| Objective To study the effects of lnc-NBN-3:1 on acquired resistance to gefitinib in human lung adenovarcinoma(LAC)cells and its potential molecular mechanism.Methods The expression profile of IncRNAs and protein-coding genes via microarrays analysis of paired gefitinib-sensitive HCC827 cells and gefitinib-resistant HCC827-8-1 cells was identified.Then we selected the target IncRNA,lnc-NBN-3:1,by bioinformatics analyses and Quantitative PCR(qRT-PCR).Specific targeted siRNAs were designed and synthesized to silence the lnc-NBN-3:1 expression in HCC827-8-1 and HCC827-7-4cells.CCK8 assay and colony formation assay were used to observe impacts of lnc-NBN-3:1 on cell proliferation in LAC.Also,flow cytometry examined the change of cell cycle and apoptosis after lnc-NBN-3:1 silence.Western blot was used to detect the protein expression level changes of cleaved caspase-3,CCND1,AKT and p-AKT,Erk and p-Erk,EGFR and p-EGFR when cells were given different treatments.Results We identified the 1476 IncRNAs(703 up-regulation,773 down-regulation)and 1026 protein-coding genes(516 up-regulation,510 down-regulation)were differentially expressed between gefitinib-sensitive HCC827 cells and gefitinib-resistant HCC827-8-1 cells(fold change>3,p<0.05).Lnc-NBN-3:1 was identified the interesting IncRNA via GO,KEGG analysis and qRT-PCR.It was obviously and steadily over-expressed in HCC827-8-1 and may be involved in the process of cell apology,cell cycle ect..Compared with the control group,the gefitinib IC50 decreased when HCC827-8-1 cells were transfected with siRNA(p<0.05).Flow cytometry showed that lnc-NBN-3:1 silence significantly incresaed HCC827-8-1 cells apoptosis and induced cell cycle arrest at the G0/G1 phase in HCC827-8-1 cells.Moreover,the level of cleaved caspase-3 protein of the cells transfected with siRNA was up-regulated while the level of CCND1 was down-regulated in HCC827-8-1 cells.The similar result could be observed in gefitinib-resisitant HCC827-7-4 cells.Then,down-regulation of lnc-NBN-3:1 inhibited the protein levels of p-EGFR and p-AKT in HCC827-8-1 cells.Conclusion Lnc-NBN-3:1 silence could partly reverse acquired resistance to gefetinib via regulating EGFR/AKT pathway.Then,it might represent a new biomarker of poor response to gefitinib and could be described as a novel target for overcoming the resistance to gefitinib. |
