Summary： The effects of RNAi-mediated gene silencing of LR1G3 expression on cell cycle and survival of human glioma cell line GL15 and the possible mechanisms were explored. The plasmids pGenesil2-LRIG3-shRNA1 and pG...Summary： The effects of RNAi-mediated gene silencing of LR1G3 expression on cell cycle and survival of human glioma cell line GL15 and the possible mechanisms were explored. The plasmids pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2 were transfected into GLI 5 glioma cells respectively by using Metafectine, and the transfected cells that stably suppressed LR1G3 expression were selected by G418. The control cells were transfected with negative control shRNA. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blot. The apoptosis rate and cell cycle were analyzed by flow cytometry. As compared with the negative shRNA-transfected GL 15 cells, LRIG3 mRNA expression in GLI 5 cells transfected with pGenesil2-LRIG3-shRNA 1 and pGenesil2-LRlG3-shRNA2 was silenced by 52.4%, 63.8%, and LRIG3 protein expression was reduced by 50.9% and 67.4% respectively. The LRIG3-specific siRNA transfected cells had higher proliferation rate than control cells. Cell cycle analysis showed that silencing LRIG3 increased the percentage of G2/M phase cells and the proliferation index significantly （P〈0.01）. Silencing LRIG3 could inhibit the apoptosis of GL15 cells （P〈0.05）. These findings suggest that the siRNA targeting LRIG3 gene shows a dramatic inhibitory effect on RNA transcription and protein expression, then promoting the proliferation of GL15 cells, arresting GL15 cells in G2/M phase, and suppressing apoptosis of GL15 cells.展开更多
This study examined the effect of silencing LRIG3 expression on the proliferation and apoptosis of bladder cancer T24 cells and explored the role of LRIG3 in the tumorigenesis of bladder cancer. Bladder cancer T24 cel...This study examined the effect of silencing LRIG3 expression on the proliferation and apoptosis of bladder cancer T24 cells and explored the role of LRIG3 in the tumorigenesis of bladder cancer. Bladder cancer T24 cells were routinely cultured and pSilencer plasmids were employed to construct LRIG3 eukaryotic expression vector of LRIG3-siRNA, i.e., pSilencer-LRIG3-siRNA. After confirmation, the vector was transfected into HEK293 cells to make a replication-deficient adenovirus, pAd-LRIG3-siRNA, which was then introduced into bladder cancer T24 cells. RT-PCR, Western- blotting were performed to detect the levels of LRIG3 mRNA and proteins. Cells number was determined by using MTT test. Hoechst33258 staining, transmission microscopy, flow cytometery were conducted to examine the cell apoptosis. Three groups included a blank control group, a negative control group （containing non-interfering plasmids） and a pAd-LRIG3-siRNA group. Our results showed that the recombinant pAd-LRIG3-siRNA was successfully transfected into the bladder cancer T24 cells. The siRNA formed by the transcription of the recombinant plasmids resulted in significantly reduced expressions of LRIG3 gene and protein and significantly decreased cell proliferation and growth in the pAd-LRIG3-siRNA group as compared with the control group （P0.01）. The siRNA also caused apoptotic changes of some cells, with the apoptosis rate being （17.69±0.75）%, which was significantly different from that of the control group （P0.01）. It was concluded that recombinant pAd-LRIG3-siRNA plasmids could effectively decrease the expression of LRIG3 mRNA and proteins and, to some extent, inhibit the proliferation and promote the apoptosis of bladder cancer T24 cells. Silencing LRIG3 gene might be a novel alternative for the treatment of bladder cancer.展开更多
The purpose of this study was to investigate the impact of leucine-rich repeats and immu- noglobulin-like domains 3 （LRIG3） on the biological features of bladder cancer cell lines. The plasmids of over-expressed LRI...The purpose of this study was to investigate the impact of leucine-rich repeats and immu- noglobulin-like domains 3 （LRIG3） on the biological features of bladder cancer cell lines. The plasmids of over-expressed LRIG3 and the blank plasmid serving as control were transfected into the bladder cancer cell lines, T24, EJ and BIU-87, and the expression levels of LRIG3 mRNA and protein were de- tected by using real-time PCR and Western blotting. The changes in the cell cycle and apoptosis were examined by using flow cytometry. The invasive ability was measured by Transwell assay, and CCK-8 assays were used to measure the proliferation of cells. As compared with the control group, the LRIG3 mRNA and protein expression levels in LRIG3 cDNA-transfected group were raised significantly （P〈0.05）. The average number of cells with up-regulated LRIG3 passing through the inserted filter was decreased significantly as compared with the control group （P〈0.05）. Up-regulation of LRIG3 also could inhibit proliferation and induce apoptosis of T24, EJ and BIU-87 cells. Except BIU-87, the T24 and EJ cells transfected with LIRG3 eDNA were arrested in G0/G1 phase compared to the control group （P〈0.05）. In conclusion, the over-expression of LRIG3 could influence the cell cycle and invasion, in- hibit proliferation and induce apoptosis in the three bladder cancer cell lines.展开更多
基金supported by a grant from National Natu-ral Sciences Foundation of China (No. 3.500521 No. 30872653).
文摘Summary： The effects of RNAi-mediated gene silencing of LR1G3 expression on cell cycle and survival of human glioma cell line GL15 and the possible mechanisms were explored. The plasmids pGenesil2-LRIG3-shRNA1 and pGenesil2-LRIG3-shRNA2 were transfected into GLI 5 glioma cells respectively by using Metafectine, and the transfected cells that stably suppressed LR1G3 expression were selected by G418. The control cells were transfected with negative control shRNA. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blot. The apoptosis rate and cell cycle were analyzed by flow cytometry. As compared with the negative shRNA-transfected GL 15 cells, LRIG3 mRNA expression in GLI 5 cells transfected with pGenesil2-LRIG3-shRNA 1 and pGenesil2-LRlG3-shRNA2 was silenced by 52.4%, 63.8%, and LRIG3 protein expression was reduced by 50.9% and 67.4% respectively. The LRIG3-specific siRNA transfected cells had higher proliferation rate than control cells. Cell cycle analysis showed that silencing LRIG3 increased the percentage of G2/M phase cells and the proliferation index significantly （P〈0.01）. Silencing LRIG3 could inhibit the apoptosis of GL15 cells （P〈0.05）. These findings suggest that the siRNA targeting LRIG3 gene shows a dramatic inhibitory effect on RNA transcription and protein expression, then promoting the proliferation of GL15 cells, arresting GL15 cells in G2/M phase, and suppressing apoptosis of GL15 cells.
基金supported by a grant from Hubei Provincial Natural Sciences Foundation (No. 2004ABA-205)
文摘This study examined the effect of silencing LRIG3 expression on the proliferation and apoptosis of bladder cancer T24 cells and explored the role of LRIG3 in the tumorigenesis of bladder cancer. Bladder cancer T24 cells were routinely cultured and pSilencer plasmids were employed to construct LRIG3 eukaryotic expression vector of LRIG3-siRNA, i.e., pSilencer-LRIG3-siRNA. After confirmation, the vector was transfected into HEK293 cells to make a replication-deficient adenovirus, pAd-LRIG3-siRNA, which was then introduced into bladder cancer T24 cells. RT-PCR, Western- blotting were performed to detect the levels of LRIG3 mRNA and proteins. Cells number was determined by using MTT test. Hoechst33258 staining, transmission microscopy, flow cytometery were conducted to examine the cell apoptosis. Three groups included a blank control group, a negative control group （containing non-interfering plasmids） and a pAd-LRIG3-siRNA group. Our results showed that the recombinant pAd-LRIG3-siRNA was successfully transfected into the bladder cancer T24 cells. The siRNA formed by the transcription of the recombinant plasmids resulted in significantly reduced expressions of LRIG3 gene and protein and significantly decreased cell proliferation and growth in the pAd-LRIG3-siRNA group as compared with the control group （P0.01）. The siRNA also caused apoptotic changes of some cells, with the apoptosis rate being （17.69±0.75）%, which was significantly different from that of the control group （P0.01）. It was concluded that recombinant pAd-LRIG3-siRNA plasmids could effectively decrease the expression of LRIG3 mRNA and proteins and, to some extent, inhibit the proliferation and promote the apoptosis of bladder cancer T24 cells. Silencing LRIG3 gene might be a novel alternative for the treatment of bladder cancer.
文摘The purpose of this study was to investigate the impact of leucine-rich repeats and immu- noglobulin-like domains 3 （LRIG3） on the biological features of bladder cancer cell lines. The plasmids of over-expressed LRIG3 and the blank plasmid serving as control were transfected into the bladder cancer cell lines, T24, EJ and BIU-87, and the expression levels of LRIG3 mRNA and protein were de- tected by using real-time PCR and Western blotting. The changes in the cell cycle and apoptosis were examined by using flow cytometry. The invasive ability was measured by Transwell assay, and CCK-8 assays were used to measure the proliferation of cells. As compared with the control group, the LRIG3 mRNA and protein expression levels in LRIG3 cDNA-transfected group were raised significantly （P〈0.05）. The average number of cells with up-regulated LRIG3 passing through the inserted filter was decreased significantly as compared with the control group （P〈0.05）. Up-regulation of LRIG3 also could inhibit proliferation and induce apoptosis of T24, EJ and BIU-87 cells. Except BIU-87, the T24 and EJ cells transfected with LIRG3 eDNA were arrested in G0/G1 phase compared to the control group （P〈0.05）. In conclusion, the over-expression of LRIG3 could influence the cell cycle and invasion, in- hibit proliferation and induce apoptosis in the three bladder cancer cell lines.
