中华病理学杂志
中華病理學雜誌
중화병이학잡지
Chinese Journal of Pathology
2014年
3期
177-183
,共7页
前列腺肿瘤%基因沉默%基质,肿瘤抑制
前列腺腫瘤%基因沉默%基質,腫瘤抑製
전렬선종류%기인침묵%기질,종류억제
Prostatic neoplasms%Gene silencing%Genes,tumor suppressor
目的 探讨LASS2/TMSG1基因沉默对人前列腺癌细胞增殖、侵袭和转移能力的影响及其分子作用机制.方法 建立稳定表达LASS2/TMSG1-shRNA的低转移潜能人前列腺癌PC-3M-2B4细胞系,然后采用四甲基偶氮唑蓝(MTT)掺入实验、软琼脂集落形成实验、流式细胞术、Matrigel穿膜侵袭实验和裸鼠体内成瘤实验研究LASS2/TMSG1的细胞生物学功能;采用Western blot法、明胶酶谱法检测基质金属蛋白酶2和9(MMP-2和MMP-9)的表达、分泌及活性;并采用V-ATPase活性检测试剂盒检测细胞内V-ATPase活性,以及采用BCECF H+敏感荧光探针检测细胞外H+浓度以研究LASS2/TMSG1抑制前列腺癌转移的分子作用机制.结果 转染LASS2/TMSG1-shRNA后,PC-3M-2B4细胞的增殖能力、锚定不依赖生长能力及体外侵袭能力明显提高(P＜0.05),而细胞凋亡率明显下降(P＜0.01),但对细胞周期无影响(P＞0.05);LASS2/TMSG1基因沉默组移植瘤的体积、质量及淋巴结转移率(5/6)和核增殖指数(0.53±0.05)明显大于空白对照组(0/6,0.13 ±0.02)和无关阴性对照组(1/6,0.10 ±0.03;P＜0.05);LASS2/TMSG1基因沉默组的V-ATPase酶活性(P＜0.01)及细胞外H+浓度明显升高(P＜0.01),而MMP-2及MMP-9的表达量和分泌量无明显变化(P＞0.05),但分泌的MMP-2和MMP-9活性增加(P＜0.05).结论 特异性shRNA沉默LASS2/TMSG1基因能促进人前列腺癌细胞增殖、侵袭和转移能力,其机制可能是沉默LASS2/TMSG1基因后激活液泡型ATPase,从而使细胞外H+浓度升高,进而激活MMP-2和MMP-9有关,提示LASS2/TMSG1基因是一种新的肿瘤转移抑制基因.
目的 探討LASS2/TMSG1基因沉默對人前列腺癌細胞增殖、侵襲和轉移能力的影響及其分子作用機製.方法 建立穩定錶達LASS2/TMSG1-shRNA的低轉移潛能人前列腺癌PC-3M-2B4細胞繫,然後採用四甲基偶氮唑藍(MTT)摻入實驗、軟瓊脂集落形成實驗、流式細胞術、Matrigel穿膜侵襲實驗和裸鼠體內成瘤實驗研究LASS2/TMSG1的細胞生物學功能;採用Western blot法、明膠酶譜法檢測基質金屬蛋白酶2和9(MMP-2和MMP-9)的錶達、分泌及活性;併採用V-ATPase活性檢測試劑盒檢測細胞內V-ATPase活性,以及採用BCECF H+敏感熒光探針檢測細胞外H+濃度以研究LASS2/TMSG1抑製前列腺癌轉移的分子作用機製.結果 轉染LASS2/TMSG1-shRNA後,PC-3M-2B4細胞的增殖能力、錨定不依賴生長能力及體外侵襲能力明顯提高(P＜0.05),而細胞凋亡率明顯下降(P＜0.01),但對細胞週期無影響(P＞0.05);LASS2/TMSG1基因沉默組移植瘤的體積、質量及淋巴結轉移率(5/6)和覈增殖指數(0.53±0.05)明顯大于空白對照組(0/6,0.13 ±0.02)和無關陰性對照組(1/6,0.10 ±0.03;P＜0.05);LASS2/TMSG1基因沉默組的V-ATPase酶活性(P＜0.01)及細胞外H+濃度明顯升高(P＜0.01),而MMP-2及MMP-9的錶達量和分泌量無明顯變化(P＞0.05),但分泌的MMP-2和MMP-9活性增加(P＜0.05).結論 特異性shRNA沉默LASS2/TMSG1基因能促進人前列腺癌細胞增殖、侵襲和轉移能力,其機製可能是沉默LASS2/TMSG1基因後激活液泡型ATPase,從而使細胞外H+濃度升高,進而激活MMP-2和MMP-9有關,提示LASS2/TMSG1基因是一種新的腫瘤轉移抑製基因.
목적 탐토LASS2/TMSG1기인침묵대인전렬선암세포증식、침습화전이능력적영향급기분자작용궤제.방법 건립은정표체LASS2/TMSG1-shRNA적저전이잠능인전렬선암PC-3M-2B4세포계,연후채용사갑기우담서람(MTT)참입실험、연경지집락형성실험、류식세포술、Matrigel천막침습실험화라서체내성류실험연구LASS2/TMSG1적세포생물학공능;채용Western blot법、명효매보법검측기질금속단백매2화9(MMP-2화MMP-9)적표체、분비급활성;병채용V-ATPase활성검측시제합검측세포내V-ATPase활성,이급채용BCECF H+민감형광탐침검측세포외H+농도이연구LASS2/TMSG1억제전렬선암전이적분자작용궤제.결과 전염LASS2/TMSG1-shRNA후,PC-3M-2B4세포적증식능력、묘정불의뢰생장능력급체외침습능력명현제고(P＜0.05),이세포조망솔명현하강(P＜0.01),단대세포주기무영향(P＞0.05);LASS2/TMSG1기인침묵조이식류적체적、질량급림파결전이솔(5/6)화핵증식지수(0.53±0.05)명현대우공백대조조(0/6,0.13 ±0.02)화무관음성대조조(1/6,0.10 ±0.03;P＜0.05);LASS2/TMSG1기인침묵조적V-ATPase매활성(P＜0.01)급세포외H+농도명현승고(P＜0.01),이MMP-2급MMP-9적표체량화분비량무명현변화(P＞0.05),단분비적MMP-2화MMP-9활성증가(P＜0.05).결론 특이성shRNA침묵LASS2/TMSG1기인능촉진인전렬선암세포증식、침습화전이능력,기궤제가능시침묵LASS2/TMSG1기인후격활액포형ATPase,종이사세포외H+농도승고,진이격활MMP-2화MMP-9유관,제시LASS2/TMSG1기인시일충신적종류전이억제기인.
Objective To explore the effects of LASS2/TMSG1 silencing on the growth,invasion and metastasis of prostate carcinoma cells and to investigate the related molecular mechanisms.Methods LASS2/TMSG1 expression of human prostate carcinoma cell line with low metastatic potentiality (PC-3M-2B4 cells) was knocked down using DNA vector-based small interfering RNA (shRNA),followed by evaluations of tumor cell invasion and metastasis.Results A stable PC-3M-2B4 cell line with expression of LASS2/TMSG1-shRNA was successfully established.MTT assay showed PC-3M-2B4 cells exhibited a strong proliferation after transfection of LASS2/TMSG1-shRNA.LASS2/TMSG1-shRNA transfected clones demonstrated an increased clonogenicity by soft agar colony formation assay and a significant increase of tumor cell invasion by matrigel invasion study.Flow cytometry showed that after LASS2/TMSG1 gene silencing,the apoptotic rate of PC-3M-2B4 cell significantly decreased (P ＜ 0.01) without significant cell cycle change (P ＞ 0.05).Eight weeks after implantation into subcutaneous tissues in BAL B/c (nu +) mice,the size and weight of sh-LASS2/TMSG1 xenografts were significantly larger than those of the control group (P ＜ 0.05).Nuclear proliferation index of the subcutaneous tumor was also higher in the LASS2/ TMSG1 shRNA group than those in the control group.Lymph node metastasis was observed in 5 of 6 mice of LASS2/TMSG1 shRNA group and only 1 of 6 of the control group.V-ATPase activity,activities of secreted MMP-2 and MMP-9 and extracellular hydrogen ion concentration were significantly increased in LASS2/ TMSG1-shRNA group compared with the control group (P ＜ 0.05).Conclusion Silencing of LASS2/ TMSG1 promotes the growth,invasion and metastasis of prostate cancer cells through up-regulation of V-ATPase activity,indicating that LASS2/TMSG1 is a tumor metastasis suppressor gene.
