过表达PAX6抑制肝癌细胞生长和促进自然杀伤细胞杀伤能力的机制  

Mechanism of PAX6 overexpression in inhibiting the growth of hepatocellular carcinoma cells and promoting the killing ability of the natural killer cells

作　　者：朱权[1] 黄柏胜 邬力祥 罗奇志[1] ZHU Quan;HUANG Baisheng;WU Lixiang;LUO Qizhi(Department of Immunology,School of Basic Medicine,Central South University,Changsha 410008;Department of Physiology,School of Basic Medicine,Central South University,Changsha 410008,China)

机构地区：[1]中南大学基础医学院免疫学系,长沙410008 [2]中南大学基础医学院生理学系,长沙410008

出　　处：《中南大学学报（医学版）》2023年第7期947-956,共10页Journal of Central South University ：Medical Science

基　　金：湖南省自然科学基金(2019JJ40398,2020JJ4768);长沙市指导性科技计划项目(kzd22002)。

摘　　要：目的:配对盒基因6(paired box gene 6,PAX6)主要在胚胎发育中发挥调控作用,其异常表达与多种肿瘤的发生、发展有关,在不同肿瘤中可发挥促癌或抑癌的作用。本研究旨在观察过表达PAX6对肝癌细胞生长及自然杀伤(natural killer,NK)细胞对肝癌细胞杀伤能力的影响及其分子机制。方法:采用ELISA技术检测68例肝细胞癌(hepatocellular carcinoma,HCC)患者和10例健康人外周血的PAX6、可溶性主要组织相容性复合体I类样蛋白A(soluble major histocompatibility complex class I-like protein A,sMICA)和可溶性UL16结合蛋白2(soluble UL16 binding protein 2,sULBP2)的表达水平;体外培养肝癌细胞HepG2和LM3及人正常肝细胞LO2,将PAX6过表达质粒(PAX6-OE)和空载体(NC)转入HepG2和LM3细胞中构建稳定细胞株,分别采用real-time PCR、蛋白质印迹法、免疫荧光等方法检测HepG2和LM3细胞中PAX6的表达水平;在HepG2和LM3细胞中过表达PAX6,采用CCK-8法和细胞划痕实验检测细胞生长和迁移能力,ELISA法检测其上清液中sMICA和sULBP2的分泌水平,蛋白质印迹法检测基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)、基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)、去整合素样金属蛋白酶10(disintegrin and metalloproteinase 10,ADAM10)的蛋白质表达水平;采用流式细胞术检测NK细胞对2种肝癌细胞的杀伤能力。结果:与健康人相比较,HCC患者血清中PAX6表达水平显著降低(P=0.002),而sMICA和sULBP2的表达水平显著增高(P=0.004和P<0.001)。Real-time PCR、蛋白质印迹法结果显示:与正常肝细胞LO2相比,HepG2和LM3细胞PAX6的mRNA和蛋白质表达水平均显著降低(均P<0.05)。免疫荧光结果亦可见肝癌细胞HepG2和LM3中PAX6的表达均低于正常肝细胞LO2。与NC组比较,PAX6-OE组肝癌细胞HepG2和LM3的增殖及迁移能力下降(均P<0.05);PAX6-OE组的HepG2和LM3细胞中MMP2、MMP9、ADAM10的蛋白质表达水平均显著降低(均P<0.05),且PAX6-OE组的HepG2�Objective:Paired box gene 6(PAX6)plays a major role in the regulation of embryonic development.Abnormal expression of PAX6 is associated with the development of various tumors.PAX6 can play a role in promoting or suppressing cancer in different tumors.This study aim to observe the effect of overexpression of PAX6 on the growth of hepatocellular carcinoma cells,and the killing of hepatocellular carcinoma cells via natural killer(NK)cell and the possible mechanism.Methods:The protein levels of PAX6,soluble major histocompatibility complex class Ilike protein A(sMICA)and soluble UL16 binding protein 2(sULBP2)in peripheral blood from 68 cases of hepatocellular carcinoma(HCC)patients and 10 healthy volunteers were detected by ELISA.Hepatocellular carcinoma cell line(HepG2,LM3)and human normal liver cells(LO2)were cultured at 37℃and 5%CO2 condition in vitro.The PAX6 overexpressed plasmid(PAX6-OE)and empty vector(NC)were transferred into HepG2 and LM3 cells to construct stable cell lines.The mRNA and protein expression levels of PAX6 in HepG2 and LM3 cells were detected by real-time PCR,Western blotting and immunofluorescence,respectively.PAX6 was overexpressed in HepG2 and LM3 cells,the cell growth and migration ability were detected by CCK-8 method and cell scratch assay,and the levels of sMICA and sULBP2 in the supernatant were detected by ELISA.Matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9)and disintegrin and metalloproteinase 10(ADAM10)in HepG2 and LM3 cells were detected by Western blotting.The killing ability of NK cells against these 2 HCC cells was detected by flow cytometry.Results:Compared with the healthy volunteers,the expressions of PAX6 in the HCC patients were significantly decreased(P=0.002),while the expression of sMICA and sULBP2 were significantly increased(P=0.004 and P<0.001,respectively).Real-time PCR and Western blotting results showed that compared with LO2 cells,mRNA and protein expressions of PAX6 in HepG2 and LM3 cells were significantly decreased(all P<0.05).Immunofluore

关 键 词：配对盒基因6 肝细胞癌 细胞生长 金属蛋白酶 自然杀伤细胞 

分 类 号：R735.7[医药卫生—肿瘤]