CAJ | 학술논문

目的探讨CCL5-siRNA对高转移性人乳腺癌细胞生物学行为的影响.方法合成特异性CCL5-siRNA并克隆入慢病毒表达载体pGCSIL-GFP,将重组CCL5-siRNA慢病毒感染高转移性人乳腺癌细胞系MDA-MB-231设为KD组,另设阴性对照组和未感染组;分别采用实时定量聚合酶链反应和Western blot方法检测CCL5-siRNA对CCL5表达的作用;用噻唑蓝比色法、流式细胞术、克隆形成试验和Boyden小窒穿透试验观察细胞的增殖、周期、体外聚集和侵袭力的改变.结果 CCL5-siRNA慢病毒可显著降低MDA-MB-231细胞CCL5的表达,改变细胞形成克隆和运动侵袭的能力,但对细胞增殖的影响不明显.与阴性对照组(1.00±0.07)和未感染组(0.88±0.15)比较,KD组细胞CCL5 mRNA的表达下降为(0.18±0.03),P<0.01.在上样量相同的条件下,与阴性对照组(1.82±0.18)比较,KD组CCL5蛋白表达降低为(0.33±0.13),P<0.01.细胞克隆计数:与阴性对照组(0.97±0.09)和未感染组(1.04±0.07)比较,KD组细胞的克隆数降为(0.33±0.10),P<0.05;穿膜细胞计数:KD组(38±15)明显少于阴性对照组(77±11)和末感染组(69±9),P<0.05.嚷唑蓝比色实验和流式细胞分析提示:KD组细胞的增殖情况与阴性对照组、未感染组相比无明显差别.三组PI值分别为0.48±0.02、0.44±0.05及0.47±0.02(P>0.05).结论慢病毒介导的CCL5-siRNA可显著抑制高转移性人乳腺癌细胞的克隆形成和运动侵袭能力,但对其增殖活性无明显影响.
목적 탐토CCL5-siRNA대고전이성인유선암세포생물학행위적영향.방법 합성특이성CCL5-siRNA병극륭입만병독표체재체pGCSIL-GFP,장중조CCL5-siRNA만병독감염고전이성인유선암세포계MDA-MB-231설위KD조,령설음성대조조화미감염조;분별채용실시정량취합매련반응화Western blot방법검측CCL5-siRNA대CCL5표체적작용;용새서람비색법、류식세포술、극륭형성시험화Boyden소질천투시험관찰세포적증식、주기、체외취집화침습력적개변.결과 CCL5-siRNA만병독가현저강저MDA-MB-231세포CCL5적표체,개변세포형성극륭화운동침습적능력,단대세포증식적영향불명현.여음성대조조(1.00±0.07)화미감염조(0.88±0.15)비교,KD조세포CCL5 mRNA적표체하강위(0.18±0.03),P<0.01.재상양량상동적조건하,여음성대조조(1.82±0.18)비교,KD조CCL5단백표체강저위(0.33±0.13),P<0.01.세포극륭계수:여음성대조조(0.97±0.09)화미감염조(1.04±0.07)비교,KD조세포적극륭수강위(0.33±0.10),P<0.05;천막세포계수:KD조(38±15)명현소우음성대조조(77±11)화말감염조(69±9),P<0.05.양서람비색실험화류식세포분석제시:KD조세포적증식정황여음성대조조、미감염조상비무명현차별.삼조PI치분별위0.48±0.02、0.44±0.05급0.47±0.02(P>0.05).결론 만병독개도적CCL5-siRNA가현저억제고전이성인유선암세포적극륭형성화운동침습능력,단대기증식활성무명현영향.
Objective To study the effect of lentivirus-mediated CCL5-RNAi on the biological behaviors of human breast cancer cells. Methods CCL5-specific siRNA gene was synthesized and cloned into the recombinant lentiviral vector, pGCSIL-GFP. Human high-metastatic breast cancer cells, MDA-MB-231, were infected by CCL5-siRNA recombinant lentivirus, which was set as KD group. Cells infected with CCL5-NC was as NC group, and cells cultured was as CON group. The expression of CCL5 mRNA and protein in MDA-MB-231 cells was detected by RT-PCR and western blot, respectively. Cell growth suppression and cell cycle was observed by MTT assay and fluorescence activated cell sorting (FACS). Colony formation and migration ability were determined by colony-rorming assay and Boyden chamber method. Results After infection of CCL5-siRNA recombinant lentivirus, the expression level of CCL5 mRNA and protein in MDA-MB-231 cells as well as the colony formation and migration ability decreased significantly, but cell's proliferation was not affected obviously. Compared with MDA-MB-231 (0.88± 0.15) and MDA-MB-231/CCL5-NC (1.00±0.07) cells, the expression of CCL5 mRNA in MDA-MB-231/ CCL5-siRNA decreased to 0.18±0.03, P<0.01. Compared with MDA-MB-231/CCL5-NC (1.82±0.18) cells, the expression of CCL5 protein in MDA-MB-231/CCL5-siRNA decreased to 0.33±0.13, P <0.01. Colony-forming assay and Boyden chamber method showed that the colony formation and migration ability of MDA-MB-231/CCL5-siRNA decreased markedly (P<0.05). The clone count in KD group was (0.33± 0.10), which was a significant decrease from (0.97±0.09) (NC group) and (1.04±0.07) (CON group), P<0.05. The number of cells that migrated through the chamber membrane of KD group (38± 15) was less than that of NC group (77±11, P <0.05) and CON group (69±9, P <0.05). However, MTT assay and FACS revealed that the proliferation of MDA-MB-231/CCL5-siRNA was not different from MDA-MB-231/CCL5-NC and MDA-MB-231 (P>0.05), the proliferation index (PI) of group KD, NC and CON were (0.48±0.02), (0.44±0.05) and (0.47±0.02) respectively. The difference was not statistically significant by multiple comparison (P>0.05). Conclusion CCL5-specific siRNA can specifically suppress the colony formation and migration of human high-matastatic breast cancer cells.