The Effect Of Interfering TGF-β Receptor Ⅱ Expression On NB4Cells Proliferation And Response To ATRA And ATO

Object: The purpose of the research was to evaluate the effect of stablyexpressing interfered TGF-βRII on NB4cells proliferation and the response of ATRAon inducing differentiation and ATO on inducing apoptosis in NB4cells.Methods:1. RNAi technology was used to design and build four kinds oflentivirus vector and effective LV-TGFBR2-RNAi (9759-1) lentivirus vector wasfiltered and certified, which was then packaged and transfected with NB4cells.Antibiotic selection was used to obtain stable interfered TβRII genes of NB4cells,which was named TβRⅡ-shRNA NB4cells. RT-qPCR assay was used to identify theexpression of TβR Ⅱm RNAin the NB4cells.2. trypan blue test and CCK-8assaywere used to see the ability of proliferation of NB4cells and TβRⅡ-shRNA NB4cells.3. FCM testing the expression of CD11b and Wright staining were used tomonitor the effects of ATRA on its differentiation of TβRⅡ-shRNA NB4cells andNB4cells.4. Double staining(AnnexinV-FITC/PI) and AO/EB staining were used tomonitior the effects of ATO on its apoptosis of TβRⅡ-shRNA NB4cells and NB4cells.Results:1. TβRII genes could be steady interfered in NB4cells, which wasvalidated by RT-qPCR.2. trypan blue exclusion test and CCK-8assay showed that the proliferation ofTβRⅡ-shRNA NB4cells was significantly higher than that of NB4cells. After96hours, the OD value of TβRⅡ-shRNA NB4cells and NB4cells were1.859±0.029,1.096±0.006(P<0.0001).3. After ATRA treatment, the above two kinds of cells had corresponding celldifferentiation phenomenon(minitored by Wright staining). The expression of CD11bon TβRⅡ-shRNA NB4cells was lower than that of NB4cells, demonstratingdose-dependent effect. At the concentration of0.1μM for96h, the expression ofCD11b on TβRⅡ-shRNA NB4cells and NB4cells were 79.133±0.85,89.333±0.55(P<0.0001).4.Apoptosis of the above two kinds of cells was inducible when ATO treatmentat the concentration of2uM for24h(minitored by AO/EB). The apoptosis rate ofTβRⅡ-shRNA NB4cells was lower than that of NB4cells, demonstratingdose-dependent effect as well. At the concentration of8μM for24h, the rates ofapoptosis in TβRⅡ-shRNA NB4cells and NB4cells were49.15±2.05,66.85±2.41(P<0.01).Conclusion: the research further confirmed that TβRⅡwas a negativeregulation receptor in NB4cells proliferation and participated in the process ofATRA inducing differentiation and ATO inducing apoptosis in NB4cells.