摘要
背景与目的我们的前期研究结果表明nm23-H1基因的肺癌转移抑制作用可能与上调Wnt信号通路中关键激酶GSK-3β的活性、进而抑制Wnt信号通路相关。本研究的目的是探讨nm23-H1点突变对人高转移大细胞肺癌细胞株L9981中胞质和胞核的GSK-3β激酶活性的影响,为阐明nm23-H1基因调控肺癌转移相关信号传导通路的分子机制提供实验依据。方法将原代人低转移大细胞肺癌细胞株NL9980、原代人高转移大细胞肺癌细胞株L9981、空载体转染细胞株L9981-pEGFP、稳定转染nm23-H1基因的细胞株L9981-nm23-H1-pEGFP以及稳定转染在44、96、118、120号位点发生突变后的nm23-H1基因的转基因肺癌细胞株L9981-nm23-H1S44A-pEGFP、L9981-nm23-H1P96S-pEGFP、L9981-nm23-H1H118F-pEGFP和L9981-nm23-H1S120G-pEGFP作为研究对象,应用免疫共沉淀同位素闪烁计数法测定上述肺癌细胞株胞质和胞核中的GSK-3β激酶活性。结果nm23-H1基因发生点突变后,所有转基因肺癌细胞株细胞胞质和胞核中的GSK-3β活性均较突变前有所下降。L9981-nm23-H1S44A-pEGFP、L9981-nm23-H1P96S-pEGFP、L9981-nm23-H1H118F-pEGFP、L9981-nm23-H1S120G-pEGFP分别与L9981-nm23-H1-pEGFP比较,胞质与胞核中GSK-3β激酶活性均存在显著性差异(P<0.05),其中L9981-nm23-H1P96S-pEGFP与L9981-nm23-H1-pEGFP胞质中GSK-3β活性存在极显著差异(P<0.01),L9981-nm23-H1S44A-pEGFP、L9981-nm23-H1P96S-pEGFP和L9981-nm23-H1H118F-pEGFP与L9981-nm23-H1-pEGFP的胞核中GSK-3β活性存在极显著差异(P<0.01)。结论①nm23-H1基因突变能显著影响其对人高转移大细胞肺癌细胞株L9981胞质和胞核中GSK-3β激酶活性的调控作用;②nm23-H1基因可能通过调控L9981中GSK-3β激酶活性来抑制L9981细胞株中Wnt信号传导。
Background and objective The results of our previous studies have proven that nm23-H1 gene can suppress metastasis of lung cancer, which may be associated with suppression of the Wnt signal pathway through up-regulating the activity of glycogen synthase kinase 3β （GSK-3β）, the key kinase of the Wnt signal pathway. The aim of this study is to investigate the effect of point mutation of nm23-H1 gene on GSK 3β activity in cytoplasm and nucleus in human high-metastatic large cell lung cancer cell line L9981. Methods Using immunoprecipitation and a radioactive isotope scintillation counter, the activity of GSK-3β was detected in cytoplasm and nucleus of human low-metastatic large cell lung cancer cell line NL9980, human high-metastatic large cell lung cancer cell line L9981, L9981-pEGFP （transfected with vector）, L9981-nm23-Hl-pEGFP （transfected with wild type nm23-H1）, L9981-nm23-H1^S44A-pEGFP mutant （transfected with serine 44 to alanineon of nm23-H1 gene）, L9981-nm23-H1^P96S-pEGFP mutant （transfected with proline 96 to serine of nm23H1 gene）, L9981-nm23-H1H118aF-pEGFP mutant （transfected with histidine 118 to phenylalanine of nm23-H1 gene） and L9981-nm23-H1S120G-pEGFP mutant （transfected with serine 120 to glycine of nm23-H1 gene）. Resuits The GSK-3β activity in cytoplasm and nucleus was remarkably decreased in the transgene lung cancer cell lines transfeeted with mutant nm23-H1 cDNA （L9981-nm23-H1^S44A-pEGFP, L9981-nm23-H1P96S-pEGFP, 1.9981-nm23-H1^H118F-pEGFP and 1.9981-nm23-HlS120G-pEGFP）. Significant differences of GSK-3β activity in cytoplasm and nucleus were observed （P（0.05） when L9981-nm23-H1-pEGFP cell fine was compared with L9981-nm23-H1^S44A-pEGFP, L9981-nm23-H1P96S-pEGFP, L9981-nm23-H1H118F-pEGFP and L9981-nm23H1S120G-pEGFP lung cancer cell lines. There was a highly significant difference in GSK-3β activity in the cytoplasm between 1.9981-nm23-H1-pEGFP cell line and L9981-nm23-H1^P96S-pEGFP lung cancer ceil line （P〈 0.01）. A highly significant difference in GSK-3β activity in the nucleus was observed （P＊0.01） when L9981- nm23-H1-pEGFP lung cancer cell line was compared with L9981-nm23-H1^S44A-pEGFP, L9981-nm23-H1^P96S-pEGFP and L9981-nm23-H1^H118F-pEGFP lung cancer cell lines. Conclusion ① Point mutation of nm23-H1 gene can significantly influence the regulating effects on the GSK-3β activity in the cytoplasm and nucleus in the human high-metastatic large cell lung cancer cell line L9981. ② The effects of nm23-H1 gene on metastatic phenotype may be related to the upregulation of GSK-3β activity in human high-metastatic large eell lung cancer cell line L9981.
出处
《中国肺癌杂志》
CAS
2006年第1期30-34,共5页
Chinese Journal of Lung Cancer
基金
国家自然科学基金重点项目(No.30430300)
国家自然科学基金(No.30070333)
高等学校博士学科点专项基金(No.20040610060)资助~~
