摘要
该文目的为探讨蛋白质精氨酸甲基转移酶5(protein arginine methyltranserase 5,PRMT5)对肝癌细胞增殖、凋亡的影响。设计PRMT5短发夹核糖核酸(sh RNA)序列,装入p LKO.1-TRC vector质粒,进行慢病毒包装,感染肝癌细胞SMMC-7721和BEL-7402,嘌呤霉素筛选并培养稳定低表达PRMT5细胞株。Western blot、q PCR检测sh RNA干扰效率;CCK-8检测细胞增殖活性;平板克隆法检测细胞克隆形成能力;流式细胞术检测细胞凋亡;Western blot检测细胞增殖、凋亡相关蛋白的影响。结果显示,成功构建慢病毒重组质粒sh-PRMT5并感染肝癌细胞。Western blot、q PCR结果表明,筛选出的稳转细胞株能低表达PRMT5;在肝癌细胞中下调PRMT5的表达,可抑制细胞增殖(P<0.05),降低cyclin B1、cyclin D1、cdc20和cdc25B蛋白质水平(P<0.05),降低细胞克隆形成能力(P<0.05),促进细胞凋亡(P<0.05),同时能诱导Caspase-3表达,降低Bcl-2/Bax的比值(P<0.05)。由此说明,下调PRMT5的表达能抑制肝癌细胞增殖,诱导细胞凋亡,为进一步研究体内PRMT5的作用提供基础。
The paper aimed at investigating the effects of PRMT5 on cell proliferation and apoptosis in hepatocellular carcinoma cells. PRMT5 short hairpin RNA (shRNA) sequence was designed and inserted into pLKO. 1-TRC vector, then packaging lentivirus to infect SMMC-7721 and BEL-7402 cells. Stable infected cells were obtained by puromycin screening, shRNA silencing efficiency was tested by Western blot and qPCR. Cell pro- liferation activity was detected by CCK-8. Colony formation ability was detected by plate colony assay. Apoptosis was observed by flow cytometry. The expressions of cell proliferation- and apoptosis-related protein were detected by Western blot. The specific shRNA against PRMT5 plasmid (sh-PRMTS) was successfully constructed and infected into SMMC-7721 and BEL-7402 cells. The results of Western blot and qPCR demonstrated that the expression of PRMT5 was efficientlv inhibited at both protein and mRNA levels (P〈0.05) It revealed that PRMT5 depletiondecreased cell proliferation (P〈0.05). It was found that knockdown of PRMT5 reduced the protein levels of cy- clin B1, cyclin D1, cdc20 and cdc25B, respectively (P〈0.05). It showed that knockdown of PRMT5 inhibited cell colony formation ability. It was found that knockdown of PRMT5 induced the apoptosis (P〈0.05). It indicated that knockdown of PRMT5 activated Caspase-3 and reduced the ratio of Bcl-2 to Bax in protein levels (P〈0.05). Taken together, downregulation of PRMT5 inhibited cell proliferation and induced the apoptosis ofhepatocellar carcinoma cells. It may provide a new therapeutic target for the clinical treatment of hepatocellular carcinoma.
出处
《中国细胞生物学学报》
CAS
CSCD
2015年第12期1639-1645,共7页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81472333)
江苏省自然科学基金(批准号:BK2013247)资助的课题
重点实验室开放课题资助(批准号:90-13-05)
江苏大学高级人才启动基金(批准号:1281270058)~~
关键词
蛋白质精氨酸甲基转移酶5(PRMT5)
肝癌
增殖
凋亡
protein arginine methyltranserase 5 (PRMT5)
hepatocellular carcinoma
proliferation
apoptosis