CAJ | 학술논문

目的探讨紧密连接蛋白claudin-6过表达对乳腺癌细胞MCF-7生物学行为的影响.方法用脂质体法将含有claudin-6的真核表达载体转染乳腺癌细胞系MCF-7后,采用RT-PCR、Western印迹和细胞免疫荧光的方法进行稳定克隆的筛选和鉴定;采用MTT法和克隆形成实验检测细胞生长情况;采用划痕法和Boyden小室体外侵袭实验检测细胞迁移和侵袭性.结果建立了稳定表达claudin-6基因的4株单克隆(C1,C2,C3,C4);细胞免疫荧光结果显示,claudin-6的表达主要定位于细胞膜;与对照组和空载体组相比,稳定表达claudin-6的MCF-7细胞生长缓慢,转染6 h时吸光度A值CI为1.62,C2为1.25,C3为1.30,C4为1.58与对照组A值1.78及空载组1.82比较明显降低.其克隆形成率CI为0.25,C2为0.17,C3为0.26,C4为0.21明显低于对照组(0.53)和空载组(0.45);细胞的迁移性、侵袭性被抑制.结论 claudin-6的过表达能够抑制MCF-7细胞的生长和转移等恶性表型,说明紧密连接蛋白claudin-6作为乳腺癌表型抑制基因在乳腺癌发生及转移中起负性调控作用.
목적 탐토긴밀련접단백claudin-6과표체대유선암세포MCF-7생물학행위적영향.방법 용지질체법장함유claudin-6적진핵표체재체전염유선암세포계MCF-7후,채용RT-PCR、Western인적화세포면역형광적방법진행은정극륭적사선화감정;채용MTT법화극륭형성실험검측세포생장정황;채용화흔법화Boyden소실체외침습실험검측세포천이화침습성.결과 건립료은정표체claudin-6기인적4주단극륭(C1,C2,C3,C4);세포면역형광결과현시,claudin-6적표체주요정위우세포막;여대조조화공재체조상비,은정표체claudin-6적MCF-7세포생장완만,전염6 h시흡광도A치CI위1.62,C2위1.25,C3위1.30,C4위1.58여대조조A치1.78급공재조1.82비교명현강저.기극륭형성솔CI위0.25,C2위0.17,C3위0.26,C4위0.21명현저우대조조(0.53)화공재조(0.45);세포적천이성、침습성피억제.결론 claudin-6적과표체능구억제MCF-7세포적생장화전이등악성표형,설명긴밀련접단백claudin-6작위유선암표형억제기인재유선암발생급전이중기부성조공작용.
Objective To explore the effect of over-expression of tight junction protein claudin-6 upon the biological characters of breast cancer cell line MCF-7. Methods The MCF-7 subline expressing a high level of claudin-6 was established by transfection with a pcDNA3. l-claudin-6 expression vector. The expression of claudin-6 in mRNA and its protein level were confirmed by RT-PCR, Western blot and immunofluorescent assays. Then the effect of claudin-6 upon cell proliferation was examined by MTT assay. Colony-forming assays were used to examine 2-D and 3-D colony-forming capacities. Invasive and migratory traits of claudin-6 expressing cells were determined by Boyden chamber invasion assay and monolayer wound-healing assay. The structure and function of tight junctions in both parental and claudin-6 expression MCF-7 cells were evaluated by measuring transepithelial electrical resistance. Results Immunofluorescent assays showed that transfected cells expressed claudin-6 on their membrane. Cells with a high level expression of claudin-6 grew slowly than control cells. Anchorage-independent growth, invasive and migratory traits also decreased substantially in cells with claudin-6 expression; whereas the transepithelial electrical resistance increased in the claudin-6 transfected cells. Conclusion The up-regulation of claudin-6 expression in MCF-7 breast cancer cells suppresses their malignant phenotypes with a correlation with the restoration of tight junction integrity. Claudin-6 may function as a cancer suppressor whose down-regulation contributes to the malignant progression of certain types of breast cancers.