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乙型肝炎病毒X蛋白对肝癌细胞侵袭与迁移能力的作用及机制

曹宽, 鲍仲明, 周新宇, 贾广宇, 张斌, 温泉, 王人颢

曹宽, 鲍仲明, 周新宇, 等. 乙型肝炎病毒X蛋白对肝癌细胞侵袭与迁移能力的作用及机制[J]. 中华消化外科杂志, 2017, 16(2): 177-182. DOI: 10.3760/cma.j.issn.1673-9752.2017.02.013
引用本文: 曹宽, 鲍仲明, 周新宇, 等. 乙型肝炎病毒X蛋白对肝癌细胞侵袭与迁移能力的作用及机制[J]. 中华消化外科杂志, 2017, 16(2): 177-182. DOI: 10.3760/cma.j.issn.1673-9752.2017.02.013
Cao Kuan, Bao Zhongming, Zhou Xinyu, et al. Effects and mechanisms of hepatitis B virus X protein on invasion and migration of hepatocellular carcinoma cells[J]. Chinese Journal of Digestive Surgery, 2017, 16(2): 177-182. DOI: 10.3760/cma.j.issn.1673-9752.2017.02.013
Citation: Cao Kuan, Bao Zhongming, Zhou Xinyu, et al. Effects and mechanisms of hepatitis B virus X protein on invasion and migration of hepatocellular carcinoma cells[J]. Chinese Journal of Digestive Surgery, 2017, 16(2): 177-182. DOI: 10.3760/cma.j.issn.1673-9752.2017.02.013

乙型肝炎病毒X蛋白对肝癌细胞侵袭与迁移能力的作用及机制

基金项目: 江苏省卫生厅基金(H201429);徐州市科技计划项目(KC14SX001);江苏省研究生科研创新计划(KYLX14-1453)

Effects and mechanisms of hepatitis B virus X protein on invasion and migration of hepatocellular carcinoma cells

  • 摘要:

    目的:探讨乙型肝炎病毒X(HBx)蛋白对肝癌细胞侵袭与迁移能力的作用及机制。
    方法:采用回顾性队列研究方法。收集2014年7月至2015年7月徐州医科大学附属医院收治的30例肝肿瘤患者[20例肝细胞癌(简称肝癌)、10例肝脏良性肿瘤)]的临床病理资料。收集20例肝癌患者(均有HBV感染史)行手术切除的肝癌组织及10例肝脏良性肿瘤患者(均无HBV感染史)的瘤旁组织(瘤体包膜外组织)。采用免疫组织化学染色检测肝癌组织与瘤旁组织中人类表皮生长因子受体3(ErbB3)蛋白表达。采用Western blot检测肝癌组织与瘤旁组织中ErbB3蛋白和HBx蛋白相对表达量,以及转染绿色荧光蛋白(GFP)质粒的肝癌细胞株HepG2和转染GFPHBx质粒的HepG2中ErbB3蛋白相对表达量。采用RTPCR检测转染GFP质粒的HepG2和转染GFPHBx质粒的HepG2中ErbB3 mRNA相对表达量。采用铺基质胶Transwell法检测HepG2侵袭能力,不铺基质胶Transwell法检测HepG2迁移能力。正态分布的计量资料采用±s表示,组间比较采用独立样本t检验。采用Pearson检验进行相关性分析。
    结果:(1)免疫组织化学染色检测ErbB3蛋白表达情况:20例原发性肝癌患者的肝癌组织及10例肝脏良性肿瘤患者的瘤旁组织中ErbB3蛋白平均光密度(MOD)相对值分别为2.54±1.33和0.99±0.29,两者比较,差异有统计学意义(t=6.542, P<0.05)。(2)Western blot检测ErbB3蛋白和HBx蛋白表达情况:10例原发性肝癌患者的肝癌组织及 10例肝脏良性肿瘤患者的瘤旁组织中ErbB3蛋白相对表达量分别为0.79±0.13和1.10±0.28,HBx蛋白相对表达量分别为1.07±0.17和0,两者上述指标比较,差异均有统计学意义(t=3.229,19.486,P<0.05)。Pearson检验结果显示:肝癌组织中ErbB3蛋白与HBx蛋白表达成正相关(r=0.637,P<0.05)。(3)Western blot和RTPCR检测HepG2中ErbB3蛋白表达及转录水平情况:转染GFP质粒的HepG2和转染GFPHBx质粒的HepG2中ErbB3蛋白相对表达量分别为0.75±0.11和1.10±0.10,两者比较,差异有统计学意义 (t=4.291,P<0.05)。转染GFP质粒的HepG2和转染GFPHBx质粒的HepG2中ErbB3 mRNA相对表达量分别为0.38±0.03和0.94±0.07,两者比较,差异有统计学意义(t=11.703,P<0.05)。(4)ErbB3蛋白对HepG2侵袭、迁移能力的影响:铺基质胶Transwell法检测转染His质粒的HepG2和转染HisErbB3质粒的HepG2穿膜细胞数分别为(271±18)个和(463±31)个,两者比较,差异有统计学意义(t=8.202,P<0.05)。不铺基质胶Transwell法检测转染His质粒的HepG2和转染HisErbB3质粒的HepG2穿膜细胞数分别为(315±38)个和(549±34)个,两者比较,差异有统计学意义(t=8.310,P<0.05)。
    结论:HBx蛋白可通过上调ErbB3蛋白表达,促进肝癌细胞侵袭与迁移。

    Abstract:

    Objective:To explore the effects and mechanisms of hepatitis B virusX protein (HBx) on invasion and migration of hepatocellular carcinoma (HCC) cells.
    Methods:The retrospective cohort study was conducted. The clinicopathological data of 30 patients with liver tumor (20 with HCC and 10 with benign tumor of liver) who were admitted to the Affiliated Hospital of Xuzhou Medical College between July 2014 and July 2015 were collected. HCC tissues of 20 patients with HCC (with history of HBV infection) were collected by surgical resection and peritumoral normal tissues (outside of tumor capsule) of 10 patients with benign tumor of liver (without history of HBV infection) were collected. The expressions of epidermal growth factor receptor 3 (ErbB3) in HCC tissues and peritumoral normal tissues were detected by immunohistochemistry (IHC). The relative expressions of ErbB3 and HBx in HCC tissues and peritumoral normal tissues were detected by Western blot, and relative expressions of ErbB3 in HepG2 of which green fluorescent protein (GFP) and GFPHBx were respectively transfected were detected. The relative expressions of ErbB3 mRNA in HepG2 transfected by GFP and GFPHBx were detected by realtime polymerase chain reaction (RTPCR). The migration and invasion of HepG2 were respectively detected by Transwell assay with and without matrix. The measurement data with normal distribution were represented as ±s. The comparisons between groups were evaluated with the independentsample t test. Correlation analysis was done by the Pearson test.
    Results:(1) The expressions of ErbB3 were detected by IHC: relative value of mean optical density (MOD) of ErbB3 in HCC tissues of 20 patients with HCC and peritumoral normal tissues of 10 patients with benign tumor of liver were 2.54±1.33 and 0.99±0.29, respectively, with a statistically significant difference (t=6.542, P<0.05). (2) The relative expressions of ErbB3 and HBx were detected by Western blot: relative expressions of ErbB3 and HBx were respectively 0.79±0.13, 1.10±0.28 in HCC tissues of 10 patients with HCC and 1.07±0.17, 0 in peritumoral normal tissues of 10 patients with benign tumor of liver, with statistically significant differences (t=3.229, 19.486, P<0.05). The results of Pearson test showed that there was a positive correlation of expression between ErbB3 and HBx in HCC tissues (r=0.637, P<0.05). (3) The relative expressions and transcriptional levels of ErbB3 were detected by Western blot and RTPCR: relative expressions of ErbB3 in HepG2 of which GFP and GFPHBx were respectively transfected were 0.75±0.11 and 1.10±0.10, respectively, with a statistically significant difference (t=4.291, P<0.05). The relative expressions of ErbB3 mRNA in HepG2 of which GFP and GFPHBx were respectively transfected were 0.38±0.03 and 0.94±0.07, respectively, with a statistically significant difference (t=11.703, P<0.05). (4) The effects of ErbB3 on migration and invasion of HepG2: numbers of transmenbrane cell in HepG2 of which His and HisErbB3 were respectively transfected by Transwell assay with matrix were respectively 271±18 and 463±31, respectively, with a statistically significant difference (t=8.202, P<0.05). Numbers of transmenbrane cell in HepG2 of which His and HisErbB3 were respectively transfected by Transwell assay without matrix were respectively 315±38 and 549±34, respectively, with a statistically significant difference (t=8.310, P<0.05).
    Conclusion:HBx protein can promote the invasion and migration of hepatocellular carcinoma cells through upregulating expressions of ErbB3 protein.

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