YTHDF1 regulates chemotherapy sensitivity of lung cancer through DSTYK/NRF2/HIF1α/VEGF signaling pathway in pulmonary endothelial cells
Objective To explore the expression of genes related to chemosensitive m6A methylation modification in lung cancer and its impact on non-small cell lung cancer and angiogenesis.Methods Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expression of m6A methylation-related genes in lung tissue of non-small cell lung cancer. The expression of YTHDF family proteins was detected by Western blot and immunohistochemistry. Immunofluorescence was used to detect the colocalization expression of YTHDF1 protein,E-cadherin (epithelial cell marker) and CD31. CCK-8,EdU,AnnexinV/PI,Transwell and scratch experiments were used to detect the effect of overexpressed YTHDF1 on A549/CR cells and H661/CR cells on the cell biological behavior. Western blot was applied to detect the expression changes of genes in epithelial-mesenchymal transition of lung cancer cells and angiogenesis of pulmonary vascular endothelial cells after YTHDF1 overexpression. m6A messenger ribonucleic acid immunoprecipitation sequencing and RNA immunoprecipitation sequencing were used to analyze the regulatory RNA of YTHDF1. Western blot was used to detect the expression of YTHDF1,DSTYK,p-NRF2S40,HIF1α and VEGF proteins.Results The expression of YTHDF1 and YTHDF2 mRNA and protein in patients with cisplatin resistant lung cancer was significantly lower than that in patients with cisplatin sensitive lung cancer;The fluorescence intensity signals of YTHDF1 protein and CD31 were consistent,and the proportion of YTHDF1+CD31+cells in resistant lung cancer patients was lower than that in cisplatin sensitive lung cancer patients. Overexpression of YTHDF1 reduced the cell viability of A549/CR cells and H661/CR cells,inhibited cell proliferation,invasion and migration,and promoted cell apoptosis. However,overexpression of YTHDF1 did not affect the expression of epithelial-mesenchymal transition markers,N-cadherin and E-cadherin. Furthermore,the angiogenesis of pulmonary artery endothelial cells was inhibited,and the protein expression of VEGF,VEGFR and Endoglin (angiogenesis markers) was significantly reduced. YTHDF1 enriched the DSTYK gene,and overexpression of YTHDF1 significantly reduced the DSTYK protein. The DSTYK and CD31 fluorescence intensity signals of lung cancer patients were consistent,and the proportion of DSTYK+CD31+cells in cisplatin resistant lung cancer patients was higher than that in cisplatin sensitive ones. After knocking down DSTYK,YTHDF1-overexpression in pulmonary vascular endothelial cells induced NRF2 serine 40 phosphorylation and HIF1α and VEGF protein expression.Conclusion The expression of YTHDF1 is related to cisplatin resistance in non-small cell lung cancer. YTHDF1 overexpression regulates the cell biological behavior of lung cancer cells,affects angiogenesis of pulmonary vascular endothelial cells,and is related to DSTYK-mediated NRF2/HIF1α/VEGF signaling.
万方数据
维普
