组织型转谷氨酰胺酶对乳腺癌侵袭转移作用的研究

Effect of transglutaminase type 2 on invasion and metastasis of breast cancer

目的探讨组织型转谷氨酰胺酶(transglutaminase type 2,tissue transglutaminase,TG2或tTG)对人乳腺癌细胞的侵袭转移能力作用及可能的机制。方法构建TGM2的特异性慢病毒干扰载体(TGM2-RNAi-LV),稳定转染至乳腺癌MDA-MB-231细胞,实验设TGM2干扰组(RNAi)、空白组(Con)和阴性对照组(NC)。实时荧光定量PCR(RT-PCR)检测细胞TGM2mRNA表达。细胞侵袭迁移实验(Transwell)检测细胞侵袭迁徙能力改变。蛋白质印迹检测MDA-MB-231细胞TG2、基质金属蛋白酶-9(matrix metalloprotein 9,MMP-9)和波形蛋白(Vimentin)的表达。选择2012-05-10-2014-07-29广西医科大学第一附属医院(46例)和江西省肿瘤医院(35例)病理确诊的81例乳腺浸润性导管癌患者,采用免疫组化检测TG2表达水平,分析其与淋巴结转移关系。结果 RT-PCR检测结果显示,Con组TGM2mRNA表达量为1,RNAi组TGM2mRNA表达量为0.21±0.03,明显低于Con组的1,z=-2.087,P=0.036 9;明显低于NC组的0.94±0.05,z=-1.964,P=0.049 5。细胞侵袭实验显示,侵袭到滤膜下的细胞数RNAi组为42.33±4.04,低于Con组的93.50±7.42和NC组的87.25±5.74,z值均为-2.121,P值均为0.033 9。迁移到滤膜下的细胞数RNAi组为66.13±4.59,低于Con组116.75±7.82和NC组109.00±7.79(z值均为-2.309,P值均为0.020 9)。蛋白质印迹检测结果显示,RNAi组、NC组和Con组TG2蛋白相对表达量分别为0.34±0.06、0.87±0.10和0.95±0.03,F=66.55,P<0.001。RNAi组Vimentin相对表达量(0.45±0.05)低于NC组(0.89±0.07)和Con组(0.90±0.03)的相对表达量,F=80.56,P<0.001。RNAi组、NC组和Con组MMP-9蛋白相对表达量分别为0.09±0.02、0.18±0.03和0.19±0.04,F=12.89,P=0.007。TG2表达水平与年龄、肿瘤分化程度和月经状态无显著相关性,P值均>0.05;而与淋巴结转移(χ2=14.750,P<0.001)、ER表达水平(χ2=11.632,P=0.001)和TNM分期(χ2=13.506,P<0.001)相关。Spearman相关分析显示,TG2与Vimentin(r=0.337,P=0.002)和MMP-9(r=0.358,P=0.001)表达水平呈正相关。结论 TG2可能具有促进乳腺癌的侵袭转移作用,下调TG2表达水平可能是治疗肿瘤转移的新靶点。

OBJECTIVE To determine the effect of TG2（transglutaminase type 2）on breast cancer cell invasion and metastasis,and to explore its function in the mechanism of breast cancer.METHODS To knock down TGM2（TG2gene）,an antisense construct（TG2gene-specific lentiviral RNA interference vetor,TGM2-RNAi-LV）was designed,synthesized and stably transfected into MDA-MB-231 cell lines.The MDA-MB-231 cells were divided into three groups：the TGM2-RNAi group（RNAi group）,the negative control group（NC group）and the blank control group（Con group）.TGM2 mRNA expression was detected by Real-Time PCR assay.Transwell test was applied to detect levels of invasion and migration of the MDA-MB-231 cells.Western blotting was applied to detect expression levels of TG2,MMP-9and Vimentin.Cancer tissue samples were studied for TG2 expression from 81 patients.RESULTS RT-PCR assay showed that TGM2 mRNA expression level of RNAi group was 0.21±0.03,and was significantly reduced compared with Con group（1,z=-2.087,P=0.036 9）and NC group（0.94±0.05,z=-1.964,P=0.049 5）.The cell numbers of invasion in RNAi group（42.33±4.04）were decreased when compared with Con group（93.50±7.42）and NC group（87.25±5.74）（both z=-2.121,both P=0.0339）.The cell numbers of migration in RNAi group（66.13±4.59）were reduced when compared with Con group（116.75±7.82）and NC group（109.00±7.79;both z=-2.309,both P=0.020 9）.Analysis by Western blotting indicated that TG2 expressions of RNAi group,NC group and Con group were0.34±0.06,0.87±0.10 and 0.95±0.03（F=66.55,P〈0.001）.The expressions of Vimentin of RNAi group（0.45±0.05）were markedly reduced in contrast to those of NC group（0.89±0.07）and Con group（0.90±0.03,F=80.56,P〈0.001）.MMP-9expression levels of RNAi group,NC group and Con group were 0.09±0.02,0.18±0.03and0.19±0.04（F=12.89,P=0.007）.Between the TG2 expressions,we found no significant differences in the distribution according to age,histological grade or menopause.However,we did observe significant correlations of TG2 with lymph node metastasis（χ^2=14.75,P〈0.001）,ER（χ^2=11.632,P=0.001）and TNM stage（χ^2=13.506,P〈0.001）.Spearman correlation analysis showed that TG2 was correlated with Vimentin（r=0.337,P=0.002）and MMP-9（r=0.358,P=0.001）.CONCLUSION These observations imply that TG2 may promote invasion and metastasis of breast carcinoma and downregulation of TG2 may be a new therapeutic approach to prevent the development of the metastatic phenotype.