Kras^G12D-LOH Modulates Malignant Phenotype And Glutamine Metabolism In Pancreatic Cancer Cells With Its Related Molecular Mechanisms

Section 1Effect of KRAS^G12D-LOH on glutamine metabolism in pancreatic cancer cells[Objective]To investigate the effect of KRAS^G12D-LOH on glutamine metabolism in pancreatic cancer cells under normal and hypoxia conditions.[Methods]1.KRAS^G12D-LOH cell lines(897 cell lines)and KRAS^G12D cell lines(399 cell lines)were incubated respectively in the oxygen(5%,95%CO₂ air)and hypoxia(1%O2,5%CO₂, 94%N₂)conditions,respectively,the concentration of Gln?intracellular ROS level? NADPH/NADP+ratio were separately measured by Glutamine Colorimetric assay kit?a ROS detection kit and a NADPH/NADP+assay kit.2.mRNA and protein expression levels of Gln metabolic molecular markers in each group were determined by RT-qPCR and Western Blot.[Results]1.Under both normoxia and hypoxia conditions,glutamine consumption and NADPH/NADP+ratio of KRAS^G12D-LOH cell lines were significantly higher than those of KRAS^G12D cell lines(P<0.01),while ROS level of KRAS^G12D-LOH cell lines was significantly lower than that of KRAS^G12D cell lines(P<0.05).2.Western Blot results showed that the expression levels of GLS1,GOT2,GOT1,MDH1 and ME1 in KRAS^G12D-LOH cell lines were higher than those in KRAS^G12D cell lines under normoxia and hypoxia conditions(P<0.01).RT-qPCR results showed that mRNA expression levels of GLS1,GOT2,GOT1,MDH1 and ME1 in KRAS^G12D-LOH cell lines were significantly higher than those in KRAS^G12D cell lines(P<0.01).[Conclusion]Kras^G12D-LOH can promote glutamine metabolism in pancreatic cancer cells and increased the mRNA and protein levels of related molecular markers including GLS1?GOT2?GOT1?MDH1 and ME1 under both normoxia and hypoxia conditions.Section 2The role of REDD1 in glutamine metabolism and malignant phenotypes regulated by Kras^G12D-LOH in pancreatic cancer cells[Objective]The purpose of this study was to explore the role of REDD1 in the malignant phenotype of KRAS^G12D-LOH pancreatic cancer cells under hypoxia conditions,as well as the role of REDD1 in KRAS^G12D-LOH mediated glutamine metabolism regulation of malignant phenotype and related mechanisms[Methods]1.The REDD1-shRNA knockdown lentivirus and control lentivirus were respectively transfected into KRAS^G12D-LOH cell lines(897 and 907 cell lines)and Kras^G12D cell lines (399 and 403 cell lines),knockdown efficiency was detected by Western Blot and realtime- qPCR.The cells in each group were cultured under hypoxia conditions.The cell proliferation ability of each group was compared by using CCK-8 assay and colony formation assay.Transwell experiment and Wound healing revealed the relationship between REDD1 and KRAS^G12D-LOH and KRAS^G12D PDAC cell lines migration and invasion ability.Flow cytometry was used to determine the apoptosis and cell cycle of PDAc cell lines in each group.2.Glutamine consumption rate was detected by Glutamine Colorimetric assay kit,intracellular ROS level was detected by a ROS detection kit,and NADPH/NADP+was detected by a NADPH/NADP+assay kit to analyze the glutamine metabolism level of pancreatic cancer cells.3.Western Blot and RT-qPCR were used to detect the protein and mRNA expression of molecular markers including GLS1?GOT2?GOT1?MDH1 and ME1 of glutamine metabolism in each group.4.Correlation of mRNA expression levels of REDD1 and glutamine metabolic markers in pancreatic cancer tissues was analyzed using GEPIA database.[Results]1.In the hypoxia condition,the cell growth rate of sh-REDD1 group of KRAS^G12D-LOH cell lines(897 and 907 cell lines)were significantly decreased compared with the Control group and shRNA-NC group(P<0.01),but decreased in KRAS^G12D cell lines(399 and 403 cell lines)(P<0.05).Wound healing assay and Transwell assay indicated that the cell migration and invasion abilities of sh-REDD1 group of KRAS^G12D-LOH cell lines (897 and 907 cell lines)were lower than those of the Control group and shRNA-NC group respectively(P<0.01),while those of KRAS^G12D cell lines(399 and 403 cell lines) were higher than those of the Control group and shRNA-NC group respectively(P<0.01). The results of cell cycle and apoptosis showed that the proportion of S phase in KRAS^G12D- LOH cell lines(897 and 907 cell lines)sh-REDD1 group was significantly lower than that in the Control group and shRNA-NC group(P<0.01),and the apoptosis rate was higher than that in the Control group and shRNA-NC group(P<0.01),while KRAS^G12D cell lines(399 and 403 cell lines)had the opposite results(P<0.01).2.The results of glutamine consumption detection showed that the glutamine consumption rate in the sh-REDD1 group of KRAS^G12D-LOH cell lines(897 and 907 cell lines)was significantly lower than that of the Control group and shRNA-NC group(P<0.01), while the glutamine consumption rate in the sh-REDD1 group of KRAS^G12D cell lines (399 and 403 cell lines)was significantly higher than that of the Control group and shRNA-NC group(P<0.05).Meanwhile,ROS levels in the sh-REDD1 group of KRAS^G12D-LOH cell lines(897 and 907 cell lines)were significantly higher than those in the Control group and shRNA-NC group(P<0.05),and the NADPH/NADP+ratio was decreased(P<0.05),while the ROS levels in the sh-REDD1 group of KRAS^G12D cell lines(399 and 403 cell lines)were decreased(P<0.01),and the NADPH/NADP+ratio was increased(P<0.05).3.Western Blot and RT-qPCR results revealed that REDD1 knock-down induced an obvious repression in GOT1,GOT2,and MDH1 expression in Kras^G12D-LOH cells,both at the mRNA and protein levels(P<0.01).Of note,in PDAC cells without Kras^G12D-LOH, GOT1,GOT2,and MDH1 expression was significantly increased upon transfection with sh-REDD1 under hypoxia conditions(P<0.01).4.Correlation analysis results of GEPIA database showed that there were strong correlation between REDD1 and glutamine metabolic markers including GOT1,GOT2 and MDH1 in pancreatic cancer(P<0.05).[Conclusion]REDD1 plays an important role in regulating the malignant phenotypes of KRAS^G12D-LOH pancreatic cancer,which can be regulated through REDD1-mediated glutamine metabolism.Section 3The role of HIF-2?in glutamine metabolism and malignant phenotypes regulated by Kras^G12D-LOH in pancreatic cancer cells[Objective]The purpose of this study was to analyze the effect of HIF-2?on the malignant phenotype of KRAS^G12D-LOH in PDAC cells under normoxia conditions,and to reveal the relationship between HIF-2?and the malignant phenotype of KRAS^G12D-LOH-mediated Gln metabolism regulation and the related mechanism[Methods]1.GEPIA was used to analyze different expressions of HIF-2?between the normal tissue and tumor tissues.Western Blot and RT-qPCR were used to detect the protein and mRNA expression of HIF-2?in KRAS^G12D-LOH cell lines(897 and 907 cell lines)and KRAS^G12D cell lines(399 and 403 cell lines).2.The HIF-2?-shRNA knockdown lentivirus and control lentivirus were respectively transfected into KRAS^G12D-LOH cell lines(897 and 907 cell lines)and Kras^G12D cell lines (399 and 403 cell lines),knockdown efficiency was detected by Western Blot and realtime- qPCR.The cells in each group were cultured.The cell proliferation in each group was studied by CCK-8 assay and colony formation effiency.Wound healing and Transwell assay were used to explore the effect of HIF-2?on the migration and invasion of KRAS^G12D- LOH and KRAS^G12D PDAC cell lines.Cell cycle and apoptosis of KRAS^G12D-LOH and KRAS^G12D PDAC cell lines were determined by flow cytometry.3.Glutamine consumption rate was detected by Glutamine Colorimetric assay kit,intracellular ROS level was detected by a ROS detection kit,and NADPH/NADP+was detected by a NADPH/NADP+assay kit to analyze the glutamine metabolism level of pancreatic cancer cells.4.Western Blot and RT-qPCR were used to detect the protein and mRNA expression of molecular markers including GLS1?GOT2?GOT1?MDH1 and ME1 of glutamine metabolism in each group.5.Correlation of mRNA expression levels of HIF-2?and glutamine metabolic markers including GLS1?GOT2?GOT1?MDH1 and ME1 of glutamine metabolism in each group in pancreatic cancer tissues was analyzed using GEPIA database.6.A mouse subcutaneous xenograft model was established for study in vivo.The weight and tumor size of experimental animals were recorded,the tumor was exfoliated,and the weight was weighed.The tumor proliferation status of each group was evaluated by immunohistochemistry Ki67 staining.The expression levels of Gln metabolic markers C- MYC and GOT1 in tumor tissues of each group were measured by Western Blot and RT- qPCR.[Results]1.HIF-2?mRNA expression in pancreatic tumor tissue was significantly higher than that in normal tissues(P<0.05).Both Western Blot and RT-qPCR results showed that th expression levels of HIF-2?proteins and mRNA in KRAS^G12D-LOH cell lines(897 and 907 cell lines)were significantly higer than that in KRAS^G12D cell lines(399 and 403 cell lines) (P<0.05).2.It was found that by CCK-8 cell proliferation assay and Colony formation assay that,the proliferation ability of sh-HIF-2?groups in 399,403,897,and 907 cell lines was lower than that in Control group and shRNA-NC group(P<0.05).Transwell invasion and migration assay showed that sh-HIF-2?group was significantly lower in invasion and migration than shRNA-NC group and Control group(P<0.01).Cell cycle of sh-HIF-2?cells in each group were lower than those in Control group and shRNA-NC group(P<0.01).The apoptosis rate was higher than that in Control group and shRNA-NC group(P<0.01).3.Glutamine consumption in sh-HIF-2?group of KRAS^G12D-LOH cell lines(897 and 907 cell lines)was significantly lower than that in the Control group and shRNA-NC group(P< 0.01).However,glutamine consumption in the sh-HIF-2?group of KRAS^G12D cell lines (399 and 403 cell lines)showed no significant change compared with the Control group and shRNA-NC group(P>0.05).Meanwhile,the sh-HIF-2?group of KRAS^G12D-LOH cell lines(897 and 907 cell lines)and KRAS^G12D cell lines(399 and 403 cell lines)had higher ROS levels than the Control group and shRNA-NC group(P<0.05),and decreased the NADPH/NADP+ratio(P<0.05).4.Both Western Blot and RT-qPCR results showed that the expression levels of GOT1 and C-MYC proteins and mRNA in KRAS^G12D-LOH cell lines(897 and 907 cell lines)were significantly lower in sh-HIF-2?group than that in the Control group and shRNA-NC group (P<0.05).5.Correlation analysis results of GEPIA database showed that there were strong correlation between HIF-2?and glutamine metabolic markers including GLS1,GOT2,GOT1,MDH1 and ME1 in pancreatic cancer(P<0.01).6.The results showed that the tumor weight and tumor volume of sh-HIF-2?group in KRAS^G12D-LOH cell lines were lower than those of the shRNA-NC group(P<0.01 and P <0.01),and the tumor weight and tumor volume of sh-HIF-2?group in KRAS^G12D cell lines were also lower than those of the shRNA-NC group(P<0.05 and P<0.05).The number of Ki67 positive cells in KRAS^G12D-LOH cell lines and KRAS^G12D cell lines was significantly lower in sh-HIF-2?group than that in shRNA-NC group(P<0.01).RT-qPCR analysis revealed that HIF-2?,C-MYC and GOT1 mRNA in sh-HIF-2?group of KRAS^G12D-LOH cell lines were significantly lower than those in shRNA-NC group(P<0.01).Western Blot analysis showed that HIF-2?,GOT1 and C-MYC protein expression levels(P<0.01,P<0.01 and P<0.01)in sh-HIF-2?group of KRAS^G12D-LOH cell lines were significantly lower than those in shRNA-NC group.[Conclusion] HIF-2? plays an important role in the regulation of the malignant phenotype of Kras^G12D-LOH pancreatic cancer,which can be regulated by HIF-2?-mediated glutamine metabolism.