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目的:构建人谷胱甘肽-S-转移酶Z1(glutathione S-transferase zeta 1,GSTZ1)重组腺病毒,并初步研究GSTZ1调控粘附连接信号通路(adhesion junction pathway)靶基因SMAD3、IGF1R等影响肝癌细胞的迁移能力。方法:PCR扩增GSTZ1 cDNA序列并克隆到腺病毒载体pAd Track-TO4,构建重组腺病毒质粒p Ad Track-GSTZ1,将重组质粒用Lipofectamine 2000转染至HEK293细胞中,包装、扩增成高滴度重组腺病毒AdGSTZ1。首先,将SMMC-7721细胞设为3组:Mock组、AdGFP组和AdGSTZ1组,Mock组为空白对照组,AdGFP组为感染腺病毒AdGFP的对照组,AdGSTZ1组为感染腺病毒AdGSTZ1的实验组。然后,通过Transwell实验和MTS实验观察其对肝癌细胞迁移增殖能力的影响,进一步经qRT-PCR及Western blot技术研究GSTZ1通过调控粘附连接信号通路关键分子影响肝癌细胞的迁移。结果:成功构建了高滴度重组腺病毒AdGSTZ1,经Western blot鉴定GSTZ1在SMMC-7721细胞中的表达。Transwell实验结果表明,AdGSTZ1组与Mock组及AdGFP组相比,能够明显抑制人肝癌细胞的迁移能力;与Mock组相比,抑制率为(68.10±1.63)%(P=0.000),与AdGFP组相比,抑制率为(50.70±0.99)%(P=0.000)。MTS实验结果表明,AdGSTZ1组在72 h和96 h的吸光度值分别为(1.13±0.08)和(1.28±0.07),AdGFP组72 h和96 h的吸光度值分别为(1.28±0.03)和(1.45±0.03),AdGSTZ1组在96 h的吸光度值与对照组相比明显降低,差异有统计学意义(P<0.01)。另外q RT-PCR及Western blot结果显示,与Mock组和AdGFP组相比,AdGSTZ1感染细胞内粘附连接信号通路中靶基因SMAD3、IGF1R等的表达明显降低。q RT-PCR结果显示,AdGSTZ1组与AdGFP组相比,TGFBR1相对表达量为(0.58±0.13)(t=4.609,P=0.010),SMAD3相对表达量为(0.51±0.08)(t=8.030,P=0.000),ERBB2相对表达量为(0.46±0.09)(t=9.384,P=0.000),IGF1R相对表达量为(0.45±0.02)(t=14.480,P=0.000),FARP2相对表达量为(0.37±0.13)(t=7.082,P=0.028)。Western blot表明,SMAD3组中,AdGFP组的蛋白相对表达量为(0.96±0.18),AdGSTZ1实验组的蛋白相对表达量(0.39±0.01)(P=0.001)。IGF1R组中,AdGFP组的蛋白相对表达量为(0.97±0.05),AdGSTZ1实验组的蛋白相对表达量(0.44±0.02)(P=0.000)。结论:GSTZ1通过调控粘附连接信号通路相关靶基因的表达抑制肝癌细胞的迁移。
OBJECTIVE: To construct a recombinant adenovirus carrying human glutathione S-transferase zeta 1 (GSTZ1) and investigate the effects of GSTZ1 on SMAD3, IGF1R and other target genes of adhesion junction pathway Liver cancer cell migration ability. Methods: The cDNA sequence of GSTZ1 was amplified by PCR and cloned into pAd Track-TO4. The recombinant plasmid was transfected into HEK293 cells with Lipofectamine 2000, and then packaged and amplified into high-drop Degree recombinant adenovirus AdGSTZ1. First, SMMC-7721 cells were divided into three groups: Mock group, AdGFP group and AdGSTZ1 group. Mock group was blank control group, AdGFP group was adenovirus AdGFP control group, and AdGSTZ1 group was adenovirus AdGSTZ1 experimental group. Then, the effects of GSTZ1 on the migration and proliferation of hepatocellular carcinoma cells were observed by Transwell assay and MTS assay, and the migration of hepatocellular carcinoma cells was also investigated by qRT-PCR and Western blot. Results: High titer recombinant adenovirus AdGSTZ1 was successfully constructed and the expression of GSTZ1 in SMMC-7721 cells was identified by Western blot. Compared with Mock group and AdGFP group, AdGSTZ1 group could significantly inhibit the migration ability of human hepatocellular carcinoma cells. Compared with Mock group, the inhibition rate of AdGSTZ1 group was (68.10 ± 1.63)% (P = 0.000) The inhibition rate was (50.70 ± 0.99)% (P = 0.000). MTS results showed that the absorbance values of AdGSTZ1 group at 72 h and 96 h were (1.13 ± 0.08) and (1.28 ± 0.07), respectively, and those at 72 h and 96 h after AdGSTZ1 treatment were (1.28 ± 0.03) and ± 0.03). The absorbance at 96 h in AdGSTZ1 group was significantly lower than that in control group (P <0.01). In addition, q RT-PCR and Western blot results showed that compared with Mock group and AdGFP group, the expression of SMAD3, IGF1R and other genes in AdGSTZ1-infected intracellular adhesion signal pathway was significantly decreased. q RT-PCR results showed that the relative expression level of TGFBR1 was (0.58 ± 0.13) (t = 4.609, P = 0.010) and the relative expression level of SMAD3 was (0.51 ± 0.08) in AdGSTZ1 group compared with AdGFP group (t = 8.030, The relative expression level of ERBB2 was (0.46 ± 0.09) (t = 9.384, P = 0.000), the relative expression level of IGF1R was (0.45 ± 0.02) (t = 14.480, P = 0.000) (0.37 ± 0.13) (t = 7.082, P = 0.028). Western blot showed that the relative expression of protein in AdGFP group was (0.96 ± 0.18) and that of AdGSTZ1 group (0.39 ± 0.01) in SMAD3 group (P = 0.001). In IGF1R group, the relative expression level of protein in AdGFP group was (0.97 ± 0.05) and that in AdGSTZ1 group was 0.44 ± 0.02 (P = 0.000). Conclusion: GSTZ1 can inhibit the migration of hepatocellular carcinoma cells by regulating the expression of target genes involved in adhesion junction signaling pathway.