乙型肝炎病毒X蛋白相关lncRNA NEAT1促肝癌增殖和转移的研究

NEAT1,an lncRNA related to the X protein of the hepatitis B virus,promotes the proliferation and metastasis of hepatocellular carcinoma

目的探讨乙型肝炎病毒X蛋白(HBx)相关lncRNA NEAT1表达与肝癌患者临床特征及预后的关系,及其对肝细胞癌(HCC)增殖和转移的影响。方法从本院行肝切除术的肝癌患者的标本中收集肝癌组织和相应的邻近非肿瘤肝组织各125份。构建稳定转染HBx基因的HepG2细胞和相应阴性对照,采用微阵列分析鉴定HBx差异调控的lncRNAs。采用实时定量聚合酶链反应(RT-qPCR)检测肝癌组织和HCC细胞中NEAT1的表达,MTT法测定细胞增殖,Transwell分析细胞迁移和侵袭能力。通过功能获得和功能丧失试验确定NEAT1/miR-128-3p在HCC细胞迁移和侵袭中的作用。结果微阵列分析显示,NEAT1在稳定转染HBx基因的HepG2细胞上调。NEAT1与HBx/HBV相关,并在体外和体内HCC中上调。临床病理分析表明,NEAT1与肝血管浸润、不良肿瘤分化和HBV感染显著相关(P<0.05),并且NEAT1在HCC组织中过表达与患者预后不良相关(P<0.05)。亚组生存分析表明,HBV阴性HCC患者的预后较好(P<0.05),而HBV+/NEAT1+亚组患者的预后较差(P<0.05)。体外试验证实,NEAT1过表达显著促进HepG2增殖(P<0.05),并增强细胞的迁移和侵袭能力(P<0.01);NEAT1敲除显著抑制Hep3B增殖(P<0.05),并降低细胞的迁移和侵袭能力(P<0.01)。双荧光素酶报告基因试验证实NEAT1靶向miR-128-3p,且miR-128-3p在很大程度上逆转NEAT1对HCC细胞迁移和侵袭能力的作用。结论 NEAT1通过竞争结合miR-128-3p作为ceRNA,促进肝癌细胞的增殖、迁移和侵袭。

Objective To explore the relationship between the expression of NEAT1, an lncRNA related to the X protein of the hepatitis B virus(HBx), and the clinical characteristics of and prognosis for patients with hepatocellular carcinoma(HCC), and the effects of NEAT1 on the proliferation and metastasis of HCC. Methods One hundred and twenty-five pairs of HCC tissues and adjacent non-cancerous liver tissues were collected from patients with HCC who underwent liver resection at this Hospital. The differential expression profiles of HepG2 cells stably transfected with the HBx gene and corresponding negative controls were identified using microarray analysis. A quantitative real-time polymerase chain reaction(RT-qPCR) was used to detect NEAT1 expression in HCC tissues and HCC cells. Cell proliferation was determined using an MTT assay, and cell migration and invasion capabilities were analyzed using the Transwell assay. The effects of NEAT1/miR-128-3 p on the migration and invasion of HCC cells were determined in gain and loss of function experiments. Results Microarray analysis indicated that NEAT1 was up-regulated in HepG2 cells stably transfected with the HBx gene. NEAT1 was correlated with HBx/HBV and upregulated in HCC in vitro and in vivo. Clinicopathological analysis indicated that NEAT1 was significantly correlated with hepatic vascular invasion, poor tumor differentiation, and HBV infection(P<0.05), and NEAT1 overexpression in HCC tissues was associated with a poor prognosis(P<0.05). Subgroup survival analysis indicated that HBV-negative patients with HCC had a better prognosis(P<0.05) while HBV+/NEAT1+ patients had a poorer prognosis(P<0.05). In vitro experiments verified that NEAT1 over-expression significantly promoted the proliferation of HepG2(P<0.05) and it increased cell migration and invasion(P<0.01), while knockout of NEAT1 significantly inhibited Hep3 B proliferation(P<0.05) and reduced the migration and invasion of cells(P<0.01). A dual luciferase reporter gene experiment verified that NEAT1 targeted miR-128-3 p, and miR-128-3 p largely rescued the effects of NEAT1 on the migratory and invasive ability of HCC cells. Conclusion NEAT1 promotes the proliferation, migration, and invasion of HCC by competitively binding to miR-128-3 p as ceRNA.