Study On The Role Of MiR-873 In The Reversing Effect Of Intercalated Treatment In Lung Cancer Patients With Acquired Resistance To EGFR Tyrosine Kinase Inhibitors

Background and Objective:The incidence and mortality of lung cancer are very high all over the world.Non-small cell lung cancer(NSCLC)accounts for more than 80% of all lung cancers.Epidermal growth factor receptor(EGFR)is often overexpressed and abnormally activated in patients with non-small cell lung cancer(NSCLC).In addition,EGFR signaling pathway plays an important role in the occurrence,development,metastasis and apoptosis of NSCLC.Epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKI)are effective drugs for the treatment of advanced NSCLC.However,almost all patients initially effective after EGFR-TKI treatment will eventually obtain EGFR-TKI acquired resistance,which will affect the patient's survival.Recent studies have shown that the mechanism of acquired resistance to EGFR-TKI in NSCLC includes secondary mutation of T790 M in exon 20 of EGFR,activation of alternative genes represented by MET(mesenchymal epithelial transition factor)and HER2(human epidermal growth factor receptor-2),mutation of PIK3CA(phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha),epithelial-to-mesenchymal transition(EMT)and histology types of tumor transformed into small cell lung cancer.In addition,the resistance mechanism of 15%-30% patients still remains unknown.In the present clinical practice,there are no uniform and widely accepted regimens for the second-line treatment of NSCLC after acquired resistance to EGFR-TKI.It has been reported that in patients with acquired EGFR-TKI-resistant NSCLC,subsequent administration of pemetrexed chemotherapy can reverse the resistance of TKI and significantly improve patient survival.However,the specific mechanism,indications and optimal timing of this treatment regime are still unclear.This study was firstly designed to investigate the effects of intercalated treatment on efficacy and survival of advanced NSCLC patients with acquired resistance to the first-line EGFR-TKIs.The second purpose of this study was to explore the potential role and possible regulatory mechanisms of miR-873 in EGFR-TKIs acquired resistance of non-small cell lung cancer and its possible action molecular mechanisms.Finally,this study will explore the role of pemetrexed in EGFR-TKI acquired resistant cell lines in human lung cancer to reveal the specific mechanism by which intercalated treatment may reverse EGFR-TKI resistance.Part ? Clinical observation of intercalated treatment in advanced NSCLC patients with acquired resistance to the first-line EGFR tyrosine kinase inhibitorsMethods:A total of 103 advanced NSCLC patients who received first-line EGFR-TKI treatment in the First Affiliated Hospital of Nanjing Medical University from January 2013 to April 2017 were investigated retrospectively.According to the strategy of second-line treatment after TKI progression,the patients were divided into one group of 52 patients that receive pemetrexed chemotherapy intercalated with TKI treatment(hereinafter referred to as intercalated treatment group)and the other group of 51 patients treated with chemotherapy alone(hereinafter referred to as chemotherapy group).The treatment efficacy,progression-free survival(PFS)and overall survival(OS)of both groups were analyzed.Some patients were tested for T790 M mutation in peripheral blood after acquiring EGFR-TKI resistance.According to the test results,the cases were categorized into T790 M positive and negative subgroups and the efficacy was accordingly analyzed.Results:All 103 patients could evaluate the efficacy of second-line therapy after acquired resistance to first-line EGFR-TKI.The overall response rate(ORR)was 30.8% and 19.6%(P=0.192)and the disease control rate(DCR)was 76.9% and 52.9%(P = 0.011)respectively in two groups.The DCR in intercalated treatment group was significantly higher than that in chemotherapy group.Median PFS was 8 months and 6 months(P = 0.003)in intercalated treatment group and chemotherapy group respectively.Cox multivariate survival analysis showed that the PFS in intercalated treatment group was significantly superior to that in chemotherapy group(HR = 0.380,95% CI = 0.222-0.651,P = 0.000).The median OS was 17 months and 11 months in intercalated treatment group and chemotherapy group(P = 0.001)respectively.Cox multivariate survival analysis showed that OS in intercalated treatment group was significantly superior to that in chemotherapy group(HR = 0.319,95% CI = 0.189-0.537,P = 0.000).Among T790 M mutation negative patients,the DCRs were 100.0% and 50.0%(P <0.001)for intercalated treatment group and chemotherapy group respectively,with a median OS of 19 months and 11 months(P = 0.020)respectively.DCR and OS in intercalated treatment group were significantly superior to those in chemotherapy group.Part ? Downregulation of MiR-873 Increases the EGFR-TKI Acquired Resistance via Targeting Glioma-Associated Oncogene Homolog 1 in Non-small Cell Lung CancerMethods:1.Human non-small cell lung cancer cells(NSCLC)including EGFR-TKI resistance cell line PC9/GR and EGFR-TKI sensitive cell lines PC9 were cultured in this study.We used quantitative real-time PCR(q PCR)to detect the expression of miR-873 and GLI1 in these two cell lines.The expression correlation of miR-873 and GLI1 was also analyzed.2.MiR-873 inhibitor was transfected into PC9 cells.The expression of GLI1 was detected by q PCR and Western Blot assays.3.PC9 cells were transfected withmiR-873 mimics and GLI1-3 'UTR(or GLI1-3 'UTR mut)luciferase reporter vectors.The luciferase activity was examined by dural luciferase reporter gene detection system.4.PC9 cells were treated with gefitinib,and the cells was transfected with miR-873 inhibitor.The cell viability,angiogenesis and cell proliferation of PC9 cell line was detected by CCK-8 test,angiogenesis tube formation assay and flow cytometry analysis,respectively.5.The expression of GLI1 was detected by q PCR and Western Blot assays.Result:1.The expression of miR-873 was higher in PC9 cells than that in PC9/GR cells(P<0.05).The m RNA expression of GLI1 was lower in PC9 cells than that in PC9/GR cells(P<0.05).The expression level of miR-873 was negatively related with the expression of GLI1 in the non-small cell lung cancer cells.2.The expression of miR-873 in lung cancer PC9 cells was significantly down-regulated at the presence of miR-873 inhibitor.Results from q PCR revealed that the expression of GLI1 in lung cancer PC9 cells was significantly up-regulated at the presence of miR-873 inhibitor.The protein expression of GLI1 in lung cancer PC9 cells was increased in miR-873 inhibitor group.The difference was statistically significant(P<0.05).3.GLI1 was a target of miR-873.The results from dural luciferase reporter assay showed that the activity of luciferase was significantly declined after transfection with miR-873 mimics in GLI1 3'UTR-WT luciferase reporter assay,and the difference was statistically significant(P<0.05).Compared with the negative control group,the activity of luciferase was not changed after transfection withmiR-873 mimics in GLI1 3'UTR-mut luciferase reporter assay(P>0.05).4.Down-regulation of miR-873 promoted PC9 cell viability.The viability of transfected PC9 cells was determined by CCK-8 assay.The cell viability of PC9 cells in miR-873 inhibitor + gefitinib group was higher than that in normal control group and gefitinib group(P<0.05).5.Down-regulation of miR-873 enhanced the angiogenesis of PC9 cells.The tube formation of PC9 cells in miR-873 inhibitor + gefitinib group were significantly higher than that in the normal control group and the gefitinib group(P<0.05).6.Cell proliferation of PC9 cells was increased by the downregulation of miR-873.The number of proliferative cells in miR-873 inhibitor + gefitinib group was significantly higher than that in the normal control group and the gefitinib group(P<0.05).7.Down-regulation of miR-873 up-regulated GLI1 expression in PC9 cells.The expression of Gli1 in miR-873 inhibitor + gefitinib group was obviously higher than that in the normal control group and the gefitinib group(P<0.05).Part ? Effect and Mechanism of Pemetrexed on Human Lung Cancer EGFR-TKI Acquired Resistant Cell LinesMethods:1.Pemetrexed was applied to human lung cancer EGFR-TKI acquired drug-resistant cell line PC-9/GR for 48 h,and the pemetrexed drug was eluted.The drug-resistant cell lines were compared with therapeutic sensitivity of EGFR-TKI before and after pemetrexed treatment.2.Western blot was used to detect the effect of pemetrexed on the expression of GLI1 protein in lung cancer cells(including EGFR-TKI resistant cells PC-9/GR and EGFR-TKI sensitive cells PC-9),and q PCR was used to detect the changes of miR-873 expression before and after the effect of pemetrexed.Result:The EGFR-TKI-resistant cell PC-9/GR treated with pemetrexed was re-sensitive to EGFR-TKI therapy.Pemetrexed inhibited the expression of GLI1 protein in EGFR-TKI-resistant cells and up-regulated miR-873 expression.Conclusion:Firstly,patients with advanced NSCLC and acquired resistance to the first-line EGFR-TKI were advised to accept T790 M mutation and other resistance gene test.Pemetrexed chemotherapy intercalated with TKI treatment should be given to T790 M negative patients with the efficacy better than chemotherapy alone,in addition such therapy strategy may reverse TKI resistance with well tolerance.Secondly,the expression of miR-873 and GLI1 in NSCLC cells was negatively correlated and GLI1 is the target gene of miR-873.Downregulation of miR-873 in lung cancer PC9 cells enhanced the expression of GLI1 and inhibited the efficiency of gefitinib.Downregulation of miR-873 increased the cell viability,angiogenesis and cell proliferation of PC9 cells.Finally,pemetrexed can up-regulate the expression of miR-873 in lung cancer PC-9/GR cells,leading to a decrease in the expression of GLI1,which may reverse the EGFR-TKI acquired resistance of lung cancer PC-9/GR cells.This also provides a theoretical basis for intercalated treatment to reverse the acquired resistance of EGFR-TKI in NSCLC.