人前列腺癌细胞 PFKFB4 基因RNA干扰慢病毒构建与鉴定  

RNA interference of human PFKFB4 gene in human prostate cancer cells

作　　者：郝南[1] 农德勇 李钻 林俊浩 黄桂海 李锡明 李伟[1] HAO Nan;NONG Deyong;LI Zuan;LIN Junhao;HUANG Guihai;LI Ximing;LI Wei(Department of Surgical Urology 2,The People's Hospital of Guangxi Zhuang Autonomous Region,Nanning 530021,China)

机构地区：[1]广西壮族自治区人民医院泌尿外二科,广西南宁530021

出　　处：《西部医学》2022年第8期1115-1120,共6页Medical Journal of West China

基　　金：国家自然科学基金项目(81460387)。

摘　　要：目的构建人前列腺癌细胞PFKFB4基因短发夹RNA(shRNA)干扰慢病毒载体,建立稳定干扰的人前列腺癌细胞株。方法通过GenBank检索人PFKFB4基因序列,根据shRNA引物设计原则,设计并合成PFKFB4-shRNA序列,并与携带绿色荧光蛋白序列的LV3载体连接,获得的重组质粒与慢病毒包装质粒系统共转染293T细胞,收集病毒。使用逐孔稀释滴度测定法对慢病毒滴度进行测定。将LV3-PFKFB4-shRNA慢病毒载体感染人前列腺癌细胞设置为空载的LV3 Negative Control组(LV3-NC组)、空白对照组(Blank组)和LV3-shPFKFB4组。采用RT-PCR检测人前列腺癌细胞PFKFB4 mRNA水平。结果重组质粒测序结果显示LV3-PFKFB4-shRNA慢病毒载体构建成功,通过荧光显微镜计数荧光细胞,结合稀释倍数计算病毒滴度为1×108TU/mL。RT-PCR结果显示,与LV3-NC组和Blank组相比,LV3-shPFKFB4组PFKFB4 mRNA水平显著降低(P<0.001),而LV3-NC组与Blank组PFKFB4-mRNA水平比较差异无统计学意义(P=0.271)。结论LV3-PFKFB4-shRNA慢病毒载体构成功,同时获得稳定感染的人前列腺癌细胞株。Objective To construct a lentiviral vector for RNA interference of the human PFKFB4 gene and establish a PFKFB4 gene silencing on human prostate cancer cells.Methods According to the human PFKFB4 sequence archived in the GenBank database,a shRNA targeting PFKFB4 was designed,and cloned into a linear vector containing the green fluorescent protein(GFP)gene to produce a recombinant lentivirus plasmid.The recombinant plasmids and helper plasmids were transfected into 293T cells,and the titer of the virus was determined.PC-3 cells were infected with the constructed lentivirus,and the silencing effect on PFKFB4 was accessed by real-time PCR(RT-PCR).Results The virus titer was calculated to be 1×108 TU/mL,RT-PCR results showed that the PFKFB4 mRNA expression level of the LV3-PFKFB4-shRNA group was significantly lower than the negative control group and the blank group.Conclusion A lentivirus vector for RNA interference of human PFKFB4 was successfully constructed and PC-3 cell lines with PFKFB4 gene knockdown were manufactured,which will be useful for future research of PFKFB4 function in prostate cancer cell models.

关 键 词：PFKFB4基因 RNA干扰 短发夹RNA 慢病毒载体 前列腺癌 

分 类 号：R33[医药卫生—人体生理学] R69[医药卫生—基础医学] R737.25