摘要
目的观察卵泡抑素样蛋白1(FSTL1)对肝星状细胞生物学特性的影响,并初步探讨其促进肝癌细胞恶性转化的分子机制。方法收集2021年1月至2022年10月新乡医学院第三附属医院收治的乙肝、肝硬化、原发性肝细胞癌患者及健康志愿者的血液样本各30份,采用酶联免疫吸附法检测并比较其血清中FSTL1表达水平;收集2019年6月至2022年6月新乡医学院第三附属医院收治的原发性肝细胞癌患者的手术切除组织蜡块30份,免疫组织化学法检测其FSTL1表达水平;通过慢病毒感染构建稳定过表达及干扰FSTL1的肝星状细胞株,与肝癌细胞共培养,利用细胞划痕及Transwell实验分别观察FSTL1对肝星状细胞增殖、迁移及侵袭能力的影响;采用蛋白质免疫印迹法检测过表达FSTL1的肝星状细胞对肝癌细胞EMT相关标记物E-cadherin、N-cadherin、Vimentin、β-cadherin、snail的影响。结果健康志愿者、乙型肝炎、肝硬化、肝癌患者外周血中FSTL1表达水平逐渐升高,分别为(44.91±4.060)ng/mL、(75.47±8.149)ng/mL、(143.9±13.29)ng/mL、(255.8±13.18)ng/mL,差异均具有统计学意义(P<0.05);在肝细胞癌患者肿瘤组织中,肝癌细胞与肝星状细胞的FSTL1表达免疫组化评分分别为(2.03±0.20)分、(6.60±0.41)分,差异具有统计学意义(P<0.05)。FSTL1稳定过表达或干扰可以相应促进或抑制肝星状细胞的体外增殖及迁移能力。CCK8实验结果显示,与对照组(LX-2-Vector)比较,干扰组(LX-2-FSTL1-sh)第2、3、4、5天增殖速度的光密度(OD)值分别为0.36±0.02、0.46±0.02、0.63±0.05、0.98±0.11,差异具有统计学意义(P<0.05)。与对照组(LX-2-Vector)比较,过表达组(LX-2-FSTL1)在第2、3、4、5天增殖速度的OD值分别为0.48±0.01、1.00±0.02、1.72±0.12、2.09±0.2,差异具有统计学意义(P<0.05)。Transwell小室迁移实验结果显示,与对照组(151.3±2.404)比较,干扰组(49.00±1.732)的迁移能力明显下降,穿过小室的细胞数目显著减少,差异有统计学意义(P<0.05);与对照组(79.33±2.728)比较,过表达组(173.0±3.786)的迁移能力明显增强,穿过小室的细胞数目显著增加,差异有统计学意义(P<0.05)。FSTL1+肝星状细胞与肝癌细胞HepG2共培养与对照组比较,可以促进肝癌细胞的迁移能力和上皮间质转化;Transwell小室侵袭实验结果显示,与对照组(43.67±3.283)比较,过表达组(157.0±14.57)穿透Matrigel类基底膜的能力明显增强,穿过小室的细胞数目显著增加,差异有统计学意义(P<0.05);利用Western blotting实验检测共培养后肝癌细胞中典型的EMT标志物的表达变化,结果显示,与对照组比较,采用FSTL1-CM与HepG2细胞进行共培养处理后,E-cadherin的表达显著下调,相反,N-cadherin、Vimentin、β-cadherin、snail的表达显著上调,差异均有统计学意义(P<0.05)。结论FSTL1在肝癌组织中主要分布于肝星状细胞,且较其在肝硬化组织中的蛋白表达水平上调;FSTL1能够使肝星状细胞大量增殖、迁移能力增强,而且能够诱导肝癌细胞发生上皮-间质转化,从而促进肝癌的恶性进展。
Objective To examine the influence of follistatin-like protein 1(FSTL1)on the biological charac-teristics of hepatic stellate cells(HSCs)and to explore its molecular mechanisms in promoting the malignant transforma-tion of hepatocellular carcinoma(HCC)cells.Methods Blood samples were collected from 30 healthy volunteers and 30 patients each with hepatitis B,cirrhosis,or primary hepatocellular carcinoma treated at the Third Affiliated Hospital of Xinxiang Medical University from January 2021 to October 2022.The expression level of FSTL1 in serum was detect-ed by enzyme-linked immunosorbent assay(ELISA).Thirty paraffin-embedded surgical specimens from HCC patients ad-mitted to the Third Affiliated Hospital of Xinxiang Medical University from June 2019 to June 2022 were examined for FSTL1 expression by immunohistochemistry.Lentiviral infection was used to create HSC lines with stable FSTL1 overex-pression(LX-2-FSTL1)or knockdown(LX-2-FSTL1-sh).These cells were co-cultured with HCC cells,and scratch wound healing and Transwell assays were performed to assess proliferation,migration,and invasion.Western blotting was used to examine the effect of FSTL1 overexpression in HSCs on the expression of epithelial-mesenchymal transition(EMT)markers E-cadherin,N-cadherin,Vimentin,β-cadherin,and Snail.Results The expression levels of FSTL1 in the peripheral blood increased progressively from healthy controls to patients with hepatitis B patients,liver cirrhosis pa-tients,and hepatocellular carcinoma,measuring(44.91±4.060)ng/mL,(75.47±8.149)ng/mL,(143.9±13.29)ng/mL,and(255.8±13.18)ng/mL,respectively(all P<0.05).In the tumor tissues of hepatocellular carcinoma patients,the immuno-histochemical scores for FSTL1 expression in HCC cells and HSCs were(2.03±0.20)points and(6.60±0.41)points,re-spectively,with statistically significant differences(P<0.05).FSTL1 overexpression significantly enhanced,whereas knockdown inhibited,HSC proliferation and migration in vitro.In CCK-8 assays,OD values on days 2,3,4,and 5 for LX-2-FSTL1-sh cells were 0.36±0.02,0.46±0.02,0.63±0.05,and 0.98±0.11,versus 0.48±0.01,1.00±0.02,1.72±0.12,and 2.09±0.2 for LX-2-FSTL1 cells(P<0.05).Transwell migration assay results showed that compared with the control group(151.3±2.404),the migration ability of the knockdown group(49.00±1.732)was significantly decreased,and the number of cells passing through the chamber was significantly reduced,with statistically significant differences(P<0.05).Compared with the control group(79.33±2.728),the migration ability of the overexpression group(173.0±3.786)was significantly enhanced,and the number of cells passing through the chamber was significantly increased,with statis-tically significant differences(P<0.05).Co-culture of FSTL1+HSCs with HepG2 HCC cells promoted the migration ability and epithelial-mesenchymal transition(EMT)of HCC cells compared to the control group.Transwell invasion as-say results showed that compared to the control group(43.67±3.283),the overexpression group(157.0±14.57)exhibited significantly enhancedability to penetrate the Matrigel basement membrane,with a notable increase in the number of cells passing through the chamber,and the difference was statistically significant(P<0.05).Western blotting showed sig-nificant down-regulation of E-cadherin and up-regulation of N-cadherin,Vimentin,β-cadherin,and Snail in HepG2 cells treated with FSTL1-conditioned medium(all P<0.05).Conclusion In HCC tissues,FSTL1 is predominantly ex-pressed in HSCs at higher levels than in cirrhotic tissue.FSTL1 can promote the proliferation and migration abilities of HSCs and induce EMT in HCC cells,thereby facilitating the malignant progression of HCC.
作者
薛桦
聂宇
张成岩
杨肖莉
王艺伟
余渊博
李广申
谷川莎
XUE Hua;NIE Yu;ZHANG Cheng-yan;YANG Xiao-li;WANG Yi-wei;YU Yuan-bo;LI Guang-shen;GU Chuan-sha(Department of Pathology,the Third Affiliated Hospital of Xinxiang Medical University,Xinxiang 453003,Henan,CHINA;Department of Pathology,the First Affiliated Hospital of Henan Polytechnic University,Jiaozuo 454000,Henan,CHINA;School of Medical Technology,Xinxiang Medical University,Xinxiang 453000,Henan,CHINA;School of Medicine,Henan Polytechnic University,Jiaozuo 454000,Henan,CHINA)
出处
《海南医学》
2025年第13期1825-1831,共7页
Hainan Medical Journal
基金
河南省医学科技攻关计划联合共建项目(编号:LHGJ20220643)
河南省科技厅科技攻关项目(编号:232102310196)。
关键词
卵泡抑素样蛋白1
肝星状细胞
肝纤维化
肝癌
上皮间质转化
机制研究
Follistatin-like protein 1
Hepatic stellate cells
Liver fibrosis
Hepatocellular carcinoma
Epitheli-al-mesenchymal transition
Mechanism
