分子生物学43592842

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1、LRRC4,a Putative Tumor Suppressor Gene,Requires a Functional Leucine-rich Repeat Cassette Domain to Inhibit Proliferation of Glioma Cells In Vitro by Modulating theExtracellular Signal-regulated Kinase/Protein Kinase B/Nuclear Factor-B PathwayMolecular Biology of the CellVol.17,35343542,August 2006G

2、uiyuan Li et al.0711078 曹广超1LRRC4 gene which contains a conserved leucine-rich repeat(LRR)cassette and an immunoglobulin(Ig)IgC2 domain,is associated with glioma suppression both in vitro and in vivo.The present study provides evidence that the conspicuous absence of LRRC4 in high-grade gliomas dire

3、ctly contributes to the increasing tumor grade.The loss of LRRC4 in U251 cells is caused by the loss of homozygosity at chromosome 7q32-ter.It was also found that LRRC4 requires a functional LRR cassette domain to suppress U251 cell proliferation.In the LRR cassette domain,the third LRR motif of the

4、 core LRR is found to be indispensable for the function of LRRC4.23The inhibitory effect of LRRC4 is accompanied by a decrease in the expression of pERK,pAkt,pNF-Bp65,signal transducer and activator of transcription protein-3(STAT3),and mutant p53,and an increase in the expression of c-Jun NH2-termi

5、nal kinase(JNK)2 and p-c-Jun,suggesting that LRRC4 plays a major role in suppressing U251 cell proliferation by regulating the extracellular signal-regulated kinase(ERK)/Akt/NF-Bp65,STAT3,and JNK2/c-Jun pathways.4Tumor Samples and Cell LinesNucleic Acid Isolation and RNA AnalysisOne-Step PCR Mutagen

6、esis and ConstructsWestern Blotting AnalysisCell Proliferation AssaySoft Agar AssayCell Cycle AnalysisAcridine Orange(AO)/Ethidium Bromide(EB)StainingStatistical Analysis56Figure 1.Analysis of LRRC4 expression in gliomas and glioblastoma cell lines.(A)Northern blotting analysis of LRRC4 in glioblast

7、oma cell lines and gliomas(top).Relative levels of RNA loading are shown as methylene blue staining of 28s RNA and GAPDH(bottom).(B)RT-PCR analysis of LRRC4 expression in gliomas,primary culture glioma cells,and glioblastoma cell lines.RNA from gliomas and cell lines was amplified for 25 and 35 cycl

8、es,respectively,using GAPDH(bottom)and LRRC4(top)primers.Water was used as the negative control,and fetal brain cDNA and pcDNA3.1()-LRRC4 plasmid were used as the positive control templates.Lanes 14,primary culture tumor cells derived from grade IIIII glioma;and lane 5,primary culture tumor cells de

9、rived from grade IV glioblastoma.7LRRC4 arrests U251 cells in G0/G1 phase,it is possible that LRRC4 also induces U251 cells apoptosis.We used the analysis of flow cytometry and AO/EB dual staining to detect U251 cell apoptosis.8An AO/EB cocktail(80 l)containing 1 ml of DMEM was added in the culture

10、plate.Fields of stained cells were selected and focused using fluorescence microscopy(Nikon Eclipse E800;Nikon,Tokyo,Japan).Viable cells stained only with AO were bright green with intact structure;early apoptotic cells stained with AO/EB were bright green in the nucleus with red-orange chromatin.La

11、te apoptotic cells stained with both AO and EB were red-orange with chromatin condensation.9It was shown that LRRC4 induced necrosis,but not apoptosis10In conclusion,LRRC4 may act as a novel candidate of tumor suppressor gen（TSG）.may be involved in the pathogenesis of gliomas and the transmission of complex growth suppressive signals.Therefore,the loss of LRRC4 function may be an important event in the progression of gliomas.11Thats all.Thank you!12