中华乳腺病杂志（电子版）
中華乳腺病雜誌（電子版）
중화유선병잡지（전자판）
Chinese Journal of Breast Disease (Electronic Version)
2015年
4期
252-256
,共5页
乳腺肿瘤%癌基因%基因,肿瘤抑制%细胞系,转化
乳腺腫瘤%癌基因%基因,腫瘤抑製%細胞繫,轉化
유선종류%암기인%기인,종류억제%세포계,전화
Breast neoplasms%Oncogenes%Genes,tumor suppressor%Cell line,transformed
目的：探讨过表达转移抑制基因KAI1对人乳腺癌MDA-MB-231细胞上皮间质转化的影响。方法利用慢病毒感染乳腺癌MDA-MB-231细胞,获得稳定过表达KAI1的细胞系(过表达KAI1组),并设置空白慢病毒感染的阴性对照细胞系(阴性对照组)及未处理的人乳腺癌MDA-MB-231细胞为空白对照组。用Western blot法检测KAI1蛋白过表达的情况；采用RT-PCR法检测过表达KAI1对MDA-MB-231细胞间质标志物(波形蛋白、N-钙黏蛋白和纤粘蛋白)的 mRNA 表达水平的影响；并用Western blot检测过表达KAI1对MDA-MB-231细胞间质标志物(波形蛋白、N-钙黏蛋白)的蛋白表达水平的影响；采用明胶酶谱检测过表达KAI1对MDA-MB-231细胞基质金属蛋白酶2( MMP2)和MMP9活性的影响。多样本均数若满足方差齐性采用单因素方差分析,其两两比较采用LSD法进行,若不满足方差齐性则采用Dunnett’s T3。结果慢病毒感染MDA-MB-231细胞后,各组间AKI1蛋白表达有差异有统计学意义(F=25.610,P=0.001),并且与阴性对照组(0.575±0.065)和空白对照组(0.458±0.0500)相比,过表达KAI1组(0.953±0.034)的KAI1蛋白表达水平明显提高( P均<0.050)。 RT-PCR检测表明,细胞间质标志物波形蛋白(F=10.268,P=0.012)、N-钙黏蛋白(F=32.159,P=0.001)和纤粘蛋白的mRNA(F=38.364,P=0.000)在各组间表达有差异。与阴性对照组(0.937±0.102,0.998±0.064,1.093±0.083)和空白对照组(1.000±0.000,1.000±0.000,1.000±0.000)相比,过表达KAI1组(0.636±0.027,0.576±0.038,0.435±0.051)的波形蛋白、N-钙黏蛋白和纤粘蛋白 mRNA 表达水平明显降低( P 均<0.050),且Western blot检测表明,各组间N-钙黏蛋白(F=9.172,P=0.015)和波形蛋白(F=14.441,P=0.005)表达有差异,与阴性对照组(1.068±0.032)和空白对照组(0.957±0.103)相比,过表达KAI1组(0.597±0.032)的波形蛋白表达水平明显降低(P均<0.050)；与阴性对照组(1.452±0.036)和空白对照组(1.403±0.073)相比,过表达KAI1组(1.100±0.073)的N-钙黏蛋白表达水平相似(P=0.080,0.067)。酶活性分析发现,各组间MMP2(F=18.928,P=0.003)和MMP9(F=21.310,P=0.002)表达差异有统计学意义,与阴性对照组(1.090±0.160,1.091±0.107)和空白对照组(1.000±0.000,1.000±0.000)相比,过表达KAI1组(0.326±0.047,0.460±0.071)的 MMP2和 MMP9表达水平明显降低(P 均<0.050)。结论过表达转移抑制基因KAI1能抑制人乳腺癌MDA-MB-231细胞的间质表型。
目的：探討過錶達轉移抑製基因KAI1對人乳腺癌MDA-MB-231細胞上皮間質轉化的影響。方法利用慢病毒感染乳腺癌MDA-MB-231細胞,穫得穩定過錶達KAI1的細胞繫(過錶達KAI1組),併設置空白慢病毒感染的陰性對照細胞繫(陰性對照組)及未處理的人乳腺癌MDA-MB-231細胞為空白對照組。用Western blot法檢測KAI1蛋白過錶達的情況；採用RT-PCR法檢測過錶達KAI1對MDA-MB-231細胞間質標誌物(波形蛋白、N-鈣黏蛋白和纖粘蛋白)的 mRNA 錶達水平的影響；併用Western blot檢測過錶達KAI1對MDA-MB-231細胞間質標誌物(波形蛋白、N-鈣黏蛋白)的蛋白錶達水平的影響；採用明膠酶譜檢測過錶達KAI1對MDA-MB-231細胞基質金屬蛋白酶2( MMP2)和MMP9活性的影響。多樣本均數若滿足方差齊性採用單因素方差分析,其兩兩比較採用LSD法進行,若不滿足方差齊性則採用Dunnett’s T3。結果慢病毒感染MDA-MB-231細胞後,各組間AKI1蛋白錶達有差異有統計學意義(F=25.610,P=0.001),併且與陰性對照組(0.575±0.065)和空白對照組(0.458±0.0500)相比,過錶達KAI1組(0.953±0.034)的KAI1蛋白錶達水平明顯提高( P均<0.050)。 RT-PCR檢測錶明,細胞間質標誌物波形蛋白(F=10.268,P=0.012)、N-鈣黏蛋白(F=32.159,P=0.001)和纖粘蛋白的mRNA(F=38.364,P=0.000)在各組間錶達有差異。與陰性對照組(0.937±0.102,0.998±0.064,1.093±0.083)和空白對照組(1.000±0.000,1.000±0.000,1.000±0.000)相比,過錶達KAI1組(0.636±0.027,0.576±0.038,0.435±0.051)的波形蛋白、N-鈣黏蛋白和纖粘蛋白 mRNA 錶達水平明顯降低( P 均<0.050),且Western blot檢測錶明,各組間N-鈣黏蛋白(F=9.172,P=0.015)和波形蛋白(F=14.441,P=0.005)錶達有差異,與陰性對照組(1.068±0.032)和空白對照組(0.957±0.103)相比,過錶達KAI1組(0.597±0.032)的波形蛋白錶達水平明顯降低(P均<0.050)；與陰性對照組(1.452±0.036)和空白對照組(1.403±0.073)相比,過錶達KAI1組(1.100±0.073)的N-鈣黏蛋白錶達水平相似(P=0.080,0.067)。酶活性分析髮現,各組間MMP2(F=18.928,P=0.003)和MMP9(F=21.310,P=0.002)錶達差異有統計學意義,與陰性對照組(1.090±0.160,1.091±0.107)和空白對照組(1.000±0.000,1.000±0.000)相比,過錶達KAI1組(0.326±0.047,0.460±0.071)的 MMP2和 MMP9錶達水平明顯降低(P 均<0.050)。結論過錶達轉移抑製基因KAI1能抑製人乳腺癌MDA-MB-231細胞的間質錶型。
목적：탐토과표체전이억제기인KAI1대인유선암MDA-MB-231세포상피간질전화적영향。방법이용만병독감염유선암MDA-MB-231세포,획득은정과표체KAI1적세포계(과표체KAI1조),병설치공백만병독감염적음성대조세포계(음성대조조)급미처리적인유선암MDA-MB-231세포위공백대조조。용Western blot법검측KAI1단백과표체적정황；채용RT-PCR법검측과표체KAI1대MDA-MB-231세포간질표지물(파형단백、N-개점단백화섬점단백)적 mRNA 표체수평적영향；병용Western blot검측과표체KAI1대MDA-MB-231세포간질표지물(파형단백、N-개점단백)적단백표체수평적영향；채용명효매보검측과표체KAI1대MDA-MB-231세포기질금속단백매2( MMP2)화MMP9활성적영향。다양본균수약만족방차제성채용단인소방차분석,기량량비교채용LSD법진행,약불만족방차제성칙채용Dunnett’s T3。결과만병독감염MDA-MB-231세포후,각조간AKI1단백표체유차이유통계학의의(F=25.610,P=0.001),병차여음성대조조(0.575±0.065)화공백대조조(0.458±0.0500)상비,과표체KAI1조(0.953±0.034)적KAI1단백표체수평명현제고( P균<0.050)。 RT-PCR검측표명,세포간질표지물파형단백(F=10.268,P=0.012)、N-개점단백(F=32.159,P=0.001)화섬점단백적mRNA(F=38.364,P=0.000)재각조간표체유차이。여음성대조조(0.937±0.102,0.998±0.064,1.093±0.083)화공백대조조(1.000±0.000,1.000±0.000,1.000±0.000)상비,과표체KAI1조(0.636±0.027,0.576±0.038,0.435±0.051)적파형단백、N-개점단백화섬점단백 mRNA 표체수평명현강저( P 균<0.050),차Western blot검측표명,각조간N-개점단백(F=9.172,P=0.015)화파형단백(F=14.441,P=0.005)표체유차이,여음성대조조(1.068±0.032)화공백대조조(0.957±0.103)상비,과표체KAI1조(0.597±0.032)적파형단백표체수평명현강저(P균<0.050)；여음성대조조(1.452±0.036)화공백대조조(1.403±0.073)상비,과표체KAI1조(1.100±0.073)적N-개점단백표체수평상사(P=0.080,0.067)。매활성분석발현,각조간MMP2(F=18.928,P=0.003)화MMP9(F=21.310,P=0.002)표체차이유통계학의의,여음성대조조(1.090±0.160,1.091±0.107)화공백대조조(1.000±0.000,1.000±0.000)상비,과표체KAI1조(0.326±0.047,0.460±0.071)적 MMP2화 MMP9표체수평명현강저(P 균<0.050)。결론과표체전이억제기인KAI1능억제인유선암MDA-MB-231세포적간질표형。
Objective To investigate the impact of overexpression of non-metastatic gene KAI1 on the epithelial-mesenchymal transition in human breast cancer cell line MDA-MB-231. Methods Lentivirus vector was transfected into MDA-MB-231 cells to obtain the KAI1-overexpressed cell line ( KAI1 overexpression group) . The cell line with blank lentivirus vector infection served as negative control group and untreated cell line as control group. Western blot analysis was used to verify KAI1 protein expression. RT-PCR was used to detect the mRNA expression levels of vimentin, N-cadherin and fibronectin in KAI1-overexpressed MDA-MB-231 cells. Western blot was used to detect the protein expression levels of vimentin and N-cadherin. The gelatin zymography was used to detect MMP2 and MMP9 activities. If the means of multi-sample met the homogeneity of variance, ANOVA was used, otherwise Dunnett’s T3 test was used. Pairwise comparison was performed using LSD method. Results After lentivirus infection of MDA-MB-231 cells, KAI1 protein expression showed a significant difference among groups (F=25. 610, P=0. 001). Compared with the negative control group (0. 575 ± 0. 065) and control group (0. 458 ± 0. 0500), KAI1 protein level (0. 953±0. 034)was significantly higher in KAI1 overexpression group ( all P values <0. 050 ) . RT-PCR showed that there were significant differences in the mRNA levels of stromal cells markers vimentin (F=10. 268, P=0. 012), N-cadherin (F=32. 159, P=0. 001) and fibronectin (F=38. 364, P=0. 000) among groups. The mRNA levels of vimentin, N-cadherin and fibronectin in KAI1 overexpression group ( 0. 636 ± 0. 027, 0. 576 ± 0. 038, 0. 435 ± 0. 051 ) were significantly lower than those in negative control group (0. 937 ± 0. 102,0. 998 ± 0. 064,1. 093 ± 0. 083) and control group (1. 000 ± 0. 000,1. 000 ± 0. 000,1. 000 ± 0. 000) respectively (all P values<0. 050). Western blot analysis showed a significant difference in protein levels of N-cadherin ( F=9. 172, P=0. 015 ) and vimentin (F=14. 441, P=0. 005) among groups. Compared with the negative control group (1. 068 ± 0. 032) and control group ( 0. 957 ± 0. 103 ) , vimentin protein expression in KAI1 overexpression group ( 0. 597 ± 0. 032) was significantly lower (all P values <0. 050), while N-cadherin expression showed no significant difference (1. 452 ± 0. 036 vs 1. 403 ± 0. 073 vs 1. 100 ± 0. 073, P=0. 080, 0. 067). The gelatin zymography showed a significant difference in the expressions of MMP2 (F=18. 928, P=0. 003) and MMP9 (F=21. 310, P=0. 002) among the groups. Compared with the negative control group (1. 090 ± 0. 160, 1. 091 ± 0. 107) and control group ( 1. 000 ± 0. 000, 1. 000 ± 0. 000 ), MMP2 and MMP9 expression levels in KAI1 overexpression group (0. 326 ± 0. 047,0. 460 ± 0. 071) were significantly lower (all P values <0. 050). Conclusion The overexpression of KAI1 can inhibit the mesenchymal phenotype in human breast cancer cell line MDA-MB-231.
