中华病理学杂志
中華病理學雜誌
중화병이학잡지
Chinese Journal of Pathology
2010年
2期
88-94
,共7页
于文娟%王曰伟%谢志刚%由江峰%王洁良%崔湘琳%裴斐%郑杰
于文娟%王曰偉%謝誌剛%由江峰%王潔良%崔湘琳%裴斐%鄭傑
우문연%왕왈위%사지강%유강봉%왕길량%최상림%배비%정걸
前列腺肿瘤%肿瘤转移%肿瘤标记%生物学%寡核苷酸序列分析
前列腺腫瘤%腫瘤轉移%腫瘤標記%生物學%寡覈苷痠序列分析
전렬선종류%종류전이%종류표기%생물학%과핵감산서렬분석
Prostatic neoplasma%Neoplasm metastasis%Tumor markers%biological%Oligonucleotide array sequence analysis
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.
目的 篩選腫瘤轉移相關新基因,探討鞘精脂微結構域1相關燐痠化蛋白基因(PAG1)轉染對人前列腺癌細胞繫PC-3M的高轉移亞繫PC-3M-1E8體外生物學行為的影響.方法 利用PC-3M高轉移亞繫PC-3M-1E8、低轉移亞繫PC-3M-284,人肺巨細胞痛細胞繫(PG)高轉移亞繫PG-BE1和低轉移亞繫PG-LH7 cDNA製作4張基因芯片,篩選齣PC-3M和PG高、低轉移亞繫共同差異錶達基因.對在兩箇轉移亞繫共同錶達下調的PAG1基因做進一步研究,採用即時定量PCR及Western blot驗證PAG1在PC-3M細胞繫中的錶達.構建pcDNA3.0-PAG1真覈錶達載體,穩定轉染PC-3M-1E8細胞.MTT比色實驗及軟瓊脂集落形成實驗檢測腫瘤細胞體外增殖能力;流式細胞術檢測腫瘤細胞週期及凋亡;Matrigel穿膜實驗檢測腫瘤細胞體外侵襲能力.結果 基因芯片初步篩選齣PC-3M高、低轉移亞繫差異錶達基因共327箇,上調基因123箇,下調基因204箇.PG高、低轉移亞繫差異錶達基因共281箇,上調基因167箇,下調基因114箇.PC-3M與PG高轉移亞繫共同錶達下調基因9箇、上調基因8箇.即時定量PCR及Western blot證實PAG1在PC-3M高轉移亞繫中錶達低于低轉移亞繫.MTT比色及軟瓊脂集落形成實驗顯示轉染pcDNA3.0-PAG1組細胞增殖速度明顯低于轉染空載體組和未轉染組(均P<0.05).細胞週期檢測轉染pcDNA3.0-PAG1組比轉染空載體組和未轉染組處于G_0～G_1期的細胞百分數明顯增加(P<0.05).轉染pcDNA3.0-PAG1組與轉染空載體組和未轉染組相比凋亡細胞百分率無顯著差異(P>0.05).體外穿膜侵襲實驗結果錶明轉染pcDNA3.0-PAG1組比轉染空載體組和未轉染組穿膜細胞數目明顯減少,分彆為(35.1±4.9)、(127.6±6.6)和(135.0±5.0)箇(P<0.05).結論 利用同一母繫來源的高、低轉移亞繫製作基因芯片可以摒除轉移無關基因的榦擾,篩選齣差異錶達的腫瘤轉移相關基因.PAG1基因穩定轉染能抑製人前列腺癌高轉移亞繫PC-3M-1E8細胞的體外增殖能力和侵襲能力,PAG1基因可能是一箇潛在的腫瘤增殖、侵襲和轉移的抑製基因.
목적 사선종류전이상관신기인,탐토초정지미결구역1상관린산화단백기인(PAG1)전염대인전렬선암세포계PC-3M적고전이아계PC-3M-1E8체외생물학행위적영향.방법 이용PC-3M고전이아계PC-3M-1E8、저전이아계PC-3M-284,인폐거세포통세포계(PG)고전이아계PG-BE1화저전이아계PG-LH7 cDNA제작4장기인심편,사선출PC-3M화PG고、저전이아계공동차이표체기인.대재량개전이아계공동표체하조적PAG1기인주진일보연구,채용즉시정량PCR급Western blot험증PAG1재PC-3M세포계중적표체.구건pcDNA3.0-PAG1진핵표체재체,은정전염PC-3M-1E8세포.MTT비색실험급연경지집락형성실험검측종류세포체외증식능력;류식세포술검측종류세포주기급조망;Matrigel천막실험검측종류세포체외침습능력.결과 기인심편초보사선출PC-3M고、저전이아계차이표체기인공327개,상조기인123개,하조기인204개.PG고、저전이아계차이표체기인공281개,상조기인167개,하조기인114개.PC-3M여PG고전이아계공동표체하조기인9개、상조기인8개.즉시정량PCR급Western blot증실PAG1재PC-3M고전이아계중표체저우저전이아계.MTT비색급연경지집락형성실험현시전염pcDNA3.0-PAG1조세포증식속도명현저우전염공재체조화미전염조(균P<0.05).세포주기검측전염pcDNA3.0-PAG1조비전염공재체조화미전염조처우G_0～G_1기적세포백분수명현증가(P<0.05).전염pcDNA3.0-PAG1조여전염공재체조화미전염조상비조망세포백분솔무현저차이(P>0.05).체외천막침습실험결과표명전염pcDNA3.0-PAG1조비전염공재체조화미전염조천막세포수목명현감소,분별위(35.1±4.9)、(127.6±6.6)화(135.0±5.0)개(P<0.05).결론 이용동일모계래원적고、저전이아계제작기인심편가이병제전이무관기인적간우,사선출차이표체적종류전이상관기인.PAG1기인은정전염능억제인전렬선암고전이아계PC-3M-1E8세포적체외증식능력화침습능력,PAG1기인가능시일개잠재적종류증식、침습화전이적억제기인.
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.
