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OBJECTIVE@#To investigate the effects of LASS2/TMSG1 gene overexpression on proliferation and apoptosis of human lung cancer A549 cells and explore the possible mechanism.@*METHODS@#We examined LASS2/TMSG1 expression level in a previously constructed A549 cell line overexpressing LASS2/TMSG1 using Western blotting. The proliferation and apoptosis of the cells were detected using colony-forming assay, CCK-8 assay, Hoechst/PI double staining and flow cytometry. Fourteen nude mice were randomized into 2 groups (n=7) to receive subcutaneous injection of A549 cells with or without LASS2/TMSG1 overexpression on the back of the neck, and the cell proliferation in vivo was observed. The expression levels of p38 MAPK protein and p-p38 MAPK protein in the xenografts were detected with Western blotting. ELISA was used to detect the levels of ceramide and p38 MAPK protein in cultured A549 cell supernatants and the xenografts in nude mice.@*RESULTS@#Compared with the negative control cells, A549 cells with LASS2/TMSG1 overexpression had significantly lowered proliferation ability in vitro with increased early apoptosis rate (P < 0.05), and showed obvious growth inhibition after inoculation in nude mice(P < 0.05). Western blotting showed that in both cultured A549 cells and the xenografts in nude mice, LASS2/TMSG1 gene overexpression significantly increased the expression levels of p38 MAPK protein and p-p38 MAPK protein (P < 0.05); the results of ELISA also revealed significantly increased levels of ceramide and p38 MAPK protein in the cell supernatant andxenografts as well (P < 0.05).@*CONCLUSION@#Overexpression of LASS2/TMSG1 gene can significantly inhibit the proliferation and promote early apoptosis of human lung cancer A549 cells both in vitro and in vivo possibly by upregulating the expressions of ceramide and p38 MAPK protein to activate a signal transduction cascade.
Subject(s)
Animals , Humans , Mice , A549 Cells , Apoptosis , Cell Line, Tumor , Cell Proliferation , Lung Neoplasms , Membrane Proteins/metabolism , Mice, Nude , p38 Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolismABSTRACT
Objective:Vacuolar ATPase (V-ATPase) was found within the membranes and internal organelles of a vast array of eukaryotic cells,and was related to various kinds of highly metastatic tumors.LASS2/TMSG1 gene was a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by our laboratory.It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of V-ATPase (ATP6V0C).In this study,To use RNA interference to suppress the expression of ATP6V0C and try to further investigate the molecular mechanism of ATP6V0C in tumor metastasis and its relationship with LASS2/TMSG1 gene.Methods and Results:The expression level of ATP6V0C mRNA and protein in high metastatic potential prostate cancer cell lines (PC-3M-1E8 and PC-3M) was significantly higher than that in low metastatic potential prostate cancer cell lines (PC-3M-2B4 and PC-3),the expression level in PC-3M-1E8 being the highest.Follow-up tests selected PC-3M-1E8 cells for gene silencing.The expression and secretion of MMP-2 and the expression of MMP-9 in ATP6V0C siRNA transfected PC-3M-1E8 cells displayed no obvious change,but the activity of secreted MMP-9 was abated noticeably compared with the controls (P ＜ 0.05).Extracellular hydrogen ion concentration and V-ATPase activity in interference group were both reduced significantly compared with the controls (P ＜ 0.05).The migration and invasion capacity of ATP6V0C siRNA interfered cells in vitro were diminished significantly compared with the controls (P ＜ 0.05).Furthermore,a dramatic reduction of LASS2/TMSG1 mRNA and protein level after transfection of siRNA in PC-3M-1 E8 cells was discovered (P ＜ 0.05).Confocal immunofluorescence showed a vast co-localization of ATP6V0C protein and LASS2/TMSG1 protein in plasma and membrane.The co-localization signals of control group were much stronger than those of interference group.Conclusion:Specific siRNA silencing of ATP6V0C gene inhibits the invasion of human prostate cancer cells in vitro by mechanism of inhibiting V-ATPase activity and then reducing the extracellular hydrogen ion concentration,inhibiting MMP-9 activation and affecting ECM degradation and reconstruction.Meanwhile,ATP6V0C and LASS2/TMSG1 have interaction and it is likely that ATP6V0C functions as a feedback regulator of LASS2/TMSG1.
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Objective:To investigate microRNA-20a (miRNA-20a) expression in bladder cancer and its potential mechanism. Methods:MiRNA-20a expression was examined using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in human bladder cancer tissues and the paired adjacent non-tumor bladder tissues of 96 patients. The target gene of the miRNA-20a was predicted and validated using bioinformatics analysis and reporter gene assay, respectively. The mRNA or protein expression of the target gene in bladder cancer T24 and J82 cells transfected with miRNA-20a mimic or negative control (NC) mimics was detected via qRT-PCR, West-ern blot analysis, and cell immunofluorescence. CCK-8, Transwell chamber, and wound-healing assays were applied to test the prolifer-ation, migration, and invasion of T24 cells after miRNA-20a over-expression in vitro. Results:MiRNA-20a expression significantly in-creased in bladder cancer tissues compared with those in corresponding adjacent non-tumor tissues. High miRNA-20a expression in bladder cancer tissues was closely related to aggressive tumor phenotype, such as high histological grade, poor TNM stage, lymph node invasion, distant metastasis, and tumor recurrence (all P<0.001). Dual-luciferase reporter assay confirmed that miRNA-20a can di-rectly bind to the 3'-untranslated region (3'-UTR) of Homo sapiens longevity assurance homologue 2 (LASS2). Transfection with miRNA-20a mimics significantly inhibited mRNA and protein expression of LASS2 in T24 and J82 cells (all P<0.01) and promoted T24 cell prolif-eration, migration, and invasion in vitro. Conclusion:MiRNA-20a is highly expressed in bladder cancer tissues. MiRNA-20a enhances cell migration as well as proliferation and acts as an oncogene in bladder cancer because of the targeted inhibition of LASS2 expression.
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Objective: To investigate the effects LASS2 gene on the metastasis of human hepatocellular carcinoma cell line, HCCL3. Methods: The expression level of LASS2 mRNA in HCCLM3 cells was analyzed by northern blotting in high metastatic HCCLM3 cells and in low metastatic MHCC97-L cells. The eukaryotic expression vector pCMV-HA2-LASS2 was constructed and transfected into HCCLM3 cells. The stable clone with LASS2 overexpression was selected by G418 screening. The metastatic ability of HCCLM3 cells such as migration and invasion was observed in vitro after LASS2 overexpression. The synthesis, secretion, and activation of matrix metaloproteinase-2 (MMP-2) were examined by western blotting, zymography, and in situ zymography. Results: Northern blot analysis showed that the level of LASS2 mRNA decreased in HCCLM3 cells compared with MHCC97-L cells. LASS2 overexpression inhibited the ability of migration and invasion of HCCLM3 cells (P <0. 001 ). The synthesis of MMP-2 was not different but the secretion and activation of MMP-2 were down-regulated in HCCLM3 cells afterLASS2 overexpression. Conclusion: Overexpression of LASS2 down-regulated the secretion and activation of MMP-2 in HCCLM3 cells and inhibited the cell ability of migration and invasion indicating that LASS2 gene had inhibitory effects on the metastasis of HCCLM3 cells.