。
结果:CircUCK2水平在HCC中升高,和circUCK2耗竭抑制肝癌细胞增殖,菌落形成,迁移和侵袭和加速细胞凋亡。机械上,circUCK2可以通过与miR-149-5p相互作用来正向调节UCK2的表达。此外,通过UCK2过表达或miR-149-5p抑制,circUCK2敲低对HCC细胞恶性行为的抑制作用得以缓解.通过UCK2沉默或miR-149-5p导入减轻circUCK2过表达对HCC细胞恶性的促进作用。此外,circUCK2敲低阻碍了体内肿瘤的生长。
结论:CircUCK2通过靶向miR-149-5p/UCK2轴在体外和体内促进HCC恶性进展,证明circUCK2可能是肝癌的新治疗靶点。