[1]王宇锋,朱怡凤,殷国志.肝细胞癌中microRNA-1469表达及其对肝癌细胞生物学行为的影响[J].新乡医学院学报,2019,36(9):806-814.[doi:10.7683/xxyxyxb.2019.09.002]
　WANG Yu-feng,ZHU Yi-feng,YIN Guo-zhi.Expression of microRNA-1469 and its function in hepatocellular carcinoma cells[J].Journal of Xinxiang Medical University,2019,36(9):806-814.[doi:10.7683/xxyxyxb.2019.09.002]
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肝细胞癌中microRNA-1469表达及其对肝癌细胞生物学行为的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
36
期数:
2019年9
页码:
806-814
栏目:
基础研究
出版日期:
2019-09-05

文章信息/Info

Title:
Expression of microRNA-1469 and its function in hepatocellular carcinoma cells
作者:
王宇锋; 朱怡凤; 殷国志
（西安交通大学第一附属医院肝胆外科，陕西　西安　710061）
Author(s):
WANG Yu-feng; ZHU Yi-feng; YIN Guo-zhi
（Department of Hepatobiliary Surgery，the First Affiliated Hospital of Xi′an Jiaotong University，Xi′an 710061，Shaanxi Province，China）
关键词:
microRNA-1469; 肝细胞癌; 细胞增殖; 细胞周期; 细胞侵袭
Keywords:
microRNA-1469; hepatocellular carcinoma; cell proliferation; cell cycle; cell invasion
分类号:
R735.7
DOI:
10.7683/xxyxyxb.2019.09.002
文献标志码:
A
摘要:
目的　探讨microRNA-1469（miR-1469）在肝细胞癌（HCC）中的表达及其对肝癌细胞生物学行为的影响。方法　选取2010年1月至2012年12月于西安交通大学第一附属医院手术切除的HCC组织和对应的癌旁组织（距肿瘤边缘>2 cm）标本各76例，采用实时荧光定量聚合酶链反应检测HCC组织及癌旁组织中miR-1469的表达水平，分析miR-1469表达与HCC临床病理特征的关系，Kaplan-Meier生存曲线分析miR-1469表达与HCC患者3 a总体生存率的关系。采用实时荧光定量聚合酶链反应检测HCC细胞系HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402及正常肝细胞系LO2中miR-1469的表达水平；培养Hep3B和SMMC-7721细胞，当细胞融合度达到70%时，分别应用miR-1469 mimics和相应的阴性对照脂质体转染Hep3B细胞，应用miR-1469 inhibitors和相应的阴性对照脂质体转染SMMC-7721细胞。转染48 h后，采用实时荧光定量聚合酶链反应检测各组细胞中miR-1469的表达水平，采用四甲基偶氮唑盐（MTT）法和平板克隆形成实验观察miR-1469对Hep3B和SMMC-7721细胞增殖及克隆形成能力的影响，流式细胞术检测miR-1469对Hep3B和SMMC-7721细胞周期的影响，Transwell实验观察miR-1469对Hep3B和SMMC-7721细胞侵袭及迁移能力的影响，Western blot法检测Hep3B和SMMC-7721细胞中NDRG1蛋白表达。结果　miR-1469在HCC组织和癌旁组织中的相对表达量分别为0.71±0.03、5.49±0.04，HCC组织中miR-1469相对表达量显著低于癌旁组织（P<0.05）。miR-1469表达与肿瘤直径、肿瘤数目、TNM分期、组织分化程度及微血管侵犯相关（P<0.05），而与患者的年龄、性别、血清甲胎蛋白水平、静脉侵犯、Edmondson病理分级、肿瘤部位无相关性（P>0.05）。Kaplan-Meier生存分析显示，miR-1469高表达HCC患者的3 a总体生存率显著高于低表达者（P<0.05）。miR-1469在LO2、HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402细胞中的相对表达量分别为1.01±0.02、0.45±0.01、0.05±0.01、0.61±0.02、0.14±0.01、0.29±0.02；miR-1469在HepG2、Hep3B、SMMC-7721、Huh7、Bel-7402细胞中的相对表达量显著低于LO2细胞（P<0.05）；其中Hep3B细胞中miR-1469表达量最低，SMMC-7721细胞中miR-1469表达量最高。miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞中miR-1469相对表达量分别为4.47±0.15、0.05±0.01，miR-1469 mimics组Hep3B细胞中miR-1469相对表达量显著高于miR-1469 control mimics组（P<0.05）。miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞中miR-1469相对表达量分别为0.20±0.11、1.00±0.00，miR-1469 inhibitors组SMMC-7721细胞中miR-1469相对表达量显著低于miR-1469 control inhibitors组（P<0.05）。MTT结果显示，培养24、48、72 h时，miR-1469 mimics组Hep3B细胞增殖能力显著低于miR-1469 control mimics组，miR-1469 inhibitors组SMMC-7721细胞增殖能力显著高于miR-1469 control inhibitors组（P<0.05）。平板克隆实验结果显示，miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞的克隆数分别为17.00±1.73、65.67±2.33，miR-1469 mimics组Hep3B细胞的克隆形成能力显著低于miR-1469 control mimics组（P<0.05）；miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞的克隆数分别为93.00±2.08、27.67±1.45，miR-1469 inhibitors组SMMC-7721细胞的克隆形成能力显著高于miR-1469 control inhibitors组（P<0.05）。流式细胞术结果显示，与miR-1469 control mimics组比较，miR-1469 mimics组G1期Hep3B细胞显著增多，S期Hep3B细胞显著减少（P<0.05），但2组G2期细胞比例比较差异无统计学意义（P>0.05）。与miR-1469 control inhibitors组比较，miR-1469 inhibitors组G1期SMMC-7721细胞显著减少，S期SMMC-7721细胞显著增多（P<0.05）；但2组G2期SMMC-7721细胞比例比较差异无统计学意义（P>0.05）。Transwell实验结果显示，miR-1469 mimics组Hep3B细胞迁移数和侵袭数分别为59.00±2.08、29.00±2.08，miR-1469 control mimics组Hep3B细胞迁移数和侵袭数分别为35.00±1.53、20.33±1.45；miR-1469 mimics组Hep3B细胞的迁移和侵袭能力显著高于miR-1469 control mimics组（P<0.05）。miR-1469 inhibitors组SMMC-7721细胞迁移数和侵袭数分别为26.00±1.16、17.33±0.88，miR-1469 control inhibitors组SMMC-7721细胞迁移数和侵袭数分别为56.33±3.18、44.67±1.45；miR-1469 inhibitors组SMMC-7721细胞的迁移和侵袭能力显著低于miR-1469 control inhibitors组（P<0.05）。Western blot结果显示，miR-1469 mimics组和miR-1469 control mimics组Hep3B细胞中NDRG1蛋白相对表达量分别为0.23±0.04、1.00±0.00，miR-1469 mimics组Hep3B细胞中NDRG1蛋白相对表达量显著低于miR-1469 control mimics组（P<0.05）；miR-1469 inhibitors组和miR-1469 control inhibitors组SMMC-7721细胞中NDRG1蛋白相对表达量分别为3.90±0.17、1.00±0.00，miR-1469 inhibitors组SMMC-7721细胞中NDRG1蛋白相对表达量显著高于miR-1469 control inhibitors组（P<0.05）。结论　MiR-1469在HCC中表达下调，且其表达与肿瘤数目、肿瘤直径、TNM分期、组织分化程度、微血管侵犯及患者预后密切相关；miR-1469可能通过靶向结合NDRG1并抑制其表达而抑制HCC细胞的增殖、周期、侵袭和迁移。
Abstract:
Objective　To observe the expression of miR-1469 in hepatocellular carcinoma（HCC），and investigate the effect of microRNA-1469（miR-1469） on biological behaviors of HCC cells.Methods　The HCC tissues and paraneoplastic tissues （from the edge of the tumor >2 cm） from 76 patients with HCC underwent surgical resection were selected in the First Affiliated Hospital of Xi′an Jiaotong University from January 2010 to December 2012.The expression of miR-1469 in HCC tissues and paraneoplastic tissues was detected by real-time fluorescence quantitative polymerase chain reaction.The relationship between the expression of miR-1469 and the clinicopathological characteristics of HCC was analyzed.The relationship between the expression of miR-1469 and the 3-year overall survival rate of HCC patients was analyzed by Kaplan-Meier survival curve.The expression of miR-1469 in HCC cell lines HepG2，Hep3B，SMMC-7721，Huh7，Bel-7402 and normal hepatocyte LO2 was detected by real-time fluorescence quantitative polymerase chain reaction.When the Hep3B and SMMC-7721 cells were cultured to 70% cell fusion，the mi-1469 mimics and corresponding negative control liposomes were transfected into Hep3B cells，and mi-1469 inhibitors and corresponding negative control liposomes were transfected into SMMC-7721 cells.Forty-eight hours after transfection，the expression of miR-1469 in the cells was detected by real-time fluorescence quantitative polymerase chain reaction，the effects of miR-1469 on the proliferation and cloning ability of Hep3B and SMMC-7721 cells were observed by methyl thiazolyl tetrazolium(MTT) method and plate cloning experiment，the effects of miR-1469 on cell cycle of Hep3B and SMMC-7721 were detected by flow cytometry，the effect of miR-1469 on invasion and migration of Hep3B and SMMC-7721 cells was observed by Transwell experiment，and the expression of NDRG1 protein in Hep3B and SMMC-7721 cells was detected by Western blot method.Results　The relative expression of miR-1469 in HCC tissues and adjacent tissues was 0.71±0.03 and 5.49±0.04，respectively.The relative expression of miR-1469 in HCC tissues was significantly lower than that in paraneoplastic tissues （P<0.05）.The expression of miR-1469 was significantly correlated with tumor diameter，number of tumors，TNM stage，tissue differentiation degree and microvascular invasion （P<0.05），but it was not correlated with the age，gender，serum alpha-fetoprotein level，venous invasion，Edmondson′s pathological grade and location of tumor （P>0.05）.Kaplan-Meier survival analysis showed that the 3-year overall survival rate of HCC patients with high expression of miR-1469 was significantly higher than that of patients with low expression of miR-1469 （P<0.05）.The relative expression of miR-1469 in LO2，HepG2，Hep3B，SMMC-7721，Huh7 and Bel-7402 cells was 1.01±0.02，0.45±0.01，0.05±0.01，0.61±0.02，0.14±0.01 and 0.29±0.02，respectively.The relative expression of miR-1469 in HepG2，Hep3B，SMMC-7721，Huh7 and Bel-7402 cells was significantly lower than that in LO2 cells （P<0.05）.The expression of miR-1469 was the lowest in Hep3B cells and the highest in SMMC-7721 cells.The relative expression of miR-1469 in Hep3B cells of miR-1469 mimics group and miR-1469 control mimics group was 4.47±0.15 and 0.05±0.01，respectively.The relative expression of miR-1469 in Hep3B cells of miR-1469 mimics group was significantly higher than that of miR-1469 control mimics group （P<0.05）.The relative expression of miR-1469 in SMMC-7721 cells of miR-1469 inhibitors group and miR-1469 control inhibitors group was 0.20±0.11 and 1.00±0.00，respectively.The relative expression of miR-1469 in SMMC-7721 cells of miR-1469 inhibitors group was significantly lower than that of miR-1469 control inhibitors group （P<0.05）.MTT results showed that the proliferation ability of Hep3B cells in miR-1469 mimics group was significantly lower than that in miR-1469 control mimics group，and the proliferation ability of SMMC-7721 cells in miR-1469 inhibitors group was significantly higher than that in miR-1469 control inhibitors group when cultured for 24，48 and 72 h （P<0.05）.The results of cell plate cloning experiment showed that the number of clones of Hep3B cells in miR-1469 mimics group and miR-1469 control mimics group was 17.00±1.73 and 65.67±2.33，respectively.The cloning ability of Hep3B cells in miR-1469 mimics group was significantly lower than that in miR-1469 control mimics group （P<0.05）.The number of clones of SMMC-7721 cells in miR-1469 inhibitors group and miR-1469 control inhibitors group was 93.00±2.08 and 27.67±1.45，respectively.Conclusion　MiR-1469 is down-regulated in HCC，and its expression is closely related to the number of tumors，tumor diameter，TNM stage，tissue differentiation，microvascular invasion and prognosis of patients.Mir-1469 may inhibit the proliferation，cycle，invasion and migration of HCC cells by targeting NDRG1 and inhibiting its expression.

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更新日期/Last Update: 2019-09-05