AIM:To profile expression of micro RNAs(mi RNAs)in gastric cancer cells and investigate the effect of mi R-374b-5p on gastric cancer cell invasion and metastasis.METHODS:An mi RNA microarray assay was performed to ide...AIM:To profile expression of micro RNAs(mi RNAs)in gastric cancer cells and investigate the effect of mi R-374b-5p on gastric cancer cell invasion and metastasis.METHODS:An mi RNA microarray assay was performed to identify mi RNAs differentially expressed in gastric cancer cell lines(MGC-803 and SGC-7901)compared with a normal gastric epithelial cell line.Upregulation of mi R-374b-5p was newly identified and confirmed via quantitative real-time reverse transcriptionPCR(q RT-PCR).MGC-803 cells were transfected with a synthesized anti-mi R-374b-5p sequence or a control vector using Lipofectamine reagent,or treated with transfection reagent alone or phosphate-buffered saline as controls.Rate of transfection was verified after 48 h by q RT-PCR.Cells were then subjected to transwell migration,wound scratch and cell counting kit-8 assays.A bioinformatic analysis to identify mi R-374b-5p target genes was performed using mi Randa,Pic Tar and Target Scan software.A dual luciferase reporter assay was performed to evaluate the influence of mi R-374b-5p on target gene activation,and q RT-PCR and Western blot were used to evaluate the levels of target m RNA and protein following transfection with mi R-374b-5p antisense oligonucleotides.RESULTS:The microarray profiling revealed downregulation of 14(fold change<0.667;P<0.05)and upregulation of 12(fold change>1.50;P<0.05)mi RNAs in MGC-803 and SGC-7901 cells compared with GES-1 controls.The upregulation of mi R-374b-5p(fold change=1.75 and 1.64 in MGC-803 and SGC-7901,respectively;P<0.05)was confirmed by q RT-PCR.Compared with the control groups,the restoration of mi R-374b-5p expression with anti-mi R-374b-5p significantly suppressed the metastasis,invasion and proliferation of MGC-803 cells.The bioinformatic analysis predicted that the 3’untranslated region(UTR)of reversion-inducing cysteine-rich protein with Kazal motif(RECK)contains three mi R-374b-5p target sequences.RECK was verified as a target gene in a dual luciferase reporter assay showing that activation of RECK 3’UTR-pmir GLO was increased by co-transfection with mi R-374b-5p.Finally,transfection of mi R-374b-5p antisense oligonucleotides increased m RNA and protein levels of RECK in MGC-803cells(P<0.05).CONCLUSION:These findings indicate that upregulation of mi R-374b-5p contributes to gastric cancer cell metastasis and invasion through inhibition of RECK expression.展开更多
基金Supported by National Natural Science Foundation of China,No.81071965
文摘AIM:To profile expression of micro RNAs(mi RNAs)in gastric cancer cells and investigate the effect of mi R-374b-5p on gastric cancer cell invasion and metastasis.METHODS:An mi RNA microarray assay was performed to identify mi RNAs differentially expressed in gastric cancer cell lines(MGC-803 and SGC-7901)compared with a normal gastric epithelial cell line.Upregulation of mi R-374b-5p was newly identified and confirmed via quantitative real-time reverse transcriptionPCR(q RT-PCR).MGC-803 cells were transfected with a synthesized anti-mi R-374b-5p sequence or a control vector using Lipofectamine reagent,or treated with transfection reagent alone or phosphate-buffered saline as controls.Rate of transfection was verified after 48 h by q RT-PCR.Cells were then subjected to transwell migration,wound scratch and cell counting kit-8 assays.A bioinformatic analysis to identify mi R-374b-5p target genes was performed using mi Randa,Pic Tar and Target Scan software.A dual luciferase reporter assay was performed to evaluate the influence of mi R-374b-5p on target gene activation,and q RT-PCR and Western blot were used to evaluate the levels of target m RNA and protein following transfection with mi R-374b-5p antisense oligonucleotides.RESULTS:The microarray profiling revealed downregulation of 14(fold change<0.667;P<0.05)and upregulation of 12(fold change>1.50;P<0.05)mi RNAs in MGC-803 and SGC-7901 cells compared with GES-1 controls.The upregulation of mi R-374b-5p(fold change=1.75 and 1.64 in MGC-803 and SGC-7901,respectively;P<0.05)was confirmed by q RT-PCR.Compared with the control groups,the restoration of mi R-374b-5p expression with anti-mi R-374b-5p significantly suppressed the metastasis,invasion and proliferation of MGC-803 cells.The bioinformatic analysis predicted that the 3’untranslated region(UTR)of reversion-inducing cysteine-rich protein with Kazal motif(RECK)contains three mi R-374b-5p target sequences.RECK was verified as a target gene in a dual luciferase reporter assay showing that activation of RECK 3’UTR-pmir GLO was increased by co-transfection with mi R-374b-5p.Finally,transfection of mi R-374b-5p antisense oligonucleotides increased m RNA and protein levels of RECK in MGC-803cells(P<0.05).CONCLUSION:These findings indicate that upregulation of mi R-374b-5p contributes to gastric cancer cell metastasis and invasion through inhibition of RECK expression.
