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朱权，Email: firstname.lastname@example.org, ORCID: 0000-0002-7568-4916
罗奇志，Email: email@example.com, ORCID: 0000-0003-1868-9363
朱权, 黄柏胜, 邬力祥, 等. 过表达
ZHU Quan, HUANG Baisheng, WU Lixiang, et al. Mechanism of
朱权, 黄柏胜, 邬力祥, 等. 过表达
ZHU Quan, HUANG Baisheng, WU Lixiang, et al. Mechanism of
目的,2,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,2,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,2,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和LM3细胞上清液中sMICA和sULBP2的分泌水平均显著低于NC组(均,P,<,0.05)。流式细胞术显示：与NC组比较，PAX6-OE组NK细胞对HepG2和LM3细胞的杀伤率显著增高(均,P,<,0.05)。,结论,2,PAX6在HCC患者血清及肝癌细胞系中表达降低，过表达,PAX6,可抑制肝癌细胞生长，增强NK细胞对肝癌细胞的杀伤效率，其机制与PAX6抑制金属蛋白酶的表达、降低sMICA和sULBP2的分泌水平有关。
Objective,2,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,2,The protein levels of PAX6, soluble major histocompatibility complex class I-like 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% CO,2, 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,2,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). Immunofluorescence results also showed that the expressions of PAX6 in HepG2 and LM3 were lower than those of LO2 cells. Compared with the NC group, the ability of proliferation and migration of HepG2 and LM3 cells were decreased (both ,P,<,0.05). The protein expressions of MMP2, MMP9 and ADAM10 in HepG2 and LM3 cells in the PAX6-OE group were significantly decreased, and the levels of sMICA and sULBP2 in superneant of HepG2 and LM3 cells in the PAX6-OE group were significantly lower than those in the NC group (all ,P,<,0.05). Flow cytometry results showed that compared with the NC group, the proportion of NK cells killing HepG2 and LM3 cells in PAX6-OE group was significantly increased (both ,P,<,0.05).,Conclusion,2,The expression of PAX6 is decreased in serum of HCC patients and hepatocellular carcinoma cell lines. Overexpression of ,PAX6, can inhibit the growth of hepatocellular carcinoma cells, enhance the killing efficiency of NK cells against hepatoma cells. The mechanism is related to the inhibition of the expression of metalloproteinase via PAX6 and the decrease of the secretion levels of sMICA and sULBP2.
paired box gene 6hepatocellular carcinomacell growthmetalloproteinasenatural killer cells
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