| Objective: Lung cancer is one of the most common malignancies and the leading cause of cancer-related deaths worldwide,of which adenocarcinoma is the predominant subtype.In patients with lung adenocarcinoma,EGFR mutations are frequently observed such as inframe deletions in exon 19 and a nucleotide substitution within codon 858 of exon 21.For lung adenocarcinoma(LAD)patients who harbor EGFR-sensitizing mutations,icotinib has been commonly used in first-line treatment.Unfortunately,most of the responding patients eventually acquire EGFR-TKIs resistance within 10–16months.Therefore,icotinib resistance is one of the major obstacles in the treatment of LAD.Although some molecular mechanisms of EGFR-TKIs resistance have been revealed including MET or ERBB2 gene amplification and EGFR gate keeper mutation(T790M),other potential mechanisms still need to be elucidated,which will provide novel therapeutic targets for LAD treatment.Long non-coding RNAs(lncRNAs),which are a novel class of transcripts lacking of protein-coding potential and with the size than 200 nucleotidesare involved in epigenetic,transcriptional,post-transcriptional,and translation regulation,as well as post-translational modification.LncRNAs localized in cytoplasm often functions as a competitive endogenous RNA(ceRNAs)to exert their regulatory functions at post-transcriptional level.Recently,accumulative studies have proved that lncRNAs play pivotal roles in the regulation of cancer development and biological progression.Several studies also demonstrated that lncRNA participated in regulating drug resistance.However,most EGFR-TKI-resistance-related lncRNAs and their functional mechanisms are still largely unknown.In the current study,icotinib resistance-related lncRNAs were screened using transcriptome sequencing and differential lncRNA expression analysis in icotinib resistant LAD cells,and a novel icotinib resistance-related lncRNA,APCDD1L-AS1(ENSG00000231290)was identified.Further study showed that lncRNA APCDD1L-AS1 could promote icotinib resistance of LAD cells by up-regulating the expression of SIRT5 via sponging miR-1322/ miR-1972/ miR-324-3p.Finally,SIRT5 enhanced EGFR expression and activated EGFR phosphorylation by inhibiting autophagic degradation of EGFR to affect icotinib resistance.This study demonstrated that lncRNAs were involved in the novel mechanism of icotinib resistance,which was that APCDD1L-AS1 could inhibit autophagic degradation of EGFR to promote icotinb resistance via miR-1322/ miR-1972/ miR-324-3p/SIRT5 axis in LAD cells.Therefore,it further elucidated that APCDD1L-AS1,miR-1322,miR-1972,miR-324-3p and SIRT5 may serve as a promising biomarker for overcoming icotinb resistance in treating LAD.Methods: 1.LncRNA APCDD1L-AS1 was identified using transcriptome sequencing and differential lncRNA expression analysis of icotinib-resistant LAD cells and their parental cells.Similar result was also verified by qRT-PCR.2.Fluorescence in situ hybridization(FISH)and nuclear/cytoplasmic RNA separation analysis was used to investigate the localization of APCDD1L-AS1.3.The ceRNA relationship of APCDD1L-AS1,miR-1322/miR-1972/miR-324-3p and SIRT5 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation(RIP).4.The results of qRT-PCR confirmed that the expression of miR-1322/ miR-1972/ miR-324-3p and SIRT5 in icotinib-resistant cells and their parental cells.5.Icotinib-induced apoptosis in APCDD1L-AS1-KD and SIRT5-KD cotinib-resistant LAD cells was assessed by flow cytometry and western blot。 6.The sensitivity of icotinib in APCDD1L-AS1-KD、miR-1322/miR-1972/miR-324-3p mimics and SIRT5-KD icotinib-resistant LAD cells and their parental cells was assessed by MTT assay.7.The effect of SIRT5 on EGFR expression and autophagy was observed by western blot and immunofluorescence staining in vitro and in vivo.8.Immunohistochemical staining showed the expression of SIRT5 and EGFR in Lv-sh RNA-APCDD1L-AS1 group.Results: 1.APCDD1L-AS1 was significantly upregulated in icotinib-resistant LAD cells.To screen the lncRNA related to icotinib resistance,the global expression profiles analysis of lncRNA were performed in PC9,PC9/Ico RL and PC9/Ico RH cells.Similar result was also verified by qRT-PCR.2.APCDD1L-AS1 contributed to icotinib resistance by up-regulating EGFR and inhibiting apoptosis in LAD.The result of MTT assay and western blot showed that APCDD1L-AS1-KD in icotinib-resistant LAD cells significantly enhanced the icotinib sensitivity and decreased the protein and phosphorylation level of EGFR,whereas APCDD1L-AS1-OE increased EGFR expression and activation.Additionally,the results of flow cytometry and western blot confirmed that APCDD1L-AS1-KD promoted icotinib-induced apoptosis in icotinib-resistant cells.3.Mi R1322/ miR1972/ miR324-3p increased the sensitivity of lung adenocarcinoma-resistant cells to icotinib by inhibiting EGFR expression and promoting apoptosis.The mimics of miR-1322,miR-1972 and miR-324-3p were separately transfected into icotinib-resistant LAD cells followed by icotinb treatment.Then the results showed that all these mimics significantly enhanced icotinib sensitivity.Simultaneously,the mimics of all three miRNAs not only alleviated the protein and phosphorylation level of EGFR,but also promoted apoptosis in icotinib-resisitant cells.4.APCDD1L-AS1 up-regulated SIRT5 expression by sponging miR1322/ miR1972/miR324-3p.The dual luciferase reporter assays and RIP assay showed that APCDD1L-AS1 could sponge with miR-1322,miR-1972 and miR-324-3p.The results of qRT-PCR and western blot showed that both the m RNA and protein expression level of SIRT5 was decreased by APCDD1L-AS1-KD,but increased by APCDD1L-AS1-OE.Additionally,APCDD1L-AS1-KD also inhibited EGFR expression and its activation,as well as promoted the autophagic flux,which were partially rescued by the inhibitors of miR-1322,miR-1972 and miR-324-3p.5.SIRT5 induced icotinib resistance in icotinib-resistant LAD cells by up-regulating EGFR and inhibiting apoptosis.MTT assay showed that SIRT5-KD significantly reduced the IC50 values for icotinib in icotinib-resistant cells.Meanwhile,both protein and phosphorylation level of EGFR was significantly decreased in SIRT5-KD icotinib-resistant cells.In addition,the results of flow cytometry and western blot showed that SIRT5-KD significantly increased apoptotic proportion in icotinib-resistant cells.6.SIRT5 upregulated EGFR by inhibiting its autophagic degradation.The result of western blot showed that the expression of p62 was decreased,whereas the expression LC3B-Ⅱ was increased in SIRT5-KD cells.Two autophagy inhibitors,CQ and 3-MA,partially rescued SIRT5-KD-induced EGFR degradation,as well as the following inhibition of EGFR activation and increase of PARP cleavage.On the contrary,the combination of autophagy initiator rapamycin with icotinib partially reversed icotinib resistance of LAD cells.7.APCDD1L-AS1 induced drug resistance by inhibiting autophagy degradation of EGFR through miR1322/ miR1972/ miR324-3p-SIRT5 axis.The result of western blot showed that APCDD1L-AS1-KD also inhibited EGFR expression and its activation,as well as promoted the autophagic flux,which were partially rescued by the inhibitors of miR-1322,miR-1972 and miR-324-3p.The results of subcutaneous tumor formation in nude mice proved to be consistent with the aforementioned results in vitro.Conclusion: 1.A novel icotinib resistance-related lncRNA APCDD1L-AS1,promoted icotinib resistance in LAD cells.2.APCDD1L-AS1 contributed to icotinib resistance by upregulating SIRT5 and EGFR via sponging miR1322/miR1972/miR324-3p and inhibiting apoptosis.3.SIRT5 contributed to icotinib resistance by inhibiting autophagic degradation of EGFR,and the combination icotinib with autophay initiator perfectly reversed icotinib resistance in LAD cells.4.APCDD1L-AS1 promoted icotinib resistance by inhibiting autophagic degradation of EGFR via miR-1322/ miR-1972/miR-324-3p-SIRT5 axis. |
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