Knockdown of UHRF1 by lentivirus-mediated shRNA inhibits ovarian cancer cell growth.
Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has been reported to be over-expressed in many cancers, but its role in ovarian cancer remains elusive. Here, we determined whether knockdown of UHRF1 by lentivirus-mediated shRNA could inhibit ovarian cancer cell growth. Lentivirus- mediated short hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference (RNAi) pathway. The efficiency of lentivirus-mediated shRNA infection into HO-8910 and HO-8910 PM cells was determined using fluorescence microscopy to observe lentivirus-mediated GFP expressionand was confirmed to be over 80 percent. UHRF1 expression in infected HO-8910 and HO-8910 PM was evaluated by real-time PCR and Western blot analysis. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability; flow cytometry and Hoechst 33342 assay was applied to measure cell cycle arrest and apoptosis. Cell invasion was assessed using transwell chambers. Our results demonstrated that the loss of UHRF1 promoted HO-8910 and HO-8910 PM cell apoptosis, while inhibiting cell proliferation. In addition, UHRF1 knockdown significantly inhibited the invasion of human ovarian cancer cells. In the present study, we also showed that depleting HO-8910 cells of UHRF1 caused activation of the DNA damage response pathway, with the cell cycle arrested in G2/M-phase. The DNA damage response in cells depleted of UHRF1 was illustrated by phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser 216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr 15.
College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, China E-mail : xyhu@yzu.edu.cn, yanfeng1895@163.com.
Feng
F
Yan
Li-Jia
LJ
Shao
Xiao-Ya
XY
Hu
D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000235:genetics*; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle; D002465:Cell Movement; D049109:Cell Proliferation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases
D014408:Biomarkers, Tumor; D022762:CCAAT-Enhancer-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C406364:UHRF1 protein, human; D044767:Ubiquitin-Protein Ligases