摘要
目的探讨THUMPD3反义RNA 1(THUMPD3⁃AS1)通过靶向微小RNA⁃877(miR⁃877)调控肝细胞癌(HCC)细胞增殖、迁移和侵袭的潜在机制。方法采用实时荧光定量PCR检测THUMPD3⁃AS1在正常肝细胞LO2和HCC细胞(BEL⁃7404、BEL⁃7404、Hep3B、Huh⁃7、SMMC⁃7721)的表达情况。培养SMMC⁃7721细胞并分为si⁃NC组(转染阴性对照序列)、si⁃THUMPD3⁃AS1组(转染THUMPD3⁃AS1干扰序列)、si⁃THUMPD3⁃AS1+miR⁃NC组(转染THUMPD3⁃AS1干扰序列+miR⁃877无关序列)和si⁃THUMPD3⁃AS1+miR⁃877 inhibitor组(转染THUMPD3⁃AS1干扰序列+miR⁃877抑制物inhibitor)。在线预测结合双荧光素酶报告基因实验验证THUMPD3⁃AS1和miR⁃877的靶向关系。采用MTT法、划痕实验和Transwell小室实验分别评估细胞的增殖、迁移和侵袭能力。Western blotting检测磷酸化Janus激酶1(p⁃JAK1)和磷酸化信号传导及转录激活因子3(p⁃STAT3)的表达。结果与LO2细胞相比,HCC细胞的THUMPD3⁃AS1表达升高而miR⁃877表达降低(P<005)。THUMPD3⁃AS1能够结合并负调控miR⁃877表达。与si⁃NC组相比,si⁃THUMPD3⁃AS1组细胞增殖、迁移和侵袭能力受到抑制(P<005)。与si⁃THUMPD3⁃AS1组相比,si⁃THUMPD3⁃AS1+miR⁃877 inhibitor组细胞增殖、迁移和侵袭能力增强(P<005)。si⁃THUMPD3⁃AS1组的p⁃JAK1和p⁃STAT3水平低于si⁃NC组,而si⁃THUMPD3⁃AS1+miR⁃877 inhibitor组的p⁃JAK1和p⁃STAT3水平高于si⁃THUMPD3⁃AS1组(P<005)。结论THUMPD3⁃AS1通过海绵化miR⁃877促进HCC细胞增殖、迁移和侵袭并激活JAK/STAT信号,成为HCC治疗的潜在靶点。
Objective To investigate the underlying mechanism of THUMPD3 antisense RNA1(THUMPD3-AS1)regulating proliferation,migration and invasion of hepatocellular carcinoma(HCC)cells through targeting microRNA-877(miR-877).Methods The expressions of THUMPD3-AS1 and miR-877 in normal liver cell line LO2 and HCC cells including BEL-7404,BEL-7404,Hep3B,Huh-7 and SMMC-7721 were examined by quantitative real-time polymerase chain reaction.SMMC-7721 cells were cultured and divided into si-NC group(cells transfected with negative control sequence),si-THUMPD3-AS1 group(cells transfected with THUMPD3-AS1 interference sequence),si-THUMPD3-AS1+miR-NC group(cells transfected with THUMPD3-AS1 interference sequence and miR-877 irrelevant sequence miR-NC)and si-THUMPD3-AS1+miR-877 inhibitor group(cells transfected with THUMPD3-AS1 interference sequence and miR-877 inhibitor).Targeting relationships between THUMPD3-AS1 and miR-877 was predicted by online software and confirmed by dual-luciferase reporter assay.Proliferation,migration,and invasion abilities of cells were evaluated using MTT assay,scratch assay,and Transwell chamber assay,respectively.The phosphorylated Janus-activated kinase 1(p-JAK1)and phosphorylated signal transducers and activators of transcription 3(p-STAT3)levels were detected by Western blotting.Results Compared with LO2 cells,the expression of THUMPD3-AS1 in HCC cells increased,while the expression of miR-877 decreased(P<005).THUMPD3-AS1 could bind and negatively regulate miR-877 expression.Compared with the si-NC group,the proliferation,migration and invasion abilities of cells in the si-THUMPD3-AS1 group were inhibited(P<005).Compared with the si-THUMPD3-AS1 group,the si-THUMPD3-AS1+miR-877 inhibitor group showed increased cell proliferation,migration,and invasion abilities(P<005).The levels of p-JAK1 and p-STAT3 in the si-THUMPD3-AS1 group were lower than those in the si-NC group,while the levels of p-JAK1 and p-STAT3 in the si-THUMPD3-AS1+miR-877 inhibitor group were higher than those in the si-THUMPD3-AS1 gro
作者
卢宏全
黄国定
林影
张诚胜
LU Hongquan;HUANG Guoding;LIN Ying;ZHANG Chengsheng(Department of Medical Oncology,Hainan Western Central Hospital,Danzhou 571700,China)
出处
《临床肿瘤学杂志》
CAS
2023年第9期778-784,共7页
Chinese Clinical Oncology
基金
海南省卫生健康行业科研项目(20A200112)。
