中华病理学杂志
中華病理學雜誌
중화병이학잡지
Chinese Journal of Pathology
2011年
8期
542-548
,共7页
龚苗子%由江峰%裴斐%崔湘霖%李刚%郑杰
龔苗子%由江峰%裴斐%崔湘霖%李剛%鄭傑
공묘자%유강봉%배비%최상림%리강%정걸
肿瘤转移%转录,遗传%基因,肿瘤抑制%细胞系,肿瘤
腫瘤轉移%轉錄,遺傳%基因,腫瘤抑製%細胞繫,腫瘤
종류전이%전록,유전%기인,종류억제%세포계,종류
Neoplasm metastasis%Transcription,genetic%Genes,tumor suppressor%Cell line,tumor
目的 探讨肿瘤转移抑制基因1(TMSG-1)的基因转录调控机制.方法 PCR扩增TMSG-1转录起始位点上游-271 bP与下游+303 bp之间不同长度片段,连入荧光素酶报告基因质粒pGL3-basic,各重组质粒转染后对表达的荧光素酶活性进行检测;对重组质粒pGL3-237插入DNA序列的潜在转录因子结合位点进行定点突变,然后检测其荧光素酶活性的变化;运用凝胶迁移阻滞和染色质免疫共沉淀验证转录因子KLF6和Sp1与TMSG-1DNA调控区的相互作用;通过免疫共沉淀实验分析转录因子KLF6和Sp1之间的相互作用;对人前列腺癌PC-3M不同转移潜能亚系PC-3M-1E8、PC-3M-2B4,人肺巨细胞癌PG不同转移潜能亚系PG-BE1、PG-LH7通过荧光定量PCR分析TMSG-1和野生型KLF6 mRNA水平;将KLF6真核表达质粒转染PC-3M-2B4后检测TMSG-1蛋白水平及细胞侵袭能力的变化.结果 通过荧光素酶报告基因实验鉴定出TMSG-1基因第1号外显子内存在明显促进基因转录的调控区域+59～+123 bp.对该区域序列定点突变后重组荧光素酶报告基因表达的荧光素酶活性下降,而共转染未突变的荧光素酶报告基因质粒和KLF6或Sp1的真核表达质粒则荧光素酶活性上升,差异具有统计学意义(P＜0.01).凝胶迁移阻滞和染色质免疫共沉淀结果显示,转录因子KLF6和Sp1与该区域特定序列存在相互作用.免疫共沉淀结果验证转录因子KLF6与Sp1之间存在相互作用.荧光定量PCR结果显示前列腺癌细胞低转移亚系PC-3M-2B4中TMSG-1和野生型KLF6 mRNA水平均高于高转移亚系PC-3M-1E8(P＜0.05,P＜0.01),同样肺巨细胞癌低转移亚系PG-LH7中TMSG-1和野生型KLF6 mRNA水平均高于高转移亚系PG-BE1(均P＜0.01).PC-3M-2B4细胞转染KLF6真核表达质粒后检测到TMSG-1蛋白的增加和细胞侵袭能力的下降.结论 前列腺癌细胞中转录因子KLF6与Sp1共同作用促进肿瘤转移抑制相关基因TMSG-1的转录激活;不同转移潜能前列腺癌和肺巨细胞癌细胞亚系之间TMSG-1的差异表达与其野生型KLF6差异表达相关.
目的 探討腫瘤轉移抑製基因1(TMSG-1)的基因轉錄調控機製.方法 PCR擴增TMSG-1轉錄起始位點上遊-271 bP與下遊+303 bp之間不同長度片段,連入熒光素酶報告基因質粒pGL3-basic,各重組質粒轉染後對錶達的熒光素酶活性進行檢測;對重組質粒pGL3-237插入DNA序列的潛在轉錄因子結閤位點進行定點突變,然後檢測其熒光素酶活性的變化;運用凝膠遷移阻滯和染色質免疫共沉澱驗證轉錄因子KLF6和Sp1與TMSG-1DNA調控區的相互作用;通過免疫共沉澱實驗分析轉錄因子KLF6和Sp1之間的相互作用;對人前列腺癌PC-3M不同轉移潛能亞繫PC-3M-1E8、PC-3M-2B4,人肺巨細胞癌PG不同轉移潛能亞繫PG-BE1、PG-LH7通過熒光定量PCR分析TMSG-1和野生型KLF6 mRNA水平;將KLF6真覈錶達質粒轉染PC-3M-2B4後檢測TMSG-1蛋白水平及細胞侵襲能力的變化.結果 通過熒光素酶報告基因實驗鑒定齣TMSG-1基因第1號外顯子內存在明顯促進基因轉錄的調控區域+59～+123 bp.對該區域序列定點突變後重組熒光素酶報告基因錶達的熒光素酶活性下降,而共轉染未突變的熒光素酶報告基因質粒和KLF6或Sp1的真覈錶達質粒則熒光素酶活性上升,差異具有統計學意義(P＜0.01).凝膠遷移阻滯和染色質免疫共沉澱結果顯示,轉錄因子KLF6和Sp1與該區域特定序列存在相互作用.免疫共沉澱結果驗證轉錄因子KLF6與Sp1之間存在相互作用.熒光定量PCR結果顯示前列腺癌細胞低轉移亞繫PC-3M-2B4中TMSG-1和野生型KLF6 mRNA水平均高于高轉移亞繫PC-3M-1E8(P＜0.05,P＜0.01),同樣肺巨細胞癌低轉移亞繫PG-LH7中TMSG-1和野生型KLF6 mRNA水平均高于高轉移亞繫PG-BE1(均P＜0.01).PC-3M-2B4細胞轉染KLF6真覈錶達質粒後檢測到TMSG-1蛋白的增加和細胞侵襲能力的下降.結論 前列腺癌細胞中轉錄因子KLF6與Sp1共同作用促進腫瘤轉移抑製相關基因TMSG-1的轉錄激活;不同轉移潛能前列腺癌和肺巨細胞癌細胞亞繫之間TMSG-1的差異錶達與其野生型KLF6差異錶達相關.
목적 탐토종류전이억제기인1(TMSG-1)적기인전록조공궤제.방법 PCR확증TMSG-1전록기시위점상유-271 bP여하유+303 bp지간불동장도편단,련입형광소매보고기인질립pGL3-basic,각중조질립전염후대표체적형광소매활성진행검측;대중조질립pGL3-237삽입DNA서렬적잠재전록인자결합위점진행정점돌변,연후검측기형광소매활성적변화;운용응효천이조체화염색질면역공침정험증전록인자KLF6화Sp1여TMSG-1DNA조공구적상호작용;통과면역공침정실험분석전록인자KLF6화Sp1지간적상호작용;대인전렬선암PC-3M불동전이잠능아계PC-3M-1E8、PC-3M-2B4,인폐거세포암PG불동전이잠능아계PG-BE1、PG-LH7통과형광정량PCR분석TMSG-1화야생형KLF6 mRNA수평;장KLF6진핵표체질립전염PC-3M-2B4후검측TMSG-1단백수평급세포침습능력적변화.결과 통과형광소매보고기인실험감정출TMSG-1기인제1호외현자내존재명현촉진기인전록적조공구역+59～+123 bp.대해구역서렬정점돌변후중조형광소매보고기인표체적형광소매활성하강,이공전염미돌변적형광소매보고기인질립화KLF6혹Sp1적진핵표체질립칙형광소매활성상승,차이구유통계학의의(P＜0.01).응효천이조체화염색질면역공침정결과현시,전록인자KLF6화Sp1여해구역특정서렬존재상호작용.면역공침정결과험증전록인자KLF6여Sp1지간존재상호작용.형광정량PCR결과현시전렬선암세포저전이아계PC-3M-2B4중TMSG-1화야생형KLF6 mRNA수평균고우고전이아계PC-3M-1E8(P＜0.05,P＜0.01),동양폐거세포암저전이아계PG-LH7중TMSG-1화야생형KLF6 mRNA수평균고우고전이아계PG-BE1(균P＜0.01).PC-3M-2B4세포전염KLF6진핵표체질립후검측도TMSG-1단백적증가화세포침습능력적하강.결론 전렬선암세포중전록인자KLF6여Sp1공동작용촉진종류전이억제상관기인TMSG-1적전록격활;불동전이잠능전렬선암화폐거세포암세포아계지간TMSG-1적차이표체여기야생형KLF6차이표체상관.
Objective To investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1. Methods Luciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay(EMSA) and chromatin immunoprecipitation(CHIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation(ColP) was performed to analyze the interaction between KLF6 and Spl. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay. Results A 63 bp inducible regulatory region ( +59 bp - + 123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Spl binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Spl interacted with this region. CoIP also indicated a possible interaction between KLF6 and Spl proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability. Conclusions Transcription factor complex of KLF6 and Spl may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.
