摘要
目的探讨LASS2／TMSGl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／TMSGl-shRNA的低转移潜能人前列腺癌PC-3M-2134细胞系，然后采用四甲基偶氮唑蓝（MTT）掺人实验、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／TMSGl的细胞生物学功能；采用Westernblot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BCECFH＋敏感荧光探针检测细胞外H＋浓度以研究LASS2／TMSGl抑制前列腺癌转移的分子作用机制。结果转染LASS2／TMSGl-shRNA后，PC-3M-284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／TMSGl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／TMSGl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／TMSGl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。
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/TMSGI-shRNA was successfully established. MTT assay showed PC-3M-2B4 cells exhibited a strong proliferation after transfection of LASS2/TMSGI-shRNA. LASS2/TMSGI-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-2P,4 cell significantly decreased （ P 〈 0. 01 ） without significant cell cycle change （ P 〉 O. 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/ TMSGI-shRNA group compared with the control group （ P 〈 0. 05 ）. Conclusion Silencing of LASS2/ TMSGI 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 ene.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2014年第3期177-183,共7页
Chinese Journal of Pathology
基金
国家自然科学基金面上项目（30971142）
关键词
前列腺肿瘤
基因沉默
基质
肿瘤抑制
Prostatic neoplasms
Gene silencing
Genes,tumor suppressor
