Research Of The Biological Function And Mechanism Of B4GALT6 In Non-small Cell Lung Cancer Resistance To Osimertinib

Objective:Induce the resistance of HCC827 Osimertinib resistant cell line(HCC827OR)and verify the resistance of HCC827 OR and NCI-H1975 Osimertinib resistant cell line(NCI-H1975OR).Make clear the biological function and mechanism β-1,4-galactosyltransferase 6(B4GALT6)in NSCLC Osimertinib-resistant cells,expecting to provide new molecular markers and therapeutic targets for the diagnosis and treatment of NSCLC Osimertinib-resistant patients.Methods: 1.Induce the resistance of HCC728 Osimertinib resistant cell line(HCC827OR)and verify the resistance of HCC827 OR and H1975 Osimertinib resistant cell line(H1975OR): High dose drug shock method,using 0.5μM Osimertinib to expose to HCC827 every 3 days,and then use CCK-8 to detect the half inhibition rate(IC50).The proliferation ability of HCC827 OR,NCI-H1975 OR cells and their parent strains were verified by CCK-8 experiment and plate cloning experiment,and q RT-PCR verify the relative m RNA expression of drug resistant protein.2.Screening of genes affecting cell drug resistance and validation of target gene expression and cell location: Seven genes with large differences were selected(B4GALT6,CSF1,e EF1 A,CXCL1,IL6,ITGB4 and ZNF300).q RT-PCR tested the target gene and the expression of the target gene B4GALT6 in the Osimertinibresistant cell line and its parent cells were verified by Western Blot.The location of B4GALT6 in the cell was determined by cell immunofluorescence.3.To study the effect of B4GALT6 expression on the cell function and drug resistance: Using B4GALT6 si RNA and si RNA NC to transiently transfect HCC827 OR and NCI-H1975 OR cell lines,q RT-PCR and Western Blot to evaluate the transfection efficiency,and using CCK-8 test and plate cloning test to detect the proliferation ability of B4GALT6 in Osimertinib resistant cells.Further test whether B4GALT6 affects the drug resistance of NSCLC cells to Osimertinib by CCK-8experiment.The expression of differential genes of non-small cell lung cancer cell lines NCI-H1975,HCC827 and their drug resistant strains and the immunofluorescence cell localization of B4GALT6 were detected by q RT-PCR,immunofluorescence and other experiments.4.Explore signal path: Analyze the pathways and functions that may be regulated by B4GALT6 through bioinformatics technology,and combine with literature research to screen the most likely pathway that B4GALT6 can promote drug resistance,and verify the expression changes of key proteins on the pathway through Western Blot experiment.(Presumably is PI3K-AKT signal pathway).Results: 1.High dose drug shock method,using 0.5μM Osimertinib to expose to HCC827 every 3 days,and then use CCK-8 to detect the half inhibition rate(IC50).IC50 of HCC827 OR after induction for 6 months was 2.468 ± 0.12 μM.The IC50 of HCC827 was 0.4084 ± 0.11 n M,and the resistance index was greater than 5.The construction of the resistant cell line was successful.The results of CCK-8experiments and plate cloning experiments showed that compared with the parent strain,HCC827 OR and NCI-H1975 OR had stronger proliferative ability(P<0.05).The expression of drug resistant proteins in HCC827 OR and NCI-H1975 OR was upregulated by q RT PCR.2.The transcriptome sequencing data of NSCLC Osimertinib resistant cell line and the parent line in this study were obtained from GEO database through bioinformatics technology,and seven genes with large differences were selected from them(B4GALT6,CSF1,e EF1 A,CXCL1,IL6,ITGB4 and ZNF300).The expression results of 7 genes detected by q RT PCR showed that B4GALT6 was significantlyupregulated in the Osimertinib resistant cell line compared to its parent lines(P<0.0001),and Western blot analysis showed that B4GALT6 was significantly upregulated in the Osimertinib resistant cell line compared to its parent strain.And through cell immunofluorescence confirmed that B4GALT6 is highly expressed in Osimertinib resistant cells,and B4GALT6 is located in the cytoplasm.3.q RT-PCR and Western Blot evaluation of transient transfection efficiency showed that using B4GALT6 si RNA knockdown had a better effect.The results of the CCK-8 experiment and plate cloning experiment showed that after downregulating B4GALT6 in the Osimertinib resistant cell line,the cell proliferation ability of the B4GALT6 si RNA group was significantly reduced compared to the si RNA NC group(P<0.05).Further CCK-8 experiments showed that downregulating B4GALT6 can reverse the resistance of Osimertinib resistant cell lines to Osimertinib.4.Through bioinformatics technology analysis,it was found that B4GALT6 may affect the resistance of NSCLC Osimertinib resistant cells to Osimertinib through the PI3K-AKT signaling pathway.Western Blot confirmed that knockdown B4GALT6 can inhibit the expression of PI3K-AKT signaling pathway proteins.This part of the results showed that B4GALT6 can promote the activation of the PI3K-AKT pathway in Osimertinib resistant cells,thereby affecting the drug resistance of Osimertinib resistant cells.Conclusion: This study found that HCC827 OR and NCI-H1975 OR have stronger proliferation ability compare to their parent lines,and exhibit strong resistance to Osimertinib.B4GALT6 can promote the proliferation ability of Osimertinib resistant cells,thereby promoting drug resistance in NSCLC cells.Its mechanism may be through the activation of the PI3K-AKT signaling pathway.Targeted treatment of B4GALT6 may help NSCLC reverse drug resistance in patients with acquired resistance to Osimertinib,prolong drug resistance time,and enable patients to achieve better prognosis.