NDRG1基因与乳腺癌转移的关系及其转染对乳腺癌细胞株增殖及侵袭力的影响

Overexpression of NDRG1:relationship with proliferative activity and invasiveness of breast cancer cell line and breast cancer metastasis

目的探讨NDRG1基因与乳腺癌转移的关系及其转染对乳腺癌细胞株增殖及侵袭力的影响。方法免疫组织化学SP法、即时荧光定量逆转录聚合酶链反应(real time RT-PCR)检测10例新鲜乳腺浸润性导管癌和27例石蜡包埋乳腺癌组织中NDRG1蛋白和mRNA的表达。脂质体介导NDRG1基因瞬时转染高侵袭高转移性人乳腺癌细胞株MDA-MB-231;用5-溴-2-脱氧尿苷(BrdU)掺入法、流式细胞术观察NDRG1基因转染后对细胞增殖和细胞周期的影响;细胞体外侵袭实验和迁移实验观察转染后细胞侵袭和迁移能力的变化。结果NDRG1蛋白和mRNA表达分别为:无转移的乳腺癌组织阳性率为100%(14/14)和0·82±0·14,而有转移的乳腺癌组织中为69·2%(9/13)和0·17±0·11,均明显降低;对两株细胞的检测中,高侵袭高转移的MDA-MB-231细胞株中NDRG1mRNA的表达水平(0·14±0·02)明显低于低侵袭低转移的MCF7细胞株(1·51±0·11)。NDRG1基因瞬时转染MDA-MB-231细胞后,与未转染组和pEGFP-N3组相比,pEGFP-NDRG1-N3转染组中,细胞BrdU掺入率降低,细胞周期G0/G1期比例增高,S期比例明显减低;同时细胞体外侵袭能力下降,而体外迁移能力则差异无统计学意义。结论NDRG1基因的表达与乳腺癌的转移呈负相关关系,提示该基因可望成为早期预测乳腺癌转移的分子生物学标记物之一;NDRG1基因通过脂质体转染高侵袭高转移性人乳腺癌MDA-MB-231细胞株,可抑制肿瘤细胞增殖、降低其侵袭能力,提示其可作为一个候选的肿瘤转移抑制基因,并有望成为乳腺癌基因治疗的新的候选基因。

Objective To investigate the relationship between NDRG1 and metastasis of breast cancer and the effects of NDRG1 overexpression on the proliferation and invasion of breast cancer cells. Methods NDRG1 was detected at its protein level by immunohistochemistry （IHC） and its messenger RNA （mRNA） was detected by real-time reverse transcriptase-polymeraae chain reaction （ real time RT-PCR） in clinical breast cancer specimens. Lipoaome was used to transiently transfer NDRG1 into MDA-MB-231, a highly invasive human breast cancer cell line. The proliferation of MDA-MB-231 was measured by Bromodeoxy Uridine （BrdU） incorporation assay and the transfection effect on cell cycle distribution was determined by fluorescence assisted cell sorting （FACS）. The invasive ability of the transfected cells was investigated by reconstituted matrigel invasion and polycarbonate filters migration experiments. Results NDRG1 expressions at protein and mRNA levels in tumors of patients with lymph node metastases were significantly lower as compared with those with localized breast cancers （ P〈0.01 ）. The amount of NDRG1 mRNA in MCF7, a relatively non-invasive breast cancer cell line, was 10.8 times higher than that in MDA- MB-231 cells （ P〈0.01 ）. The BrdU incorporation rate declined significantly （ P〈0.05 ） in NDRG1 overexpressing MDA-MB-231 cells. An increase of the cell population at G0/G1 phase was observed 48 hours post-transfection along with a decrease of cell population at S phase. Overexpression of NDRG1 significantly retarded the invasiveness of MDA-MB-231 cells in matrigel-coated invasion chambers （P〈0.05）, when compared to cells transfected with control vectors. However, the migration abilities of cells with or without the transfection were virtually identical. Conclusions NDRG1 expression reversely correlates with breast cancer metastasis and progression, and may serve as a prognostic biomarker for predicting early metastasis.The inhibition of proliferation and invasion demonstrated by our MDA-MB-231 transfection experiments implies that NDRG1 is a tumor metastasis suppressor gene and may be a new candidate for gene therapy against human breast cancer.