Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation.
Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.
Pang
Shi-Feng
SF
The Key Laboratory of Gene Engineering of Education Ministry, Zhongshan University, Guangzhou 510275, People's Republic of China.
Li
Xiao-Kun
XK
Zhang
Qiang
Q
Yang
Fang
F
Xu
Peilin
P
D018797:Cell Cycle Proteins / Q000494:pharmacology / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D009687:Nuclear Proteins / Q000494:pharmacology / Q000502:physiology*; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism; D011123:Polyploidy; D034622:RNA Interference; D016372:RNA, Antisense / Q000235:genetics; D053628:Receptors, Thrombopoietin / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018797:Cell Cycle Proteins; C512648:NUDC protein, human; D009687:Nuclear Proteins; D040201:Platelet Membrane Glycoprotein IIb; D016372:RNA, Antisense; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; C082218:MPL protein, human