MicroRNA-181a Regulates Gefitinib Sensitivity In Non-small Cell Lung Cancer Through Targeting GAS7

BackgroundWith the discoveries of many actionable driver mutations, molecular targeted therapies have become a great milestone for the treatment of Non-small cell lung cancer (NSCLC). Molecular targeted therapy based on epidermal growth factor receptor (EGFR) mutation is one of the most representative treatment options. Data from clinical research have confirmed that NSCLC patients with EGFR mutations are highly responsive to tyrosine kinase inhibitors (TKIs), and the objective response rate was71.2%with Gefitinib in the EGFR mutation-positive subgroup. However, almost all the patients with initial responses to TKIs inevitably became resistant to them mostly within12months and ultimately lead to tumor progression, which has been defined as "acquired resistance".T790M secondary mutation and MET amplification are the two major mechanisms of acquired resistance. Approximately50%of the acquired resistance is linked to T790M mutation. About22%of the acquired resistant have been demonstrated to possess MET amplification. EGFR T790M and MET amplification accounts for60%of all known mechanisms of acquired resistance. In addition, loss of PTEN、epithelial-mesenchymal transition、activation of Axl and small cell lung cancer transformation have also been shown to contribute to TKIs acquired resistant. Therefore, the early diagnosis and the effective intervention for TKIs acquired resistant are emergent issue in NSCLC patients.MicroRNAs (miRNAs) are small, non-coding RNA moleculars of22nucleotides that intrinsically suppress mRNA by pairing with the3’-untranslated region (UTR) of the mRNA. It has been shown that miRNAs could negatively modulate expression of targeted genes by degrading target mRNA or inhibiting the transcription of target mRNA. A large body of evidence has demonstrated miRNAs play an important role in a wide range of biological process in cancer, including proliferation, apoptosis, metabolism and differentiation by directly degrading messenger RNA or repressing protein translation. Most recently, the involvement of miRNAs in TKIs acquired resistant has been reported, which suggest that miRNAs could be an useful molecular tool for solving TKIs acquired resistant. For example, Garofalo et al. investigated the role of miRNAs regulated by MET receptor tyrosine kinases in Gefitinib resistance.In light of miRNAs in regulation of tumor biological process and in the role of TKIs acquired resistant, did miRNAs also result in TKIs acquired resistant that is caused by other mechanisms? Furthermore, how miRNAs regulates TKIs acquired resistant are still not understood. Therefore, the aim of this study was to investigate the role of miRNAs in Gefitinib resistance in our experimental model. We obtain the miRNAs associated likely with Gefitinib sensitivity through screening miRNAs expression profile in PC9and PC9GR using microarray, and the effects and mechanisms of miRNAs on Gefitinib sensitivity was further investigated.Method1. Using Amplification refractory mutation system (ARMS), we first investigated the EGFR mutation of PC9GR. PC9and PC9GR cell viability to different concentrations of Gefitinib was assessed using the Cell Counting Kit-8(CCK-8). After exposing to different concentrations of Gefitinib, western blot was used to explore the p-AKT and p-ERK protein expression. Using microarray and qRT-PCR, we screened the miRNAs associated with Gefitinib sensitivity. CCK8、 Transwell assays、western blot and qRT-PCR were performed to investigate the effect of miR-181a on PC9and PC9GR.2. To investigate the expression difference between NSCLC tumor tissues and adjacent non-tumor tissues and the association of miR-181a and NSCLC clinical characteristics, the expression of miR-181a was examined by qRT-PCR in47NSCLC patients.3. Using bioinformatics analysis、qRT-PCR and western blot, we found potential human target mRNA of miR-181a. To construct luciferase reported plasmids, the3’UTR sequence of GAS7and mutant were inserted into the luciferase vector, respectively. After miR-181a mimics or miR-181a inhibitor was co-transfected with reporter plasmids, the luciferase activity was assessed to explore if miR-181a direct target the3’UTR of GAS7.4. Western blot was performed to investigate the GAS7protein expression between PC9and PC9GR. In addition, the effect of siRNA-GAS7on PC9and the effect of pcDNA3.1-GAS7on PC9GR were also investigated by western blot. BrdU、Flow Cytometry、IHC and western blot were performed to investigate the effect of GAS7over-expression after transfection of miR-181a mimics in PC9. The same methods were performed to investigate the effect of GAS7under-expression after transfection of miR-181a inhibitor in PC9GR.5. To investigate the association of GAS7and NSCLC clinical characteristics, IHC method was performed to assess the GAS7protein expression in59NSCLC tumor formalin-fixed paraffin-embedded tissues. Univariate analysis and multivariate analysis was performed to explore the prognostic factors in NSCLC patients. Using Oncomine and GEO bioinformatics analysis, we evaluated the GAS7mRNA expression in NSCLC tumor tissues and normal tissues, and the correlation between GAS7and Gefitinib resistance associated gene (VEGF, MET and PTEN) was further analyzed in GEO datasets.Results1. miR-181a regulates Gefitinib sensitivity in PC9and PC9GR cells ARMS assay demonstrated that PC9GR cell retained the delE746-A750 mutation and did not contain T790M mutation. CCK8assay showed that the proliferation of PC9was significantly inhibited in response to exposure Gefitinib, while PC9GR did not show growth suppression in response to Gefitinib. In contrast with the parental PC9cell line, PC9GR cells maintained phosphorylation of Akt and Erkl/2even upon Gefitinib. However, phosphorylation of EGFR was suppressed by Gefitinib in PC9cells as well as in PC9GR cells. Following the result of microarray between PC9and PC9GR, qRT-PCR confirmed that the expression of miR-181a in PC9GR was significantly higher than PC9cells. Transfection of miR-181a mimics in PC9reduced the sensitivity to Gefitinib, and enhanced the migration and invasion ability of PC9cells, while transfection of miR-181a inhibitor in PC9GR led to a dramatic increase in Gefitinib sensitivity, and depressed the migration and invasion ability of PC9GR cells. Transfection of miR-181a mimics in PC9activated the phosphorylation of Akt and Erkl/2, and induced the expression of makers associated with protumor metastasis. However, transfection of miR-181a inhibitor in PC9GR suppressed the phosphorylation of Akt and Erkl/2, and reduced the expression of makers associated with protumor metastasis.2. The expression and significance of miR-181a in NSCLCMiR-181a expression was investigated in47NSCLC by qRT-PCR.36out of47NSCLC (76.6%) showed increased miR-181a expression in their tumor tissues than paired normal tissues. Relationship between miR-181a expression and clinicopathological characteristics was investigated. High miR-181a expression showed significant correlations with cigarette smoke (P=0.0498), T2-4stage (P=0.0458), p53(P=0.0209) and Ki67(P=0.0222). There were no significant correlations between miR-181a expression and age, gender, differentiation, pathology, lymph node involvement and TNM-stage (P>0.05).3. GAS7is a direct target of miR-181aGAS7was predicted as a potential target of miR-181a by TargetScan and miRanda. qRT-PCR and western blot showed that transfection of miR-181a mimics in PC9reduced GAS7expression, while transfection of miR-181a inhibitor in PC9GR enhanced GAS7expression. We found that transfection of pMIR-reporter-GAS73’UTR along with miR-181a mimics or miR-181a inhibitor resulted in significant change in reporter activity. However, no change of reporter activity was observed after transfection of mutant pMIR-reporter-GAS73’UTR along with miR-181a mimics or miR-181a inhibitor. Using qRT-PCR and western blot method, analysis of matched tumors for expression of miR-181a and GAS7showed a significant and inverse correlation between miR-181a and GAS7.4. miR-181a regulates Gefitinib sensitivity in PC9and PC9GR through targeting GAS7Western blot analysis showed that the protein expression of GAS7in PC9was significantly higher than PC9GR. In PC9cells, elevated expression of miR-181a reduced the sensitivity to Gefitinib, resulted in activation of p-AKT and p-ERK, and enhanced the expression of markers associated with protumor metastasis. However, over-expression of GAS7could rescue the effect of up-regulation of miR-181a in PC9cells. In PC9GR cells, knockdown of miR-181a increased the sensitivity to Gefitinib, suppressed the expression of p-AKT and p-ERK, and decreased the expression of markers associated with protumor metastasis. However, down-regulation of GAS7could rescue the effect of knockdown of miR-181a in PC9GR cells.5. The expression and significance of GAS7in NSCLCRelationship between GAS7protein expression and clinicopathological characteristics was investigated. High miR-181a expression showed significant correlations with adenocarcinoma (P=0.001) and TNM-I stage (P=0.033). There were no significant correlations between GAS7expression and age, gender, differentiation, tumor size and lymph node involvement (P>0.05). Kaplan Meier analysis showed that the high LOX expression showed a favorable impact on NSCLC patients overall survival (P=0.0315). Multivariate analysis showed that TNM stage was an independent prognostic factor for overall survival of NSCLC patients. Then we recapitulated GAS7mRNA expression in the large cohort of NSCLC patients that available from Oncomine and GEO database, and7out8studies showed that the expression of GAS7mRNA in NSCLC tumor tissues was significantly lower than normal tissues (P<0.001). Moreover, the expression of GAS7was significantly correlated with MET (rpetason=-0.205, P=0.0020), PTEN (rpearson=0.431, P<0.0001) and VEGF (rpearson=-0.264, P<0.0001) in GSE31210database.Conclusion1. MiR-181a was upregulated in PC9GR compared to PC9cells, and upregulation of miR-181a contribute to Gefitinib acquired resistance. MiR-181a regulates PC9and PC9GR cell proliferation and metastasis through ERK and AKT pathway.2. Upregulation of miR-181a may play an important role in development and progression of NSCLC.3. GAS7is a direct target of miR-181a, and miR-181a regulates Gefitinib sensitivity in PC9and PC9GR through targeting GAS7.4. Downregulation of GAS7, which was significantly associated with poorer prognosis in NSCLC, may play an important role in development and progression of NSCLC. The potential mechanism of GAS7in Gefitinib acquired resistance may correlate with MET, PTEN and VEGF.