The Expression Of RTVP-1 In Gliomas And Study Of The Inhibition Of RTVP-1 In Glioma Cell Lines By RNA Interference

Objective:To detect the expression of RTVP-1 in gliomas and to study the inhibition of RTVP-1 gene by RNA interference in glioma cell lines u251 and SHG-44 in vitro.To investigate the expression of the aim gene and its correlation with transfection time and concentration,to observe the effects of RNA interference on cell growth,cell cycle and cell apoptosis while in different transfecting time and concentration after the inhibition of RTVP-1 by RNA interference.Methods:The immunohistochemical method was used to determine the expression of RTVP-1 protein in glioma cell lines u251 and SHG-44,as well as in formalin-fixed and paraffin-embedded clinical samples of gliomas.The siRNA targeting RTVP-1 gene mixed with liposome Lipofectamine2000 was transfected into u251 and SHG-44 cells before reverse transcription-polymerase chain reaction(RT-PCR), Western-blot and flow cytometry techniques were used respectively to examine the expression levels of mRNA and protein of the aim gene,the cell cycles and apoptosis of glioma cells.The relative amount of cells was checked by SRB.Results:Immunohistochemical method shows that RTVP-1 can be detected mainly in the cytoplasm of cell line u251 cell line and the four chosen clinical samples of gliomas, while no expression was detected in cell line SHG-44 cell line.With the transfecting time increasing,RTVP-1 gene is inhibited greatest at 24h at mRNA level,and greatest at 48h at protein level.In siRNA transfected u251 cells of different concentrations,the expressions of RTVP-1 at mRNA level are all down regulated.Moreover,the higher concentration of siRNA is used,the higher degree of inhibition effect is observed.The suppression rate reaches to 100%at transfecting concentration of 200nM.Likely,Western-blot indicates that the expression of RTVP-1 protein decreases as siRNA transfecting concentration is increased.It is indicated by flow cytometry method that the cell cycle arrest and apoptosis effects are not obvious in scrambling control group in u251 cells,which is similar to blank control group.However, the experimental group shows obvious cell cycle arrest and apoptosis effect.In the time dependent manner,we can see clear cell apoptosis at 12h and 24h after transfection,but the cell apoptosis effect declines after 24h.The greatest effect of cell cycle arrest occurs at 24h after transfection,when the G1 phase cells proportion is 72.1%.In the dosage dependent manner,although inconspicuous effect of cell cycle arrest is observed,the cell apoptosis can be seen obviously at the transfecting concentration of 25nM and 50nM.With the transfecting concentration increasing,the effects of cell cycle arrest and apoptosis increases as well.Especially,in the group of 200nM,the G0/G1 phase cells proportion is 88.4%,while the S phase cells proportion is only 0.1%with the cell apoptosis rate arriving at 12%.At different transfecting time,SRB assay shows that the cell growth of u251 is obviously delayed after transfected by siRNA,which is significantly different from the blank control group statistically.However,there is no significant difference between the scrambling control group and the blank control group statistically.The growth of SHG-44 manifests no change after transfected by siRNA statistically.RTVP-1 mRNA and protein can not be detected in SHG-44 cell line by RT-PCR and Western-blot.Conclusions:1.The cell growth of u251 cell line is obviously delayed after the silencing of RTVP-1 gene by RNA interference,while silencing RTVP-1 gene has no effect on the growth of SHG-44 cell line.â‘ At mRNA Ievel,RTVP-1 gene is silenced by the chosen siRNA in u251 cell line.The silencing efficiency is relatively high and the chosen siRNA sequence is effective.RTVP-1 mRNA and protein can not be detected in SHG-44 cell line by RT-PCR and Western-blot.â‘¡In the time dependent manner,the greatest silencing effect of RTVP-1 mRNA emerges at 24h while the most obvious declination of RTVP-1 protein expression emerges at 48h.â‘¢In siRNA transfected u251 cells of different concentrations,the RTVP-1 gene are all down regulated.Moreover,the higher concentration of siRNA is used,the higher degree of silencing effect is observed.The 25nM siRNA concentration can bring about the gene silencing already.The silencing effect of the aim gene correlates with the siRNA transfection concentration positively.Western-blot method indicates that the inhibition of aim protein expression correlates with the siRNA transfection concentration positively as well.2.Silencing RTVP-1 gene by RNA interference can induce u251 cells arrested in the G1 phase and can reduce cells in S phase with cell apoptosis increasing,which results in the detention of cell growth.3.Immunohistochemical method shows that RTVP-1 protein expresses in u251 cell line and the four chosen clinical samples of gliomas,while do not express in SHG-44 cell line.