The Role And Mechanism Of Long Non-coding RNA GRLAT1 In Gefitinib-resistant Lung Adenocarcinoma Cells

Objective: The research aims to gain insight into the role and possible mechanism of long non-coding RNA in lung adenocarcinoma cell resistance to Gefitinib.Methods: We analyzed expression patterns in EGFR-TKI-resistant HCC827 cell line(HCC827/GR)by lnc RNA microarray and compared it with its parental HCC827 cell line.By using bioinformatics analysis,we selected an up-regulated lnc RNA in HCC827/GR,NONHSAT093968,and suppressed the expression of NONHSAT093968 by interfering RNA in HCC827/GR cell.The cell viability and the half maximal inhibitory concentration(IC50)with a concentration gradient of Gefitinib were measured by a cell counting kit-8(CCK-8)assay.Cell proliferation was detected by colony formation assay.Cell apoptosis and cell cycle arrest were detected by flow cytometry analysis.Cell proliferation in vivo was examined by xenograft tumor models in nude mice using HCC827/GR transfected with si RNA.The target gene was predicted by bioinformatics analysis and the influence on target gene was determined by q RT-PCR and Western Blot.Results: 1.Several lnc RNAs were dysregulated in the HCC827/GR cells,which may be involved in the bio-pathways associated with Gefitinib-resistant through their cis-and/or trans-regulation of protein-coding genes.After some strategies of screening on Gefitinib-resistant related lnc RNAs,we identified a novel lnc RNA,NONHSAT093968.Since that NONHSAT093968 was found in the Gefitinib-resistant lung adenocarcinoma,so we named it GRLAT1(Gefitinib-resistant lung adenocarcinoma transcript 1).2.By down regulating GRLAT1,the sensitivity of HCC827/GR cells to Gefitinib was increased against the decrease of IC50.At the same time,the proliferation of HCC827/GR cells could be inhibited and apoptosis was promoted.In vivo,the subcutaneous tumorigenesis of HCC827/GR cells was inhibited by the reduction of GRLAT1 in the xenograft tumor models in nude mice.3.By analyzing the information of lnc RNA GRLAT1 neighboring coding gene,we found that CCDC50 gene existed nearby.The results of q RT-PCR and Western Blot experiments showed that the level of CCDC50 m RNA and protein expression increased significantly after the reduction of GRLAT1.We reduced CCDC50 by si RNA in HCC827/GR cells,and found that the increase of IC50 in Gefitinib result in a decrease of the sensitivity of HCC827/GR cells to Gefitinib,while promoting cell proliferation and inhibiting apoptosis.Then we carried out the CCK8 rescue experiment.The down-regulated CCDC50 could save the inhibitory effect of GRLAT1-si RNA on cell proliferation.Conclusions: 1.We identified a group of Gefitinib-resistance related lnc RNAs in lung adenocarcinoma by high-throughput lnc RNA microarrays.GRLAT1 is one of the most important lnc RNA invovled in Gefitinib-resistance.2.CCDC50 is the functional target gene of GRLAT1.Down-regulation of CCDC50 can inhence lung microarrays Gefitinib resistance.3.GRLAT1 may induce lung adenocarcinoma cell resistance by negatively regulating CCDC50.