鞘糖脂微结构域1相关磷酸化蛋白基因的筛选及其对转移性前列腺癌细胞生物学行为的影响

目的 筛选肿瘤转移相关新基因,探讨鞘精脂微结构域1相关磷酸化蛋白基因(PAG1)转染对人前列腺癌细胞系PC-3M的高转移亚系PC-3M-1E8体外生物学行为的影响.方法 利用PC-3M高转移亚系PC-3M-1E8、低转移亚系PC-3M-284,人肺巨细胞痛细胞系(PG)高转移亚系PG-BE1和低转移亚系PG-LH7 cDNA制作4张基因芯片,筛选出PC-3M和PG高、低转移亚系共同差异表达基因.对在两个转移亚系共同表达下调的PAG1基因做进一步研究,采用即时定量PCR及Western blot验证PAG1在PC-3M细胞系中的表达.构建pcDNA3.0-PAG1真核表达载体,稳定转染PC-3M-1E8细胞.MTT比色实验及软琼脂集落形成实验检测肿瘤细胞体外增殖能力;流式细胞术检测肿瘤细胞周期及凋亡;Matrigel穿膜实验检测肿瘤细胞体外侵袭能力.结果 基因芯片初步筛选出PC-3M高、低转移亚系差异表达基因共327个,上调基因123个,下调基因204个.PG高、低转移亚系差异表达基因共281个,上调基因167个,下调基因114个.PC-3M与PG高转移亚系共同表达下调基因9个、上调基因8个.即时定量PCR及Western blot证实PAG1在PC-3M高转移亚系中表达低于低转移亚系.MTT比色及软琼脂集落形成实验显示转染pcDNA3.0-PAG1组细胞增殖速度明显低于转染空载体组和未转染组(均P<0.05).细胞周期检测转染pcDNA3.0-PAG1组比转染空载体组和未转染组处于G_0～G_1期的细胞百分数明显增加(P<0.05).转染pcDNA3.0-PAG1组与转染空载体组和未转染组相比凋亡细胞百分率无显著差异(P>0.05).体外穿膜侵袭实验结果表明转染pcDNA3.0-PAG1组比转染空载体组和未转染组穿膜细胞数目明显减少,分别为(35.1±4.9)、(127.6±6.6)和(135.0±5.0)个(P<0.05).结论 利用同一母系来源的高、低转移亚系制作基因芯片可以摒除转移无关基因的干扰,筛选出差异表达的肿瘤转移相关基因.PAG1基因稳定转染能抑制人前列腺癌高转移亚系PC-3M-1E8细胞的体外增殖能力和侵袭能力,PAG1基因可能是一个潜在的肿瘤增殖、侵袭和转移的抑制基因.

Screening of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) by cDNA microarray and influence of overexpression of PAG1 on biologic behavior of human metastatic prostatic cancer cell line in vitro

Objective To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomaius 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro. Methods Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential) and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1,which was markedly dowrtregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones,vector transfected clones and non-transfected parental cells. Results A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1E8. Western blot revealed that PAG1 protein was downregulated in PC-3M-1E8 cell line which was in agreement with the gene expression at mRNA level. The proliferation ability and clonogenicity of PAG1 overexpression cells by stable transfection were markedly decreased in comparing with that of the control cells (P < 0. 05). Colonies formed in stably PAG1-transfected cells, the vector-transfected clones and parental cells were 26.7±5.2,47.2±3.2 and 52. 3±3.4 respectively(P<0.05). Flow cytometry analysis showed that the stable PAG1-transfected cells at G_0-G_1 phase were significantly more than that of the control cells (P<0.05). However,no difference of the apoptosis rate was found between PAG1-transfected cells and control cells(P>0.05).The number of cells passing through the matrigel and multipore membrane was also decreased in the stable PAGl-transfected cells(35.1±4.9) compared with those of the vector-transfected clones(127.6±6.6) and parental cells(135.0±5.0,P<0.05). Conclusions Using cDNA microarray technique and differential gene expression analysis of sublines of the parental cancer cell lines enable of revealing the metastasis-related genes, among which PAG1 represents one of those under-expressed genes in the high metastatic subline PC-3M-1E8. Transfection expression of PAG1 suppresses cell proliferation, anchorage-independent growth and invasive ability of PC-3M-1E8 cells in vitro. Conclusively, PAG1 may play an important role in inhibiting the proliferation, invasion and metastasis of the cancer cells.