摘要
目的:探讨短发夹RNA(shRNA)干扰趋化因子受体4(CXCR4)表达对卵巢癌细胞株SKOV3侵袭能力及细胞凋亡的影响。方法:将带有绿色荧光蛋白的慢病毒载体转染卵巢癌细胞株SKOV3,设计特异shRNA将其连入慢病毒载体;测序及荧光显微镜下观察绿色荧光验证慢病毒载体构建并转染,利用RT-q PCR检测转染后细胞株中CXCR4的mRNA表达,Western blot检测转染后细胞株中CXCR4、促凋亡蛋白Bax、B细胞白血病-2基因(Bcl-2)、Bcl-xl及半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的蛋白表达;Annexin V/PE双染色流式细胞术检测SKOV3细胞凋亡率,Transwell小室检测转染后卵巢癌细胞株SKOV3的侵袭力。结果:测序结果及微下见绿色荧光蛋白表达,证实成功构建慢病毒载体并转染卵巢癌细胞株SKOV3,成功将特异sh CXCR4连入载体;sh CXCR4组mRNA及蛋白表达水平明显低于NC组(P<0.05);干扰细胞中CXCR4的表达能提高SKOV3细胞中Bax及Cleaved caspase-3蛋白的表达量,降低Bcl-xl与Bcl-2的蛋白表达,干扰后,SKOV3细胞凋亡率,sh CXCR4组细胞凋亡率较CON组和NC组明显增高(P<0.05);穿膜的细胞数明显减少,抑制率为58.94%,差异有统计学意义(P<0.01)。结论:慢病毒干扰载体p LVsh CXCR4-EGFP(2A)-Puro转染卵巢癌细胞株SKOV3,可以下调细胞株中CXCR4的表达,并能促进卵巢癌细胞的凋亡,并能有效地降低卵巢癌细胞的侵袭能力。
Objective： To evaluate the influence of CXCR4-targeted short hairpin RNA on invasion a- bility and apoptosis of ovarian cancer line SKOV3. Methods： CXCR4-shRNA lentivirus with Green fluorescent protein was transfected into ovarian cancer cell line SKOV3. RT-qPCR and western blot were employed to measure the expression of CXCR4 RNA and protein expression of CXCR4, Bax, Bcl- 2, Bcl-xl and Caspase-3. Apoptosis rate of SKOV3 cell was detected through Annexin V/PE double staining flow cytometry. TransweU chamber was employed to estimate the invasiveness for SKOV3. Re- sults： Transfected pLVshCXCR4-EGFP （2A）-Puro. 2. CXCR4 RNA expression level and protein in ShCXCR4 group was （0.40 ± 0.13 ） and （ 0.45 ±0.23 ） , respectively lower than that in NC group （1. 109 ±0.21, P 〈0.05）. Interference of CXCR4 expression in cells can increase Bax and Cleaved caspase-3 protein expression in SKOV3 cells, reduced the Bcl-xl and the Bcl-2 protein expression.Flow cytometry instrument was adopted to detect cell apoptosis rate, shCXCR4 group was significantly higher than that of NC group and CON group, difference was statistically significant（ P 〈 0.05 ） ; in in- vasion test , cell number of ShCXCRd group was obviously lower than that in NC group and CON group, inhibition rate of shCXCR4 group was 58.94%, difference was statistically significant（ P 〈 0. 01 ）. Conclusion： CXCR4 RNA interference mediated with pLVshCXCR4-EGFP （2A）-Puro could ef- fectively downregulate the expression of CXCR4 RNA, and promote the cancer cell apoptosis as well as decrease the invasion ability of cancer cells.
出处
《贵阳医学院学报》
CAS
2016年第6期653-659,共7页
Journal of Guiyang Medical College
基金
贵州省科技厅联合基金[黔科合LG(2011)032号]
