Primary Research In The Function And Mechanism Of ErbB3 Activated Mutations In Malignant Progression Of PDAC

Objective1.Try to investigate ErbB3 mutation in PDAC patients by genetic testing.2.Construct ErbB3 mutant plasmid.3.To investigate the effect of ErbB3 gene activation and overexpression on cell sensitivity of EGFR-targeted therapy(erlotinib).4.To preliminarily explore the mechanism of interaction between ErbB3 activation and overexpression and EGFR-targeted therapy.Methods1.Collect 140 Clinical data and pancreatic cancer specimens of PDAC patients in ChangZheng hospital,and ErbB3 mutation was detected by using second-generation gene detection chip method.2.Construct Point mutations of ErbB3 genes A232 V,H465P by site-directed mutation.3.Construct plasmids containing the target genes and make sure whether they were directly bound to the target genes.PCR was used to detect the transfection efficiency.4.To investigate the effect of ErbB3 wild-type and mutant cell lines KP4 and PANC-1 proliferation when add erlotinib by using MTT colorimeter.5.To investigate the effect of ErbB3 wild-type and mutant cell lines KP4 and PANC-1 migration when add erlotinib by using Transwell assay.Results1.Through ErbB3 genetic testing for 140 cases of specimens,we found 1 case nonsense mutations,the mutation site is 498,codon AAA?ATA,lysine?isoleucine,PPH2 Score HumDiv:0.027 points and HumVar:0.048 points,and the predicted results are both BENIGN,SIFT Score:0.18 points,predicted results is TOLERATED;Another case is missense mutation,the mutation site is 465,codon CAC?CCC,histidine?proline,PPH2 Score HumDiv:1 and HumVar:1,and the predicted results are both PROBABLY DAMAGING,SIFT Score:0,and the predicted results is DAMAGING.2.PCR amplification of the mutant gene fragment was successful: Using cDNA as template,the gene fragments of A232 V and H465 P ErbB3 were amplified by PCR;ErbB3 mutant plasmid was successfully constructed: Two mutated plasmids of ErbB3 gene A232V and H465 P were successfully obtained by PCR,cherry-picking and plasmid extraction.The results showed that the plasmids were successfully constructed.3.In KP4 cell lines that were not transfected with ErbB3 mutant plasmids,erlotinib was added and cell activity decreased slightly,but not significantly,compared with the control group.Transfection A232 V KP4 cell line,add it for later,cell proliferation activity decreased obviously,and within the first 12 h had the greatest reduction;Transfection H465 P KP4 cell line,add it for later,cell proliferation activity decline,12 h before the fall is relatively larger,follow-up action is weak.Generally in the KP4 cell line,A232 V H465P relatively to it for more sensitive,ErbB3 mutation compared with transfection mutation plasmid KP4 cell line,for it's effect on inhibition of pancreatic cancer cells larger(p< 0.05).Not transfection ErbB3 mutation plasmid PANC-1 cell line,after joining it for his cell activity compared with the control group did not decline,even after 24 h testing is found on the proliferation activity.Transfection A232 V,H465P PANC-1 cell line,add it for later,had fallen to pancreatic cancer cell proliferation activity,but the declines are smaller,transfection H465 P PANC-1 cell line proliferation activity are generally weak than the A232V(p < 0.05)4.Compared with the control group,the migration ability of KP4 and PANC-1 in tumor cells was significantly reduced in the erlotinib group.In each group.In the KP4-transfected A232 V group,the average number of cells with erlotinib migration per field of view was 81.5±0.5,compared with 297.7± 3.0 in the control group(p < 0.05).In the H465 P group,the average number of cells with erlotinib migration per field of view was 85.5 ±1.3,compared with 197.7 ±4.4 in the control group(p < 0.05).In the PANC-1transfected A232 V group,the average number of cells with erlotinib migration per field of view was 81.3±0.7,while in the control group 295.4±2.9(p < 0.05).In the transfected H465 P group,the average number of cells with erlotinib migration per field of view was96.4±3.7,and in the control group,313.8±2.1(p < 0.05).5.In the KP4 cell lines stained with ErbB3 mutant plasmid A232 V,the proliferation activity of erlotinib was significantly decreased compared with that of the control group.After the addition of SSI into erlotinib,the inhibitory effect of erlotinib on the cell line was significantly weakened.Compared with the addition of erlotinib alone,the proliferation activity of the cell line was significantly enhanced.The addition of SSI alone had no significant effect on the proliferation activity of cells,and the change was not significant compared with the control group.6.In the erlotinib+SSI group,the average visual field on the number of cells for migration is 262.2±5.8 mm,alone to join it for group,the average visual field for migration of cell number is 78.2±1.8,join in SSI group alone,the average visual field for migration of cell number is 294.8±6.2,in the control group,the average view of migration of cell number is 301.5± 8.5(p < 0.05).Conclusions1.There are ErbB3 synonymous mutations and missense mutations in pancreatic cancer,and the missense mutation rate is low.2.ErbB3 mutant pancreatic cancer cell line proliferation was inhibited by erlotinib,and A232 V KP4 cell line showed a strong inhibitory effect.3.ErbB3 mutant pancreatic cancer cell line migration was inhibited by erlotinib.4.The role of ErbB3 in pancreatic cancer is closely related to HRG,and the inhibitory effect of EGFR-targeted drugs on mutant pancreatic cancer is related to ErbB3/HRG axis to some extent.