Effects Of AXL In Non-small Cell Lung Cancer And Its Relationship With EGFR Pathway

Lung cancer is the leading cancer killer in China and worldwide.Non-small cell lung cancer(NSCLC)is the major type of lung cancer,mainly consists of two major histological subtypes: squamous cell carcinoma and adenocarcinoma.Epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKI)are effective therapies for NSCLC patients whose tumors harbor somatic mutations in EGFR.However,the emergence of drug resistance has become a challenge for genotype-directed targeted therapy in NSCLCs.Resistance to targeted therapies is generally classified as either primary or acquired.It is therefore critical to develop new insights into the mechanisms of TKI resistance to develop more effective treatment strategies.AXL is a member of the TAM(Tyro3,Axl,and Mer)family of receptor tyrosine kinases(RTKs).From the first separation obtained from chronic myeloid leukemia patients,AXL has been found overexpressed in a variety of human cells.Recently,AXL has been a focus of research in modulating resistance to conventional and targeted therapies for several types of cancers.In NSCLC,it is reported that AXL upregulation is the second most common mechanism of EGFR-TKI acquired resistance in NSCLC specimens obtained from patients upon the development of acquired resistance.In addition,the basal level of AXL was also associated with the primary resistance to EGFR-TKI in NSCLC cells harboring wild-type EGFR.Therefore,it is of great significance to explore the mechanisms of AXL underlying intrinsic or acquired resistance to EGFR-TKI treatment in NSCLC.However,the relationship between 2 receptor tyrosine kinases,AXL and EGFR,the relevance of AXL expression with EGFR mutation status in treatment-naive human NSCLCs remain uncertain.In addition,whether AXL inhibition can increase the sensitivity to EGFR-TKI in NSCLC cells(EGFR wild-type)need to be explored further.Our present study included three sections.In section 1,we aimed to investigate the relationship of AXL and EGFR signaling in EGFR wild-type and EGFR-mutant lung adenocarcinoma patients by determining EGFR-activating mutation status(exon 19 deletions and exon 21 L858R),and the expressions of AXL,EGFR,p EGFR1068 expression in Formalin-fixed and paraffin-embedded(FFPE)sections of 109 lung adenocarcinoma patients.In section 2,we aimed to detect the biological effects of AXL on cell proliferation,cell cycle distribution,apoptosis,as well as cell motility in NSCLC cell lines(A549 and H1299).Furthermore,the possible role of AXL on the downstream signaling of EGFR(AKT and ERK)was also observed.In section 3,we next tested the efficacy of AXL on the response to EGFR-TKI and docetaxel in A549 and H1299 cells.Part one Correlation analysis of receptor tyrosine kinases AXL and EGFR pathway in primary lung adenocarcinomaObjective: To investigate the coexpression pattern of AXL and EGFR signaling in EGFR wild-type and EGFR-mutant lung adenocarcinoma patients by determining the common EGFR-activating mutation status in 109 lung adenocarcinoma patients;evaluating the clinicopathologic significance of AXL,EGFR,and p EGFR1068 expression in the 109 initial tumor specimens using immunohistochemical studies;and investigating the possible relationships between AXL and EGFR signals in EGFR-mutant and wild-type patient cohorts.Methods:1 Patients and clinical specimens:Surgical specimens of 109 patients with primary lung adenocarcinoma who were not treated with radiotherapy or chemotherapy before operation were collected.The specimens were fixed by 10% neutral formalin and paraffin-embedded(FFPE).2 Genomic DNA extraction:Genomic DNA from 109 FFPEs was extracted using a TIANamp FFPE DNA Kit according to the manufacturer’s instructions.3 PCR and sequencing analysis of EGFR exons 19 and 21 mutation:19 and 21 of the EGFR gene were amplified using a nested PCR method.The PCR products were analyzed by Sanger sequencing analysis with an ABI 3730 XL DNA analyzer.This part was provided by Invitrogen(Contract No.MB140120001B).4 ARMS PCR(amplification refractory mutation specific PCR):Human EGFR gene mutation detection kit(ADx-EG10,Amoy Diagnostics Co,LTD,China)was used to detect the EGFR mutation in 109 cases of lung cancer tissues.The kit designed for detection of 4 EGFR mutations including Ex19-mutant-1: E746_A750del(2235-2249 del15),Ex19-mutant-2: E746_A750del(2236-2250 del15),Ex21-mutant-1: L858R(2573T>G),and Ex20-mutant-1: T790M(2369C>T).5 Immunohistochemical staining:Immunohistochemical staining was used to detect the expression of AXL,EGFR and p EGFR1068 in paraffin embedded sections of 109 patients with lung adenocarcinoma.And the relationships between the expression and clinicopathological features were analyzed.6 Statistical analysis:Chi-squared tests were used to examine possible correlations between the expression of AXL,EGFR and p EGFR1068 and the clinicopathologic parameters.Spearman’s correlation analysis was used to determine the correlation between mutation and wild-type groups.Results:1 EGFR mutation analyses in 109 human lung adenocarcinoma patients 1.1 PCR direct sequencing:Exon 19 deletions were found in 25 of 109(22.9%)cases.Among these mutations,E746-A750 del was the most common type and accounted for 52.0%(13/25)of all exon 19 deletions.In addition,the L858 R point mutation on exon 21 was detected in 22 of 109(20.2%)patients.Together,the mutation rate of EGFR on exons 19 del and 21 L858 R was 43.1%(47/109),as determined by PCR direct sequencing.1.2 ARMS PCRIn order to avoid false negative results,we next used ARMS analysis to detect mutations in the 62 patients who were negative for EGFR mutations based on the PCR direct sequencing method.21(33.9%)patients were positive for EGFR mutation by ARMS assay.Combining the results of PCR direct sequencing and ARMS assays,we found that the overall frequency of the most common types of EGFR19 del and 21 L858 R mutations in human lung adenocarcinomas was 62.4%(68/109)in our study.In addition,only two cases(1.83%)harboring the T790 M mutation in the 109 lung adenocarcinomas tested.2 AXL,EGFR and p EGFR1068 expression in human lung adenocarcinoma and their relationships with clinicopathologic characteristics2.1 AXL protein expression:Immunohistochemical results showed that in 109 cases of lung adenocarcinoma,positive AXL staining was observed in 60 of 109(55%)tumor tissues,and its expression was significantly associated with lymph node metastasis(P=0.035).But there was no correlation between AXL expression and age,sex,tumor size,TNM stage,and adenocarcinoma subtypes.2.2 EGFR protein expression68 of 109(62.4%)lung adenocarcinomas showed positive EGFR expression.Further analyses demonstrated that the expression of EGFR was significantly associated with histological subtype(P=0.02),tumor size(P<0.001)and metastasis(P=0.007).But there was no correlation between EGFR expression and age,sex,TNM stage.2.3 p EGFR1068 expressionIn 109 cases of lung adenocarcinoma,the positive expression rate of p EGFR1068 protein was 52.3%(57/109).The expression of p EGFR1068 in the case of tumor diameter >3cm was significantly higher than that of diameter≤3cm(P<0.001);The expression of p EGFR1068 in lymph node metastasis group was significantly higher than that without lymph node metastasis(P<0.001).3 Relationships between AXL and EGFR/p EGFR1068 expression and EGFR19 del and/or L858 R mutation status23 patients were detected to have the EGFR19 del mutation,77 cases were 19 del negative;34 had the L858 R point mutation,75 cases were L858 R negative;68 patients were detected to have the EGFR19 del or L858 R mutation in a total,and 41 cases were negative.3.1 Correlation between AXL expression and mutationThe association between AXL expression and 19 del was of borderline statistical significance(P=0.05).There was no correlation between AXL expression and L858 R mutation alone or both the 19 del and L858 R mutations(P>0.05).3.2 Correlation between EGFR expression and mutationThe positive rate of EGFR expression was significantly higher in patients with the exon 21 L858 R mutation(76.5%,26/34)than in those without the L858 R mutation(56.0%,42/75;P=0.04).There was no correlation between EGFR expression and L858 R mutation alone or both the 19 del and L858 R mutations.3.3 Correlation between p EGFR1068 expression and mutation:In 34 cases with L858 R mutation alone,the positive expression rate of p EGFR1068 was 73.5%(25/34),which was significantly higher than that in L858 R negative cases(42.7%,P=0.003).The positive expression rate of p EGFR1068 in 68 patients with Del/L858 R mutation was significantly higher than that in the negative mutation group(60.3% v.s.14.6%,P=0.031)4 The co-expression pattern of AXL,EGFR and p EGFR1068In the 109 patients,two with tumor samples harboring EGFR T790 M mutations were eliminated from further analysis.Among the 107 lung adenocarcinoma patients,37 exhibited the co-expression of AXL and EGFR,30 patients exhibited co-expression of AXL and p EGFR1068,and 25 patients exhibited co-expression of AXL,EGFR and p EGFR1068.Further analysis indicated that the percentage of AXL+/EGFR+/p EGFR1068+co-expression in EGFR-mutation patients was significantly higher than that of EGFR wild-type patients(30.9% vs.10.3%,P=0.015).Summary:1 In 109 cases of lung adenocarcinoma,the incidence of EGFR exon 19 deletion and exon 21 L858 R mutation was higher(62.4%),while the primary mutation of exon 20 T790 M was only about 1.83%.2 In 109 cases of lung adenocarcinoma,the expression of AXL in lymph node metastasis group was significantly higher than that without lymph node metastasis;the expression of EGFR was significantly associated with histological subtype,tumor size and metastasis;the expression of p EGFR1068 was significantly correlated with tumor size(P<0.001)and lymph node metastasis.3 There was no significant correlation between AXL expression and EGFR mutation;the expressions of EGFR and p EGFR1068 were significantly correlated with exon 21 L858 R point mutation;moreover,p EGFR1068 expression was significantly increased in patients with 19 Del or L858 R mutation,suggested that the common EGFR-activating mutation cases were associated with EGFR phosphorylation on Tyr-1068.4 The co-expression rate of AXL+/EGFR+/p EGFR1068+ in EGFR mutant group was significantly higher than that in wild-type lung adenocarcinoma patients.Part two The biological effects of AXL in Non-Small cell lung cancer cells in vitroObjective: In the present study,we aimed to explore the biology effect of Axl in lung cancer by AXL inhibition using siRNA interference or Axl kinase inhibitor R428 in A549 and H1299 cell in vitro.The possible founction of AXL on cell proliferation,cycle distribution,cell apoptosis,cell migration,as well as related signaling pathways were investigated in A549 cell and H1299 cell.At the same time,the possible effect of AXL on the downstream of EGFR signaling pathway-ERK/p-ERK and AKT/p-AKT was observed.Methods:1 Cell culture and treatmentHuman NSCLC cell lines A549 and H1299,were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum,100U/m L penicillin and 100U/m L streptomycin.Both of the cells were incubated in a humidified atmosphere of 5% CO2 in air at 37?C.2 R428 treatment2×104/m L cells were inoculated on the surface of the 96 plates,starved for 24 h by cultured in RPMI-1640 culture medium containing 1% serum,followed by different concentration of R428 treatment for additional 24 h.The half inhibitory concentration(IC50)was calculated.3 siRNA transfectionH1299 and A549 cells were transfected using 100 pmol AXL specific siRNA or negtive control siRNA(NC siRNA),cells were collected for subsequent experiments after transfection.4 MTT assay2×104/m L cells were inoculated on the surface of the 96 plates,12 h after plated,cells were transfected with NC siRNA or AXL siRNA or treated with R428.MTT colorimetric method was used to calculate the cell survival rate.5 Real-time PCRTotal RNA was extracted from the above cells.The expression of AXL m RNA was detected by Real-time PCR.The relative expression level of the target gene was calculated using the comparative Ct(ΔΔCt)method provided by Applied Biosystems.6 Western Blot assay:Total protein was extracted from cells in each group,and the expressions of AXL,EGFR/p EGFR,ERK/p-ERK,AKT/p-AKT were detected by Western blot assay.7 Cell cycle distribution detection by flow cytometryCells were collected after siRNA transfection or R428 treatment,digested to single cell suspension,fixed in 70% ethanol at 4℃ overnight.Cell cycle distribution was detected by flow cytometry after stainnd with PI solution.Data was analyzed using Flow Jo 7.6.Each experiment was repeated three times.8 Apoptosis detection by flow cytometryCells from NC siRNA or AXL siRNA transfected group,control group and R428 treated group were collected.The apoptosis rates of the cells were determined by flow cytometry using Annexin-V/PI kit.Flow Jo 7.6 software was used for data analysis.9 Transwell assay2×104 cells were plated in medium without serum in the top chamber of a transwell.Cells that had migrated to the lower surface of the membrane were fixed with formalin,stained with crystal violet,and photographed under microscope.Cell numbers were counted under a light microscope.Experiments were carried out at least three times.10 Wound healing assayThe growth of cells on both sides of the scribing line was observed within 0-48 h after scratches,and the cell migration was observed under microscope.Each experiment was repeated three times.11 Statistical analysisSPSS Statistics 19.0 software was used to analyze the statistical significance.The measurement data are expressed as mean ± SD.Statistical comparisons between experimental groups were analyzed by χ2,two-sample t-test and straight line fitting.P<0.05 was taken to indicate statistical significance.Results: 1 The biological effects of AXL in A549 and H1299 cells transfected by siRNA1.1 The interference efficiency of AXL siRNABoth the Western and q PCR results showed that the three siRNA sequences can significantly inhibit the expression of AXL at protein and m RNA level,respectively.We just selected one of the siRNA1(AXL siRNA)for subsequent experiments.1.2 Effects of AXL siRNA interference on cell growthThe cell survival rate of A549 cells and H1299 cells transfected with AXL siRNA was decreased compared with the negative control group,and there was statistical significance(P < 0.05)1.3 Effects of AXL siRNA interference on cell cycleThe percentage of G0/G1 phase of A549 cell transfection by AXL siRNA group was(86.10±0.10)%,which was significantly higher than that of negative control group(74.15±0.25)%,and the proportion of S phase and G2/M phase was significantly lower than that of control group(P<0.05).However,knockdown of AXL expression by siRNA transfection had no significant effect on H1299 cell cycle distribution.1.4 Effects of AXL siRNA interference on cell apoptosisCompared with the negative control group,the percentage of apoptosis in A549 cells transfected with AXL siRNA was increased,but there was no statistically significant(P>0.05).In H1299 cells,the apoptosis rate was increased significantly in AXL siRNA transfected group compared with the NC group(P<0.05).1.5 Effects of AXL siRNA interference on cell migrationMonolayer wound healing assay and Transwell assay were performed to observe cell ability of migration.In A549 and H1299 cells,the number of cells migrated into the lower chamber was significantly higher in AXL siRNA group than that in NC group(P<0.05),respectively.In addition,wound healing assay results showed that AXL siRNA transfection group A549 cell scratch healing area(27.43±0.01%)was significantly lower than the control group(85.67±0.02)%,the difference was statistically significant(P<0.05).Similarly,AXL siRNA transfection group H1299 cell scratch healing area was significantly lower than the control group cells.The results showed that inhibition of AXL expression could decrease the migration ability of A549 cells and H1299 cells.2 The biological effects of specific AXL kinase inhibitor R428 on A549 and H1299 cells.2.1 Effects of R428 on cell growthMTT assay showed that the survival rates of both cells were decreased gradually with R428 concentration increasing.Statistical analysis showed that the IC50 of R428 in A549 and H1299 cells was 28.98 n M and 34.71 n M,respectively.We chose R428 to use the concentration of 20 n M in the follow-up experiment.2.2 Effects of R428 on cell cycleCompared with the control group,the percentage of A549 cells in the R428 treated group was significantly increased(R428:(88.37±0.15)% V.S.(68.60±0.20)%),while the proportion of S and G2/M cells decreased significantly(P<0.05).In addition,the distribution of H1299 cell cycle in R428 treatment group was consistent with the change of A549 cells.The results showed that 20 n M R428 treatment could induce G0/G1 phase arrest in A549 and H1299 cells.2.3 Effects of R428 on cell apoptosisCompared with the control group,the apoptosis rate of A549 cells was not significantly changed,however,the apoptosis rate of H1299 cells was significantly increased by treatment with 20 n M R428(P<0.05).2.4 Effects of R428 on cell migrationTranswell test showed that: In R428 treated group,A549 and H1299 cells in lower chamber were much less than that in control group(P<0.05).Wound healing assay showed that: the healing area of A549 cells in the R428 treated group(15.78±0.01%)was significantly lower than that in the control group(92.45±0.10%),and the difference was statistically significant(P<0.05).In addition,the healing area of H1299 cells in the R428 treated group was significantly lower than that in the control group.3 Effects of inhibition of AXL expression on ERK/p-ERK and AKT/p-AKT levelsCells were divided into 4 groups: NC siRNA,AXL siRNA,NC siRNA+EGF and AXL siRNA+EGF.Wstern Blot results showed that the level of p-AKT was significantly decreased after transfection with AXL siRNA in A549 and H1299 cells,but the levels of EGFR,p-EGFR,ERK and p-ERK did not change significantly.After EGF stimulation,the level of p-EGFR in cells was significantly increased,which confirmed that the EGFR pathway was activated.At the same time,the levels of p-ERK and p-AKT in the downstream of EGFR were also increased in certain extent.Compared with EGF alone treatment group,the level of p-AKT was significantly inhibited after AXL siRNA transfection combined with EGF stimulation.The above results indicated that AXL was involved in the actication of EGFR downstream signaling,and p-AKT might be the cross point between AXL and EGFR pathway.Summary:1 Downregulation of AXL expression by AXL siRNA transfection could significantly inhibit the proliferation of A549 and H1299 cells,which might be related to G0/G1 phase arrest or apoptosis.2 AXL kinase inhibitor-R428(20n M)could induce cell cycle arrested at G0/G1 phase,and then inhibited cell survival both in A549 and H1299 cells.3 AXL siRNA transfection or R428 treatment could significantly inhibit the migration ability of A549 cells and H1299 cells.4 The expression of p-AKT in A549 and H1299 cells was significantly inhibited by knockdown of AXL expression by transfection with AXL siRNA.p-AKT is a common downstream signaling molecule of AXL and EGFR,and the regulation of AXL on p-AKT may be the crossing point between AXL kinase and EGFR downstream pathway.Part three The effect of AXL on the response of lung cancer cells to Gefitinib and DocetaxelObjective: A549 cells and H1299 cells transfected with AXL siRNA or treated by R428 were intervened by docetaxel or gefitinib,respectively.The effect of AXL on the response of lung cancer cells to chemotherapeutic drugs was observed,from the aspects of cell growth,cell cycle and cell apoptosis,further understanding of the Axl specific role.Methods:1 Cell culture and siRNA transfectionHuman NSCLC cell line A549 and H1299 were used,and the protocol for cell incubation and siRNA transfection were tha same as in part two.2 siRNA cell transfection combined with drug treatment cells and grouping2.1 siRNA cell transfection combined with gefitinibThe cells were divided into Negative control siRNA(NC group),AXL siRNA,NC+gefitinib,and AXLsiRNA+gefitinib four groups.The cells were cultured and transfected according to the above method,in 24 hours after transfection,add gefitinib(10n M),continue for 24 hours to collect cells for the relevant experiments.2.2 siRNA cell transfection combined with DocetaxelThe cells were divided into Negative control siRNA(NC group),AXL siRNA,NC+Docetaxel,and AXLsiRNA+Docetaxel four groups.The cells were cultured and transfected according to the above method,in 24 hours after transfection,add Docetaxel(20n M),continue for 24 hours to collect cells for the relevant experiments.3 R428 combined with drug treatment:3.1 R428 combined with gefitinibThe cells were divided into four groups: control group,R428 treatment group,gefitinib(10n M)group,R428 combined with gefitinib group.A549 cells and H1299 cells were taken and digested with 1.5m L trypsin,dissociated into single cell suspension with RPMI-1640 complete medium,2×104/m L cells were inoculated on the surface of the 6 holes.The RPMI-1640 culture medium containing 1% serum was replaced after adherent,starved cells after 24 h,add R428(20n M)was to 24 h,and tadd Gefitinib to 24 h,then collect the cells in each group for subsequent detection.3.2 R428 combined with DocetaxelThe cells were divided into four groups: control group,R428 treatment group,Docetaxel(20n M)group,R428 combined with Docetaxel group.A549 cells and H1299 cells were taken and digested with 1.5m L trypsin,dissociated into single cell suspension with RPMI-1640 complete medium,2×104/m L cells were inoculated on the surface of the 6 holes.The RPMI-1640 culture medium containing 1% serum was replaced after adherent,starved cells after 24 h,add R428(20n M)was to 24 h,and tadd Docetaxel to 24 h,then collect the cells in each group for subsequent detection.4 MTT assay2×104/m L cells were inoculated on the surface of the 96 holes,and the adherent cells were cultured for 12 hours.Cells were transfected with different treatment measures according to the above grouping.MTT colorimetric method was used to calculate the cell survival rate.5 Cell cycle distribution detection by flow cytometryCell cycle distribution were detected by flow cytometry after stained with PI solution,data was analyzed using Flow Jo 7.6.Each experiment was repeated three times.6 Apoptosis detection by flow cytometryThe apoptosis rates of the cells were determined by flow cytometry using Annexin-V/PI kit.Flow Jo 7.6 software was used for data analysis.7 Statistical analysisSPSS Statistics 19.0 software was used to analyze the statistical significance.The measurement data are expressed as mean ± SD.The data were analyzed by ANOVA or T test.P<0.05 was taken to indicate statistical significance.Results:1 Effects of AXL siRNA transfection combined with gefitinib on the growth of A549 and H1299 cells and its mechanism1.1 MTT detectionIn A549 and H1299 cells,compared with NC group,the survival rate of AXL siRNA group,NC+gefitinib group and AXL siRNA+gefitinib group was significantly decreased(P<0.05).Compared with AXL siRNA group,and NC+gefitinib group,the survival rate of AXL siRNA+gefitinib group was significantly decreased(P<0.05).1.2 Cell cycle distribution was detected by FCMIn A549 and H1299 cells,compared with NC group,the proportion of G0/G1 increased in AXL siRNA+gefitinib group cells,and the proportion of G2/M cells and S phase decreased(P<0.05);NC+gefitinib treatment had no significant effect on cell cycle.Comparison among groups: The proportion of G0/G1 phase in AXL siRNA+gefitinib group was significantly higher than that in NC+gefitinib group(P<0.05).1.3 Cell apoptosis was detected by FCMIn A549 and H1299 cells,compared with NC group,the apoptosis rate of NC+gefitinib group and AXL siRNA+gefitinib group were significantly increased(P<0.05).Comparison among groups: the apoptosis rate of AXL siRNA+gefitinib group was significantly higher than that of AXL siRNA group and NC+gefitinib group(P<0.05).2 Effects of R428 combined with gefitinib on the growth of A549 and H1299 cells and its mechanism2.1 MTT detectionIn A549 and H1299 cells,compared with the control group,the survival rate of R428 group,gefitinib group and R428+gefitinib group was decreased(P<0.05).Comparison among groups: the survival rate of R428+gefitinib group was the lowest,which was significantly lower than that of gefitinib group and R428 group(P<0.05).2.2 Cell cycle distribution was detected by FCMIn A549 and H1299 cells,compared with the control group the proportion of G0/G1 phase in R428 group and R428+gefitinib group increased significantly,the proportion of G2/M phase decreased(P < 0.05).Comparison among groups: the G0/G1 phase proportion of R428+gefitinib group was significantly higher than that of gefitinib group(P<0.05).2.3 Cell apoptosis was detected by FCMIn A549 and H1299 cells,compared with the control group,the apoptosis rate of gefitinib group and R428 group increased significantly(P<0.05).Comparison among groups: the apoptosis rate of R428+gefitinib group was significantly higher than that of R428 group and gefitinib group(P<0.05).3 Effects of AXL siRNA interference combined with Docetaxel on the growth of A549 and H1299 cells and its mechanism3.1 MTT detectionIn A549 and H1299 cells,compared with NC group,the survival rate of AXL siRNA group,NC+docetaxel group and AXL siRNA+docetaxel group were reduced to varying degrees(P<0.05).Comparison among groups: In A549 cells,the survival rate of AXL siRNA+docetaxel group was lower than that of NC+docetaxel group and AXL siRNA group(P<0.05).In H1299 cells,there was no significant difference between the combined treatment group and separate treatment group.3.2 Cell cycle distribution was detected by FCMIn A549 and H1299 cells,compared with NC group,the proportion of the G2/M phase in NC+docetaxel group cells increased significantly,while the proportion of G0/G1 phase cells decreased significantly(P < 0.05).Comparison among groups: In A549 cells,compared with NC+docetaxel group,the proportion of the G2/M phase decreased significantly,while the proportion of G0/G1 phase cells increased significantly(P<0.05).In H1299 cells,there was no significant difference on cell cycle between the two groups.3.3 Cell apoptosis was detected by FCMIn A549 and H1299 cells,compared with NC group,the apoptosis rate of NC+docetaxel group and AXL siRNA+docetaxel group was significantly increased(P<0.05).Comparison among groups: the apoptosis rate of AXL siRNA+docetaxel group was significantly higher than that of AXL siRNA group(P<0.05).4 Effects of R428 combined with Docetaxel on the growth of A549 and H1299 cells and its mechanism4.1 MTT detectionIn A549 and H1299 cells,compared with the control group,the survival rate of three treatment group cells(R428 group,docetaxel group and R428+docetaxel group)was significantly decreased(P<0.05).Comparison among groups: the survival rate of R428+docetaxel group was the lowest,which was significantly lower than that of docetaxel group and R428 group(P<0.05).4.2 Cell cycle distribution was detected by FCMIn A549 and H1299 cells,compared with the control group,the proportion of the G2/M phase in docetaxel group cells increased significantly(P<0.05),while the proportion of G0/G1 phase cells decreased significantly(P<0.05);In R428 group,the proportion of G0/G1 phase increased significantly,and the proportion of G2/M phase and S phase decreased(P<0.05).Comparison among groups: compared with docetaxel group,the proportion of the G2/M phase in R428+docetaxel group cells decreased significantly,and the proportion of the G0/G1 phase increased significantly(P<0.05).4.3 Cell apoptosis was detected by FCMIn A549 and H1299 cells,compared with the control group,the apoptosis rate of docetaxel group and R428+docetaxel group were significantly increased(P < 0.05).Comparison among groups: the apoptosis rate of R428+docetaxel group was significantly higher than that of R428 group(P<0.05).Summary:1 Gefitinib treatment of A549 and H1299 cells had no significant effect on cell cycle,but could promote cell apoptosis and inhibit cell proliferation.2 Docetaxel treatment of A549 and H1299 cells could induce obvious G2/M cycle arrest and play a role in inhibiting cell growth.3 The inhibition effect of AXL siRNA transfection/R428 combined with gefitinib treatment on the growth of A549 and H1299 cells was higher than that of the separate treatment group,and the inhibition might be related to cell G0/G1 phase arrest or apoptosis.4 The inhibition effect of R428 combined with docetaxel treatment on the growth of A549 and H1299 cells was higher than that of the separate treatment group,and the inhibition might be related to cell G0/G1,G2/M phase arrest or apoptosis.Conclusion:1 In 109 cases of lung adenocarcinoma,the incidence of EGFR exon 19 del and exon 21 L858 R mutation was higher(62.4%),while the primary mutation of exon 20 T790 M was only about 1.83%.2 In 109 cases of lung adenocarcinoma,the expression of AXL in lymph node metastasis group was significantly higher than that without lymph node metastasis;the expression of EGFR was significantly associated with histological subtype,tumor size and metastasis;the expression of p EGFR1068 was significantly correlated with tumor size and lymph node metastasis.3 There was no significant correlation between AXL expression and EGFR mutation;the expressions of EGFR and p EGFR1068 were significantly correlated with exon 21 L858 R point mutation;moreover,p EGFR1068 expression was significantly increased in patients with 19 Del or L858 R mutation,suggested that the common EGFR-activating mutation cases were associated with EGFR phosphorylation on Tyr-1068.4 The co-expression rate of AXL+/EGFR+/p EGFR1068+ in EGFR mutant group was significantly higher than that in wild-type lung adenocarcinoma patients.In vitro experiments further indicated that p-AKT is a common downstream signaling molecule of AXL and EGFR,and the regulation of AXL on p-AKT may be the crossing point between AXL kinase and EGFR downstream pathway.5 Downregulation of AXL by siRNA transfection or R428 could significantly inhibit the proliferation of A549 and H1299 cells,which might be related to G0/G1 phase arrest or apoptosis.At the same time,inhibition of AXL could significantly inhibit the migration ability.6 The treatment of gefitinib or docetaxel alone could significantly inhibit the growth of A549 and H1299 cells.Gefitinib treatment could promote cell apoptosis and inhibit cell proliferation,while docetaxel treatment could induce obvious G2/M cycle arrest and play a role in inhibiting cell growth.7 When Axl inhibition(AXL siRNA transfection or R428)combined with gefitinib or docetaxel treatment,the growth of A549 and H1299 cells showed synergistic inhibition effects,either by G0/G1,G2/M phase arrest or apoptosis.