nm23-H1基因对肺癌细胞株L9981中Wnt信号传导通路中糖原合成激酶-3β的表达和活性的影响

摘要

目的 观察转移抑制基因nm23-H1对肺癌细胞株L9981中Wnt信号传导通路中糖原合成激酶-3β(GSK-3β)的表达量和活性的影响.方法 将稳定转染nm23-H1基因的肺癌细胞株、原代细胞株L9981和空载体转染细胞株培养传代,分别设为nm23-H1转染组、对照组和空白转染组.应用Western blot分别检测应用20 mmol/L LiCl处理前后3组肺癌细胞株胞质、胞核中GSK-3β的表达水平,测定上述肺癌细胞株胞质和胞核中的GSK-3β活性.结果 (1)nm23-H1转染组胞质和胞核中GSK-3β蛋白表达量分别为(6 639±503)和(4481 ±446)A,空白转染组分别为(645±352)和(726 ±68)A,对照组分别为(763±267)和(683 ±49)A.3组细胞株间胞质和胞核中GSK-3β表达量差异有统计学意义(P =0.025).两两比较:nm23-H1转染组胞质和胞核GSK-3β表达水平均显著高于空白转染组和对照组(P=0.017),而后两者之间比较均差异无统计学意义(P=0.094).(2)nm23-H1转染组胞质和胞核GSK-3β激酶活性分别为(29 371 ±2 492)和(9647±859)每分钟脉冲数,空白转染组分别为(11241 ±1495)和(1 492±176)每分钟脉冲数,对照组分别为(14 271±1 137)和(1 853±113)每分钟脉冲数.3组细胞株间胞质和胞核中GSK-3β酶活性差异有统计学意义(P =0.032),两两比较:nm23-H1转染组,胞质和胞核GSK-3β酶活性均显著高于空白转染组和对照组(P =0.027),而后两者之间比较均差异无统计学意义(P=0.131).(3)经20 mmol/L LiCl处理后,nm23-H1转染组胞质和胞核中GK-3β表达量分别为(5 037±427)和(823±350)A,与处理前比较均差异无统计学意义(P=0.168),空白转染组细胞株经LiCl处理后胞质和胞核中GSK-3β表达量分别为(2 174 ±126)和(248±12)A,对照组则分别为(2 273±128)和(253±14)A,两个细胞株胞质和胞核中GSK-3β表达量均显著低于处理前(P=0.012、0.015).(4)经LiCl处理后,nm23-H1转染组胞质和胞核GSK-3β激酶活性分别为(12 837±1 042)和(748 ±215)每分钟脉冲数,空白转染组分别为(4 739 ±401)和(947±126)每分钟脉冲数,对照组分别为(4 638 ±401)和(1 162±127)每分钟脉冲数,3组细胞株胞质和胞核GSK-3β激酶活性均显著低于处理前(P=0.006、0.029、0.010).结论 nm23-H1基因转染能显著上调人肺癌细胞L9981中GSK-3β的蛋白表达和活性.其可能通过上调人肺癌细胞中GSK-3β的蛋白表达和活性抑制Wnt信号传导而发挥作用.%Objective To investigate the influence of tumor metastasis suppressor gene nm23-H1 on the activity of glycogen synthase kinase-3β (GSK-3β) in lung cancer cell line L9981.Methods The levels of GSK-3β expression in cytoplasm and nucleus were determined with anti-GSK-3β antibody in lung cancer cell line control group (cell line with nm23-H1 gene deletion),nm23-H1 transfection group (cell line with nm23-H1 transfected) and blank transfection group (cell line with vector transfected) by Western blotting.The activity of GSK-3β among those three groups was detected by immunoprecipitation and analyzed by a radioactive isotope scintillation counter before and after treatment with 20 mmol/L LiCl.Results (1) The expression indensity of GSK-3β in cytoplasm and nucleus was (6 639 ±503) and (4 481 ±446) A in nm23-H1 transfection group,(645 ±352) and (726 ±68) A in blank transfection group,and (763 ± 267) and (683 ± 49) A in control group,respectively.There was very significance in GSK-3β expressive indensity of both cytoplasm and nucleus among nm23-H1 transfection group,blank transfection group and control group (P =0.025).There was significant difference between nm23-H1 transfection group and blank transfection group or control group (P =0.017),but no significant difference was observed between blank transfection group and control group (P =0.094).(2) The GSK-3β activity in cytoplasm and nucleus was (29 371 ±2 492) and (9 647 ±859) counts per minute in nm23-H1 transfection group,(11 241 ± 1 495) and (1 492 ± 176) counts per minute in blank transfection group,and (14 271 ± 1 137) and (1 853 ± 113) counts per minute in control group,respectively.There was very significant difference in GSK-3β activity of both cytoplasm and nucleus among nm23-H1 transfection group,blank transfection group and control group (P =0.032).The GSK-3β activity in nm23-H1 transfection group was significantly higher than that in blank transfection group and control group (P =0.027),but no significant difference was observed between the blank transfection group and control group (P =0.131).(3) After treatment with 20 mmol/L LiCl,the expression indensity of GSK-3β in cytoplasm and nucleus was (5 037 ±427) and (823 ±350) A in nm23-H1 transfection group,(2 174 ±126) and (248 ±12) A in blank transfection group,and (2 273 ±128) and (253 ±14) A in con trol group.No significant difference in GSK-3β expression indensity existed before and after treatment with LiCl in nm23-H1 transfection group (P =0.168).However,the GSK-3β expression indensity in cytoplasm and nucleus before treatment was remarkably higher than that after treatment in both blank transfection group and control group (P =0.012,0.015).(4) After treatment with 20 mmol/L LiCl,the GSK-3β activity in cytoplasm and nucleus was (12 837 ± 1 042) and (748 ± 215) counts per minute in nm23-H1 transfection group,(4 739 ±401) and (947 ± 126) counts per minute in control group,and (4 638 ± 401) and (1 162 ± 127) counts per minute in blank transfection group,respectively.The GSK-3β activity in cytoplasm and nucleus after treatment with LiCl was remarkably lower than that before treatment in nm23-H1 transfection group,blank transfection group and control group (P =0.006,0.029,0.010).Conclusion Transfection of nm23-H1 gene can significantly up-regulate the expression level and activity of GSK-3β in lung cancer cell line.The inhibitory effects of nm23-H1 gene on the signal transduction of Wnt pathway might be carried out through up regnlating GSK-3β expression and activity in lung cancer cell line.