前列腺癌细胞中TNS4新突变体及其作用机制初探

A novel TNS4 mutant in prostate cancer cells and its mechanism

目的:检测前列腺癌细胞中张力蛋白家族第四成员(tensin family member 4,TNS 4)基因的表达和突变情况,并探讨其潜在的作用机制。方法:采用RT-PCR法分析多种前列腺癌细胞中TNS 4基因表达;并扩增TNS 4基因的全长cDNA,进行限制性核酸内切酶水解和DNA测序分析。构建TNS 4基因野生型和突变体重组质粒,转染前列腺癌PC-3细胞,然后采用RT-PCR、蛋白质印迹法和免疫荧光染色法分别测定野生型和突变体TNS4过表达对信号转导与转录激活因子1(signal transducer and activator of transcription 1,STAT1)mRNA、蛋白表达及定位的影响。结果:与良性前列腺增生细胞BPH1相比,前列腺癌DU145、LNCaP和22Rv1细胞中TNS 4基因呈低表达(P值均<0.01),而前列腺癌PC-3细胞中几乎没有TNS 4基因表达。分析TNS 4基因的全长cDNA序列后发现,前列腺癌DU145和LNCaP细胞中TNS 4基因存在突变位点T427C和G1455A,其中T427C突变导致对应氨基酸构成发生转变(S143P)。成功构建TNS 4基因突变体重组质粒pcDNA3.1(+)-TNS4-Mut和野生型TNS 4基因重组质粒pcDNA3.1(+)-TNS4-WT并转染前列腺癌PC-3细胞后,TNS4 mRNA和蛋白表达水平明显升高(P值均<0.05),STAT 1基因转录水平未见明显变化(P>0.05),但STAT1蛋白表达水平明显升高(P值均<0.05),而且TNS 4基因突变体过表达能够明显促进STAT1蛋白进入细胞核(P<0.01)。结论:前列腺癌细胞中TNS 4基因明显低表达,且存在新的TNS4突变体(S143P);过表达TNS4能够上调STAT1蛋白的表达水平,而且TNS4突变体过表达会进一步改变STAT1蛋白的细胞定位。

Objective： To investigate the expression and mutation status of tensin family member 4 （TNS4） gene in various prostate cancer cell lines, and to explore its potential molecular mechanism.Methods： The expression of TNS4 gene in several prostate cancer cell lines was detected by R1-PCR. The full-length cDNA of TNS4 gene was amplified for the restriction endonuclease hydrolysis and DNA sequencing analysis. Meantime, the recombinant plasmids carrying wild- or mutant-type TNS4 gene were constructed and transfected into prostate cancer PC-3 cells, then the effects of TNS4 over-expression on the mRNA and protein expressions as well as the localization of signal transducer and activator of transcription 1 （STAT1） were detected by RT-PCR, Western blotting and immunofluorescence microscopy, respectively. Results： As compared with benign prostatic hyperplasia BPH1 cells, TNS4 gene was lowly expressed in prostate cancer cell lines DU145, LNCaP and 22Rvl （all P 〈 0.01）, and nearly undetectable in prostate cancer PC-3 cells. The analysis of full-length TNS4 cDNA sequencing revealed that two sites of single point mutation were identified at T427C and G1455A of TNS4 gene derived from DU145 and LNCaP cells, and the former caused the corresponding amino acid transition （$143P）. The recombinant plasmids pcDNA3.1 （＋） -TNS4-Mut （TNS4-Mut） and pcDNA3.1 （＋） -TNS4-WT （TNS4-WT） were successfully established. After transfection with TNS4-Mut or TNS4-WT, the expression levels of TNS4 mRNA and protein and STAT1 protein in PC-3 cells were significantly increased （all P 〈 0.05）, but the expression level of STAT1 mRNA was not changed （P 〉 0.05）. Additionally, the over-expression of mutant TNS4 significantly promoted nuclear translocation of STAT1 protein （P 〈 0.01）. Conclusion： TNS4 gene is lowly expressed in prostate cancer cell lines, and there is a nove TNS4 mutant （$143P）. TNS4 over-expression can up-regulate the expression of STAT1 protein, furthermore the mutant TNS4 can alter the location of STAT1 protein.