To Investigate The Effect And Mechanism Of Epidermal Growth Factor Receptor-tyrosine Kinase Inhibitor On The Expression Of IL-6 In EGFR-mutant Lung Cancer Cell Lines

Background and objective: Epidermal growth factor receptor(EGFR)signaling pathway is currently an important signaling pathway for targeted therapy of human lung adenocarcinoma.EGFR-tyrosine kinase inhibitors(TKI)are widely used in the treatment of lung adenocarcinoma patients with EGFR mutations(mainly 19 del and L858R mutations),and their therapeutic effects are better than platinum-containing chemotherapy.However,after a median of about 10 months of progression free survival(PFS),most patients develop acquired resistance to it.Studies have shown that interleukin-6(IL-6)may induce lung adenocarcinoma to develop resistance to EGFR-TKI.Therefore,studying the effect of EGFR-TKI on the expression of IL-6 in EGFR-mutant lung adenocarcinoma and its mechanism may be an important way to improve the effect of EGFR-TKI targeted therapy and reduce the occurrence of acquired drug resistance.Methods: Sequencing the exons 18-24 of the EGFR tyrosine kinase region of lung adenocarcinoma cell lines(HCC827,HCC827-GR,PC9,PC9-GR,H1975);gene detection of lung adenocarcinoma cell lines.Detection of EGFR mutation sites;CCK8 method to detect the IC50 value of EGFR-TKI on lung adenocarcinoma cell lines;detect EGFR-TKI-treated lung cancer cell lines to detect the level of IL-6 mRNA with RT-qPCR;investigate PI3K/AKT signaling pathway,MAPK/ERK signaling pathway,EGFR-STAT3 pathway related proteins and phosphorylated protein changes with Western blot;flow cytometry was used to detect the changes in IL-6 expression level after EGFR-TKI treatment of lung adenocarcinoma;by ELISA Detect the effect of EGFR-TKI treatment of lung adenocarcinoma on the level of IL-6 extracellular secretion;treat lung adenocarcinoma cell lines with Stattic(STAT3 inhibitor)or Niclosamide(STAT3 inhibitor)simultaneously with EGFR-TKI,and observe the effects of these STAT3 inhibitors to the effect of EGFR-TKI on the secretion level of IL-6 in lung adenocarcinoma;the cell line was treated with si-STAT3 to detect its effect on the expression of IL-6 mRNA and secretion in lung adenocarcinoma cell lines induced by EGFR-TKI;data analysis Graph Pad prism 8.0 is used,Image J and other software are used for image analysis,and SPSS 22.0 statistical analysis software is used for data statistics.Results: The genetic testing of lung cancer cell lines and the sequencing results of exons 18-24 of the EGFR tyrosine kinase region showed that the sequencing results of HCC827 and PC9,which are the same EGFR-19 del,also had differences in gene sequence.In HCC827-GR and PC9-GR,the occurrence of drug resistance may be related to the change of the 18-24 exon sequence;EGFR-TKI has a killing effect on EGFR-mutated lung adenocarcinoma and its IC50 value was detected.HCC827 and PC9 have higher sensitivity to gefitinib and osimertinib,with lower IC50 values.While HCC827-GR,PC9-GR and H1975 are resistant to gefitinib and are sensitive to osimertinib,but their sensitivity to osimertinib is still lower than that of HCC827 and PC9 cell lines,and their IC50 values are relatively high levels than that in HCC827 and PC9;EGFR-TKI affects the level of IL-6 mRNA in lung adenocarcinoma cell lines,EGFR-TKI up-regulates the level of IL-6 mRNA in some EGFR-mutant lung adenocarcinomas(such as HCC827).EGFR-TKI has no effect on the level of IL-6 mRNA in another part of EGFR-mutant lung adenocarcinoma(PC9);the intracellular expression level of IL-6 detected by flow cytometry showed that the intracellular IL-6 level of HCC827 treated with EGFR-TKI was increased.However,the intracellular IL-6 level of PC9 is not regulated by EGFR-TKI;p-STAT3/STAT3 in lung adenocarcinoma cell lines is regulated by EGFR-TKI,and p-STAT3 is significantly inhibited when HCC827 is treated with EGFR-TKI,When EGFR-TKI was used to treat PC9,its p-STAT3 was highly expressed.In addition,there was no significant difference between the detected p-AKT and p-ERK;inhibition of STAT3 phosphorylation can up-regulate the IL-6 mRNA level of EGFR-mutant lung adenocarcinoma cell lines(including HCC827 and PC9 cell lines)and the level of IL-6 in the cell supernatant.Expression level;si-STAT3 can up-regulate the mRNA level of IL-6 in EGFR-mutant lung adenocarcinoma cell lines(including HCC827 and PC9 cell lines)and the expression level in the cell supernatant.Conclusion: EGFR-TKI drugs can effectively kill EGFR-mutated lung adenocarcinomas,and when EGFR-TKI drugs act on EGFR-mutated lung adenocarcinomas,they can induce changes in IL-6 secretion levels.For the HCC827 cell line,EGFR-TKI drugs can increase its IL-6 expression level.For the PC9 cell line,EGFR-TKI drugs have no significant effect on its IL-6 expression level.Therefore,STAT3 can regulate the expression of IL-6 in EGFR-mutated lung adenocarcinoma,and the expression of STAT3 and IL-6 are negatively correlated.Inhibition of STAT3 can up-regulate the expression level of IL-6.