The Investigation About EZH2 Inhibitor Combined With Gefitinib In EGFR-TKIs Resistant Lung Cancer

Background and purpose: Lung cancer is one of the leading cause of malignant tumors that impair human health.With the development of molecular biology,the researchers found that in addition to specific gene mutations and activation of abnormal signaling pathways,there are some special epigenetic changes which related to the tumourgenesis.EZH2,an important epigenetic regulatory gene with Histone methyltransferase(HMT)activity,can catalyze the expression of target gene by catalyzing the lysine trimethyl(H3K27me3)of histone H3.The researchers also found that EZH2 is highly expressed in lung cancer tissues and the expression is closely related to the prognosis.At the same time,EZH2 inhibitors have also been found to enhance the sensitivity of tumor cells to various anti-tumor drugs.This purpose of the present study is: 1)to investigate the expression of EZH2 in non-small cell lung cancer and its relationship with the prognosis.2)To investigate the EZH2 inhibitor combined with gefitinib on the proliferation,apoptosis and migration of gefitinib-resistant cells.3)To investigate whether EZH2 inhibitor combide with EGFR-TKI can sensitize the lung cancer cells to gefitinib and the effect on cell proliferation,apoptosis and migration ability in EGFR wild-type non-small cell lung cancer.METHODS: To further investigate the expression of EZH2 in non-small cells,we compared the expression of EZH2 in lung squamous cell carcinoma and lung adenocarcinoma with the data from the TCGA(The Cancer Genome Atlas)database.Subsequently,qRT-PCR was used to detect the expression of EZH2 in 40 cases of non-small cell lung cancer tissuesand adjacent normal tissues.Western blot was used to detect different non-small cell lung cancer lines(H460,A549,HCC827,H1299,H1650,H1792,H1975,H2030)and the expression level of EZH2 in normal alveolar epithelial cell line B2 B cells.To further investigate the combined effects of EZH2 inhibitors and gefitinib,we used PC-9/AB2 as a drug-resistant cell model to test the effects of combination therapy on cell viability and proliferation by using CCK-8 and EdU assays.The effect of the combination of the trace and transwell chambers on cell migration ability;and the effect of combined cytometry on apoptosis;and the effect of combination therapy on EGFR signaling pathway was observed by Western blot.A549 and H1299 cells were used as wild-type cell model of EGFR.EZH2 siRNA was used to knock down the expression of EZH2,and its sensitivity to gefitinib was detected.CCK-8 and EdU were used to detect the activity and proliferation of the combination.The effect of combination therapy on cell migration ability by scratch and transwell chamber;and the effect of combined use of flow cytometry on apoptosis;and Western blot analysis of EGFR signaling pathway and its downstream signals by Western blot.RESULTS: TCGA analysis showed that the expression of EZH2 in non-small cell lung cancer(squamous cell carcinoma and lung adenocarcinoma)was significantly higher than that in adjacent normal tissues,and its expression was negatively correlated with the prognosis of patients with lung adenocarcinoma.qRT-PCR results showed that the expression of EZH2 in tumor samples of 40 patients with non-small cell lung cancer was significantly higher than that of adjacent normal tissues.The expression level of EZH2 in cell lung cancer cells(A549,H460,H1299,H1975,H1650,H1792,H1975,H2030)weresignificantly higher than that of normal alveolar epithelial B2 B cells.In primary drug-resistant PC9/AB2 cells,the combination of EZH2 inhibitor(GSK343)and gefitinib can significantly inhibit cell activity,cell migration,and induce cell apoptosis.It was shown that the combination of drugs also significantly inhibited the phosphorylation of EGFRand EZH2 expression.In EGFR wild-type cells,we found that it was more sensitive to gefitinib after siRNA knockdown of the EZH2 gene in a non-small cell lung cancer cell line.Combined use of EZH2 inhibitor(GSK343/DZNep)+ gefitinib also significantly inhibited the proliferation and migration of A549 and H1299 cells,and induced apoptosis.The results of Westernblot also showed that gefitinib was applied after knocking down EZH2,which significantly inhibited EGFR and P-EGFR before knockdown.At the same time,the combination of EZH2 inhibitor and gefitinib could significantly inhibit Expression of EGFR protein and P-EGFR protein.