Identification of tumor metastasisrelated gene TMSG-1 by mRNA differential display

To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression of two cancer sublines derived from prostate carcinoma cell PC-3M that had different metastatic potentials. The differentially expressed genes were confirmed by Northern blot, and sequenced. The full-length cDNA of a tumor metastasis suppressor gene (TMSG-1) was obtained by using EST assembling and verified by RT-PCR and sequencing. The results showed that expression levels of TMSG-1 were lower in the highly metastatic cell line 1E8, compared with the non-metastatic cell line 2B4. The difference was significant. Full-length cDNA of TMSG-1 was about 2 kb, containing an open reading frame that encoded a protein of 230 amino acids. GenBank Blastn showed no marked homology with known genes. The functional prediction of amino acids sequence encoded by TMSG-1 gene indicated TMSG-1 protein was transmembrane protein, with 3 transmembrane domains, 3 putative protein kinase phosphorylatio     

TMSG-1基因功能的初步研究

TMSG-1是用mRNA差异显示技术克隆的转移相关基因, 它在高转移肿瘤细胞系和有转移的肿瘤组织中表达下降. 以高转移的前列腺癌细胞系PC-3M-1E8为受体细胞, 通过基因转染技术观察了TMSG-1基因表达对细胞V-ATPase活性、细胞内pH值和细胞凋亡情况的影响, 同时利用GFP对TMSG-1细胞内定位进行了分析. 结果表明, V-ATPase在PC-3M-1E8细胞系, 转染空载体和转染反义TMSG-1细胞系的活性无明显差异. 在转染正义TMSG-1的细胞系中, V-ATPase的活性比PC-3M-1E8细胞系, 转染空载体和转染反义TMSG-1细胞系均有明显增高(P < 0.001). 利用pH敏感性荧光探针BCECF测定细胞内的pH值, 结果显示转染正义TMSG-1组的pHi值有明显提高. 细胞凋亡的检测结果表明, 转染正义TMSG-1组细胞凋亡明显增多(P < 0.01), BCL2的表达显著下降. TMSG-1蛋白的细胞内定位分析表明, TMSG-1是一个跨膜蛋白, 定位于内质网、线粒体等细胞质膜系统. 实验结果表明, 前列腺癌细胞系中TMSG-1的上调可以提高细胞V-ATPase的活性, 增加细胞内的pH值, 同时TMSG-1的上调还可抑制BCL2的表达, 促进细胞凋亡的发生.     

Primary functional identification of gene <Emphasis Type="Italic">TMSG-1</Emphasis>

TMSG-1 was a tumor metastasis-related gene identified using mRNA differential display, whose expression level was lower in cancer cell lines with higher metastatic potential and in tumor tissue with metastasis. TMSG-1 was transfected to prostate cancer cell line (PC-3M-1E8) with high metastatic potential to observe the effects of increased expression of TMSG-1 on V-ATPase activity, intracellular pH and cell apoptosis. Subcellular localization of the encoded protein of TMSG-1 was determined by using GFP. Results showed that there were no differences of V-ATPase activity among parental PC-3M-1E8 cell line, pcDNA3 transfectant and anti-TMSG-1 transfectant, whereas the V-ATPase activity was significantly higher in TMSG-1 transfectant than that in parental PC-3M-1E8 cell line, pcDNA3 transfectant and Anti-TMSG-1 transfectant (p<0.001). Intracellular pH (pHi) was detected by using the pH-dependent fluorescence probe BECEF. Results showed the pHi was significantly increased in TMSG-1 transfectant. Cell apoptosis assay demonstrated cell apoptosis was significantly higher in -1 transfectant (p<0.01) and BCL2 expression was down regulated. Subcellular localization of TMSG-1 protein showed TMSG-1 was a transmembrane protein, which predicted TMSG-1 protein was located in cytoplasm system, such as endoplasmic reticulum and mitochondrial. These results indicated TMSG-1 up regulation in prostate cancer cell line could promote V-ATPase activity, increase pHi and cell apoptosis, and inhibit the expression of BCL2.     

Primary functional identification of gene TMSG-1

TMSG-1 (Tumor metastasis suppressor gene-1) is a cancer metastasis-related gene cloned by means of mRNA differential display from human prostate cancer cell lines with different metas-tatic potential[1], which has higher expression in non-metastatic cell line, whereas lower expres-sion in highly metastatic cell line. In samples of primary gastric carcinoma, the TMSG-1 expres-sion markedly decreased in gastric carcinoma with lymph node metastases. It was found that protein encoded by TMS…     

应用mRNA差异显示技术克隆肿瘤转移相关基因TMSG-1

应用mRNA差异显示技术, 对比研究前列腺癌细胞系PC-3M具有不同转移潜能的亚系, 克隆差异表达片段, 经Northern杂交验证其在不同转移潜能的人类肿瘤细胞系中的表达差异, 测序, EST拼接获得cDNA全长序列, RT-PCR验证拼接结果并重复测序再次证实. 结果显示, TMSG-1在前列腺癌细胞系PC-3M不转移亚系2B4高表达, 而在高转移亚系1E8低表达, 二者差异显著. TMSG-1 cDNA全长序列2 kb, 开放读码框编码含230aa的蛋白质, GenBank Blastn检索与已知基因无显著同源性. 对TMSG-1所编码的氨基酸序列进行功能预测提示：TMSG-1是一个跨膜蛋白, 有3个跨膜区、3个蛋白激酶C磷酸化位点、 2个酪蛋白激酶Ⅱ磷酸化位点和1个N末端肉豆蔻酸酰化位点. 在6种人类常见肿瘤组织中, RT-PCR结果显示TMSG-1在前列腺癌中表达最强, 肺癌转移灶较肺癌原发灶表达显著降低, 低分化结肠癌较高分化结肠癌表达显著降低, 在复发性乳腺癌、卵巢癌、低分化胰腺癌(均无转移)中均有表达. 9例胃癌原发灶标本中, TMSG-1与b-actin RT-PCR扩增产物的灰度扫描(CNT×mm²)比值显示: TMSG-1在已发生转移的胃癌标本中的表达与无转移的胃癌标本相比有下降的趋势, t检验显示其差异有显著性(P < 0.05). 结果表明, TMSG-1基因表达水平的降低与肿瘤转移潜能的提高具有相关性.