HnRNPA2B1 Enhances The Resistance Of Pancreatic Cancer Cellls To Ferroptosis By Inhibiting Tfrc

Objective:As one of the most widespread programmed cell death,ferroptosis has shown great potential in the treatment of many cancers,including pancreatic cancer.The purpose of this study was to investigate the correlation between the basic expression level of heterogeneous nuclear ribonucleoprotein A2B1（hnRNPA2B1）and the resistance to Erastin-induced iron death in different pancreatic cancer cell lines,analyze the differential expression of hnRNPA2B1 in pancreatic cancer tissues and normal tissues,the survival prognosis of patients with differential expression of hnRNPA2B1 and the effects of hnRNPA2B1 on maligant phenotype of pancreatic cancer cells,based on the above studies,the role of hnRNPA2B1 in ferroptosis of pancreatic cancer cells and its potential mechanisms were explored.Methods:1.PaTu8988,MIA-paca2 and PANC1 cells were treated with different concentrations of Erastin for 3 days,then the CCK-8 assay was used to assay the IC₅₀of Erastin and resistance of three kinds of cells to Erastin was compared.2.qRT-PCR and Western blotting were used to detect the relative expression levels of m RNA and protein of hnRNPA2B1 in pancreatic cancer cells.3.The differential expression of the hnRNPA2B1 in pancreatic cancer tissues and normal tissues was analyzed by GEPIA,and the survival prognosis of patients with differential expression of hnRNPA2B1 was analyzed from clinical data sets from GEO database.4.Transfect PaTu8988 cells with sh-Control or sh-hnRNPA2B1 plasmid to interfere hnRNPA2B1;transfect PANC1 cells with Vector or Flag-hnRNPA2B1plasmid to overexpress hnRNPA2B1;the effects of knockdown or overexpression of hnRNPA2B1 on the migration and invasion were detected by Transwell assay,and the effects on the proliferation of pancreatic cancer cells were detected by CCK-8assay.5.The cells in sh-Control,sh-hnRNPA2B1,Vector and Flag-hnRNPA2B1group were treated with control（0μmol/L Erastin）and Erastin(IC₅₀concentration),respectively,then flow cytometry was used to determine the lipid peroxidation level,and colorimetry was used to determine the malindialdehyde（MDA）and tissue iron concentration;in addition to Erastin,the sh-hnRNPA2B1 and Flag-hnRNPA2B1groups were added ferroptosis,apoptosis,and necroptosis inhibitors,respectively,the cells activity was detected by taphenol blue staining.6.Iron metabolism and antioxidant defence related markers were detected at m RNA and protein levels by qRT-PCR and Western blotting.Results:1.The results of CCK-8 assay which analyzed by Graphpad Prism 9.0 showed that the resistance of three types of pancreatic cancer cells to Erastin from high to low were PaTu8988,MIA-paca2,PANC1,Erastin IC₅₀was 18.020,15.760 and 2.947μmol/L,respectively.2.qRT-PCR results showed that the relative expression level of m RNA of hnRNPA2B1 was the highest in PaTu8988 cells,followed by MIA-paca2 cells and the lowest in PANC1 cells;the results of western blotting were the same as qRT-PCR.3.The results of relevant pancreatic cancer online database analysis showed that the expression level of hnRNPA2B1 in pancreatic cancer was higher than that in normal tissues,and the prognosis of patients with high expression of hnRNPA2B1was poor.4.The results of transwell and CCK-8 assay showed that after the expression of hnRNPA2B1 knock-down,the migration,invasion and proliferation ability of PaTu8988 cells were reduced,after overexpression hnRNPA2B1 in PANC1 cells,the effect is the opposite.5.Taphenol blue staining showed that Erastin-induced cell death was significantly restored by ferrostatin-1;compared with sh-Control group,the level of lipid peroxidation and MDA level in sh-hnRNPA2B1 group were significantly increased;compared with Vector group,lipid peroxidation level and MDA level in Flag-hnRNPA2B1 group were significantly decreased.6.Compared with the control group,the down-regulation hnRNPA2B1expression increased the m RNA and protein expression levels of TFRC and FTH1 in PaTu8988 cells,intracellular total iron content increased significantly,and the up-regulation expression of hnRNPA2B1 decreased the m RNA and protein expression levels of TFRC and FTH1 in PANC1 cells,intracellular total iron content decreased obviously.Conclusion:As an oncogene,hnRNPA2B1 plays an important role in the occurrence and development of pancreatic cancer,and inhibits the accumulation of iron by inhibiting the expression of TFRC,thus promoting the resistance of pancreatic cancer cells to ferroptosis.