摘要
目的探讨KH型剪接调控蛋白(KSRP)对肝细胞癌(HCC)HepG2细胞增殖、周期以及凋亡的影响。方法使用GEO数据库探讨HCC患者的肿瘤组织及癌旁组织中KSRP的表达水平。实验分为3组:对照组、过表达组KSRP及敲低组shKSRP,使用慢病毒敲低或过表达KSRP,通过嘌呤霉素筛选敲低或过表达KSRP的稳定表达HepG2细胞系,Western blot检测HepG2中KSRP蛋白的表达水平;CCK-8法检测24h、48h、72h和96h时各组HepG2细胞增值率;采用流式细胞术和RNA-seq分别检测shKSRP以及对照组的HepG2细胞周期和抗原KI-67(MKI67)的表达;Annexin V-FITC/PI双染法检测shKSRP以及对照组的HepG2细胞凋亡情况。结果HCC患者癌组织中KSRP表达较癌旁组织升高(P<0.001);CCK-8法结果表明,敲低KSRP的HepG2细胞增殖明显减慢(P<0.01)。RNA-seq显示,KSRP敲低后可抑制MKI67基因的表达(P<0.05)。流式细胞术检测,KSRP敲低后,将HepG2细胞阻滞于S期(细胞周期),并可促使其凋亡,其凋亡率差异有统计学意义(P<0.05)。结论敲低KSRP可将HepG2细胞阻滞于S期,抑制细胞增殖,促进凋亡,这可能和下调KSRP与增殖相关基因MKI67的表达密切相关。
Objective To investigate the effects of KH type splicing regulatory protein(KSRP) on the proliferation, cell cycle and apoptosis of hepatocellular carcinoma(HCC) HepG2 cells. Methods The GEO database was used to investigate the expression level of KSRP in tumor tissues and adjacent tissues of patients with HCC.The experiments were divided into three groups: the control group, the KSRP overexpression group, and the shKSRP knockdown group.The lentivirus was used to knockdown or overexpress KSRP,the stably expressed HepG2 cell line with with KSRP knocked down or overexpressed was screened by puromycin, and the expression level of KSRP protein in HepG2 was detected by Western blot.The proliferation rate of HepG2 cells in each group at 24 h, 48 h, 72 h and 96 h was detected by CCK-8 assay.The cell cycle of HepG2 and the expression of the antigen KI-67(MKI67) in shKSRP and control groups were determined by flow cytometry and RNA-seq, respectively.Annexin V-FITC/PI double staining was used to detect the apoptosis of HepG2 cells in shKSRP and the control groups.Results The expression of KSRP in cancerous tissues is higher than in paracancerous tissues in patients with HCC(P<0.001).The results of CCK-8 assay showed that the proliferation of HepG2 cells with KSRP knocked down was significantly decreased(P<0.01).RNA-seq showed that the knockdown of KSRP could inhibit the expression of MKI67 gene(P<0.05).As detected by Flow cytometry, knockdown of KSRP could arrest HepG2 cells in S phase(cell cycle) and induce apoptosis, and the difference in the apoptosis rate was statistically significant(P<0.05).Conclusion Knockdown of KSRP can arrest HepG2 cells in S phase, inhibit cell proliferation and promote apoptosis, which may be closely related to the down-regulation of expression of KSRP and proliferation-related gene MKI67.
作者
邱靓琳
瞿利花
单士刚
李明轩
周蕊
QIU Jing-lin;QU Li-hua;LI Ming-xuan(School of Pharmacy,Xianning Medical College,Hubei University of Science and Technology,Xianning Hubei 437100,China)
出处
《湖北科技学院学报（医学版）》
2022年第6期486-490,497,共6页
Journal of Hubei University of Science and Technology(Medical Sciences)
基金
湖北科技学院校内科研发展基金项目(2020-22GP06)。
关键词
KH型剪接调控蛋白
肝细胞癌
细胞周期
细胞增殖
细胞凋亡
KH type splice regulatory protein
Hepatocellular carcinoma
Cell cycle
Cell proliferation
Apoptosis
