成对盒基因6（PAX6）在胚胎发育调控中起着重要作用。PAX6的异常表达与多种肿瘤的发生发展相关。PAX6在不同肿瘤中既可以促进癌症的发展，也可以抑制癌症。本研究旨在观察PAX6过表达对肝细胞癌细胞生长的影响，以及通过自然杀伤（NK）细胞杀伤肝细胞癌细胞的效果和可能的机制。用ELISA检测了68例肝细胞癌（HCC）患者和10名健康志愿者外周血中PAX6、可溶性主要组织相容性复合物类似蛋白A（sMICA）和可溶性UL16结合蛋白2（sULBP2）的蛋白水平。在37℃和5% CO2的体外条件下培养肝细胞癌细胞系（HepG2，LM3）和人正常肝细胞（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系。用实时PCR、Western blotting和免疫荧光检测HepG2和LM3细胞中PAX6的mRNA和蛋白表达水平。在HepG2和LM3细胞中过表达了PAX6，使用CCK-8法和细胞刮痕实验检测了细胞生长和迁移能力，使用ELISA检测了上清液中sMICA和sULBP2的水平。通过Western blotting检测了HepG2和LM3细胞中的基质金属蛋白酶2（MMP2），基质金属蛋白酶9（MMP9）和解离蛋白和金属蛋白酶10（ADAM10）。通过流式细胞术检测NK细胞对这两种HCC细胞的杀伤能力。与健康志愿者相比，HCC患者中PAX6的表达显著降低（P=0.002），而sMICA和sULBP2的表达显著增加（P=0.004和P＜0.001）。实时PCR和Western blotting结果显示，与LO2细胞相比，HepG2和LM3细胞中PAX6的mRNA和蛋白表达水平显著降低（所有P＜0.05）。免疫荧光结果也显示HepG2和LM3细胞中PAX6的表达低于LO2细胞。与NC组相比，HepG2和LM3细胞的增殖和迁移能力降低（均P＜0.05）。PAX6-OE组中HepG2和LM3细胞中MMP2，MMP9和ADAM10的蛋白表达显著降低，而PAX6-OE组中HepG2和LM3细胞上清液中sMICA和sULBP2的水平显著低于NC组（均P＜0.05）。流式细胞术结果显示，与NC组相比，PAX6-OE组中NK细胞对HepG2和LM3细胞的杀伤能力显著增加（均P＜0.05）。PAX6的表达在HCC患者和肝细胞癌细胞株的血清中降低。PAX6的过表达可以抑制肝细胞癌细胞的生长，增强NK细胞对肝癌细胞的杀伤效果。机制与通过PAX6抑制金属蛋白酶的表达和降低sMICA和sULBP2的分泌有关。

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.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% 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.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).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.