Insulin Like Growth Factor 2 MRNA Binding Protein 2 Promotes Aerobic Glycolysis And Cell Proliferation In Pancreatic Ductal Adenocarcinoma Via Stabilizing GLUT1 MRNA

Pancreatic ductal adenocarcinoma?PDAC?has a very high mortality rate,with a five-year survival rate of less than 6%.The prognosis is extremely poor.The main reason is that early detection is difficult,and the diagnosis is often in the advanced stage.The treatment of advanced invasive pancreatic ductal adenocarcinoma is hard,so to understand the physiological and pathological mechanism of PDAC can help to develop specific and effective targeted therapeutic drugs.The Insulin growth factor?IGF?family is recognized as a gene family that is widely involved in growth and development.Members of this family also play indispensable roles in the development of cancer.By consulting the literature,we found a family related to this family,insulin-like growth factor 2 mRNA binding protein?IGF2BP?family,plays a role at the post-transcriptional level,which regulates the expression of genes by regulating the physiological processes of mRNA.As an m6A reader gene family,this family has thousands of target genes,so its regulation of cellular physiological functions is very extensive,and more and more studies have shown the function of this family members in cancer.Further clarification of the relationship between the IGF2BP family and cancer is of great significance.This study first compared the expression of IGF2BP family members in cancer and adjacent tissues using three PDAC gene expression microarrays in the GEO?Gene Expression Omnibus?database,and we found that only IGF2BP2 showed consistently up-regulated expression.Then we used OncoLnc database for prognostic analysis and found that high expression of IGF2BP2 was most significantly associated with poor prognosis of PDAC.From the above analysis,we suspected that IGF2BP2has the potential to promote the development of PDAC.To confirm the results of the database analysis,we testes the expression of IGF2BP2 at the cellular and tissue levels,and found that the expression of IGF2BP2 was up-regulated in cancer as demonstrated by the database.Next,we immunohistochemically stained IGF2BP2 in pancreatic tumor tissue sections of LSL-Kras^G12D/+;LSL-Trp53^R172H/+;Pdx1-Cre?KPC,spontaneous PDAC models?mice.Through observing staining of different degree of malignant lesions,we found that as the degree of malignancy increased,the staining became deeper and deeper.The result indicated that IGF2BP2 may have a role in promoting the development of PDAC.Next,we studied the function of IGF2BP2 in PDAC through cell function experiments and subcutaneous tumor models.We found that the use of siRNA to interfere with IGF2BP2 in the PDAC cell line can significantly reduce the cell proliferation ability,while the subcutaneous tumor growth of the subcutaneous tumor is slower than that of normal cells after IGF2BP2 knockdown,indicating that IGF2BP2 can promote the growth of PDAC cells.To further investigate the mechanism by which IGF2BP2 promotes cell growth,we sequenced the transcriptome of IGF2BP2 knockdown cell line or untreated wild-type cell line and performed a Gene Set Enrichment Analysis?GSEA?with the transcriptome data using the Reactome gene sets,and found that the Glucose transport gene set was enriched.The result indicated that IGF2BP2 affects the transport of glucose into cells.Glucose transport is the first critical step of glucose metabolism.According to the Warburg Effect,cancer cells mainly produced ATP by glycolysis,so the knockdown of IGF2BP2 may significantly affect the cell glycolysis process.Then we carried out a glycolysis stress test and found that the glycolytic ability of PDAC cells was significantly reduced after IGF2BP2 knockdown,indicating that IGF2BP2can promote cell proliferation by promoting the glycolysis ability of cells.We then examined the mRNA level of glycolytic-related genes and found that IGF2BP2knockdown decreased GLUT1 expression greatly and significantly.The result suggested that IGF2BP2 may function by regulating GLUT1 expression.GLUT1 is the most common glucose transporter in cells and is often up-regulated in cancer cells and plays a role in promoting cancer development.Since IGF2BP2 is an mRNA-binding protein,it can stabilize mRNA by direct binding,and it is found that IGF2BP2 may bind to GLUT1 mRNA through a protein-RNA interaction database.Therefore,we performed CLIP experiments and RNA stability experiments,respectively,and verified that IGF2BP2 binds directly to GLUT1 mRNA and IGF2BP2 promotes the stability of GLUT1 mRNA.These results indicate that IGF2BP2 promotes the glycolysis and proliferation of PDAC cells by directly binding and stabilizing GLUT1 mRNA.To further confirm that IGF2BP2 regulates the expression of GLUT1 and thus affects cell function,we overexpressed GLUT1 in IGF2BP2 knockdown cell lines,and tested cell proliferation and glycolysis compared with IGF2BP2 knockdown cells and wild-type cells to find whether overexpression of GLUT1 in the IGF2BP2knockdown cell line can rescue cell function.It was found that overexpression of GLUT1 partially rescued the down-regulation of cell function caused by knockdown of IGF2BP2,indicating that IGF2BP2 can promote cell proliferation and glycolysis by promoting GLUT1 expression.In summary,IGF2BP2 is up-regulated in PDAC and its high expression leads to poor prognosis;IGF2BP2 can increase the expression of GLUT1 by directly binding and stabilizing GLUT1 mRNA,thereby promoting PDAC cell glycolysis and proliferation.