摘要
目的构建YTH N6-甲基腺苷RNA结合蛋白3(YTH N6-methyladenosine RNA binding protein F3,YTHDF3)稳定沉默的MCF-7和MDA-MB-231乳腺癌细胞株。方法数据库分析YTHDF3在乳腺癌中的表达及其与预后的相关性,选定蛋白研究靶点;利用人肾上皮细胞293T细胞产生含有YTHDF3-短发夹RNA(short hairpin RNA,shRNA)慢病毒颗粒,转染MCF-7和MDA-MB-231乳腺癌细胞系,并利用嘌呤霉素筛选不含嘌呤抗性的阴性细胞,构建YTHDF3稳定沉默的MCF-7和MDA-MB-231乳腺癌细胞株;将MCF-7和MDA-MB-231细胞分为阴性对照组(shNC组,转染无意义序列)、shY3-1组(转染沉默YTHDF3的第一条shRNA)及shY3-2组(转染沉默YTHDF3的第二条shRNA);实时荧光定量PCR技术(quantitative real-time polymerase chain reaction,RT-PCR)检测YTHDF3 mRNA表达,Western blot检测YTHDF3蛋白沉默效率。结果YTHDF3在乳腺癌不同内在分子亚型中高表达,且其高表达与不良预后显著相关;经嘌呤霉素筛选后,成功获得sh-NC组、shY3-1组和shY3-2组稳定转染的MCF-7和MDA-MB-231细胞系;与sh-NC组相比,shY3-1组和shY3-2组细胞的YTHDF3蛋白表达显著降低(P<0.05)。结论初步构建了YTHDF3稳定沉默的MCF-7和MDA-MB-231乳腺癌细胞株。
Objective To construct the MCF-7 and MDA-MB-231 cell lines with stable silencing for YTH N6-methyladenosine RNA binding protein 3(YTHDF3).Methods Database analysis of YTHDF3 expression in breast cancer was conducted and its correlation with prognosis was analyzed to select research targets.The MCF-7 and MDA-MB-231 cell lines were divided into a negative control group(shNC group,transfected with a non-targeting sequence),the shY3-1 group(transfected with the first shRNA targeting YTHDF3),and the shY3-2 group(transfected with the second shRNA targeting YTHDF3).Human embryonic kidney 293T cells were used to produce lentiviral particles containing YTHDF3 short hairpin RNA(shRNA),which were then transfected into MCF-7 and MDA-MB-231 cell lines.Puromycin was employed to kill cells lacking puromycin resistance,successfully constructing YTHDF3 stable silenced MCF-7 and MDA-MB-231 cell lines.Quantitative real-time polymerase chain reaction(RT-PCR)was used to detect YTHDF3 mRNA expression,and Western blot test was performed to evaluate the silencing efficiency of YTHDF3 protein.Results YTHDF3 was highly expressed in different intrinsic molecular subtypes of breast cancer,and this high expression was significantly correlated with the poor prognosis.YTHDF3 was thus established as the research target.After puromycin selection,stable transfection of MCF-7 and MDA-MB-231 cell lines was successfully achieved in the sh-NC,shY3-1,and shY3-2 groups.Compared to the sh-NC group,the expression of YTHDF3 protein was significantly decreased in the shY3-1 and shY3-2 groups.Conclusion Stable silenced MCF-7 and MDA-MB-231 cell lines for YTHDF3 were successfully constructed in breast cancer.
作者
龚荣府
许修颖
杨娉娉
张正花
方文
GONG Rongfu;XU Xiuying;YANG Pingping;ZHANG Zhenghua;FANG Wen(Department of Clinical Biochemistry,Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
2025年第8期1162-1168,共7页
Journal of Guizhou Medical University
基金
国家自然科学基金项目(81560481)
贵州省科技计划项目(黔科合基础ZK2022一般419)。
