YTHDF1通过肺血管内皮细胞DSTYK/NRF2/HIF1α/VEGF信号调节肺癌化疗敏感性

YTHDF1 regulates chemotherapy sensitivity of lung cancer through DSTYK/NRF2/HIF1α/VEGF signaling pathway in pulmonary endothelial cells

目的探究肺癌化疗敏感性m6A甲基化修饰相关基因表达及对非小细胞肺癌细胞和肺血管新生的影响。方法定量逆转录聚合酶链反应检测非小细胞肺癌组织m6A甲基化相关基因mRNA表达。采用免疫印迹和免疫组化检测非小细胞肺癌组织YTHDF家族蛋白表达。采用免疫荧光检测非小细胞肺癌组织YTHDF1蛋白和上皮细胞标志E-cadherin与内皮细胞标志CD31共定位表达;采用CCK-8、EdU、AnnexinV/PI、Transwell和划痕实验检测过表达YTHDF1对A549/CR细胞和H661/CR细胞的细胞生物学行为的影响。免疫印迹检测了YTHDF1过表达后肺癌细胞上皮间质转化和肺血管内皮细胞的血管生成的变化;m6A信使核糖核酸免疫沉淀测序和RNA免疫沉淀测序分析YTHDF1的调控RNA;免疫印迹检测YTHDF1、DSTYK、p-NRF2^(S40)、HIF1α和VEGF等蛋白表达。结果顺铂耐受非小细胞肺癌患者YTHDF1和YTHDF2 mRNA与蛋白表达明显低于顺铂敏感肺癌患者;且YTHDF1蛋白与CD31荧光强度信号一致,YTHDF1^(+)CD31^(+)细胞比例低于顺铂敏感非小细胞肺癌患者;过表达YTHDF1降低A549/CR细胞和H661/CR细胞活力、抑制细胞增殖、侵袭和迁移,促进细胞凋亡;过表达YTHDF1并不影响上皮间质转化标志N-cadherin和E-cadherin的表达。但抑制肺动脉内皮细胞的血管生成,VEGF、VEGFR和Endoglin(血管生成标志)蛋白表达明显降低;YTHDF1富集DSTYK基因,过表达YTHDF1明显降低DSTYK蛋白;肺癌患者的DSTYK和CD31荧光强度信号一致,且顺铂耐受DSTYK^(+)CD31^(+)细胞比例高于顺铂敏感肺癌患者;敲低DSTYK后YTHDF1过表达的肺血管内皮细胞NRF2丝氨酸40位磷酸化,HIF1α和VEGF蛋白表达明显降低。结论YTHDF1的表达与非小细胞肺癌顺铂耐受性有关,YTHDF1过表达可调节肺癌细胞的细胞生物学行为,影响肺动脉内皮细胞的血管新生,并与DSTYK介导的NRF2/HIF1α/VEGF信号有关。

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-NRF2^(S40),HIF1αand 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 HIF1αand 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.