The Role Of ASF1B And Its Potential Mechanism In The Development Of Lung Adenocarcinoma

Objective:1、Anti-silencing function 1B histone chaperone(ASF1B)was evaluated comprehend sively and systematically.2、To explore the effects of ASF1 B knockdown or overexpression on the proliferation,apoptosis and cell cycle of LUAD cells.3、To reveal the preliminary molecular mechanism of ASF1 B in LUAD.Method:1、Analysis of ASF1 B different expression in lung adenocarcinoma tissue and normal lung tissue which were attained from TCGA and GTEx database were performed;IHC technology was used to detect the expression level of ASF1 B in lung adenoc arcinoma tissue and adjacent normal tissue.The relationship between ASF1 B expr ession level and clinicopathological parameters was analyzed by non-parametric test;Kaplan-Meier(KM)was used to analyze the relationship between ASF1 B and prognosis;Spearson correlation was used to analyze the correlation between ASF1 B and methylation,copy number variation(CNV),mutation,microsatellite instability(MSI)and immune microenvironment.2、The expression level of ASF1 B in lung adenocarcinoma cell lines was detected by Western blot.The expression of ASF1 B protein was low in A549 and H1299 cell lines,and high in H1975 and H1650 cell lines.RNA Seq results of CCLE cell line database showed that the expression of ASF1 B was A549,H1299,H1975 and H1650 from low to high.ASFB knockdown and overexpression lentivirus were constructed to infect two cell lines with high expression of ASF1 B and two cell lines with low expression of ASF1 B.The efficiency of ASF1 B knockdown and overexpression was detected by Western blot.CCK-8 and Ed U were used to detect the effects of knockdown or overexpression of ASF1 B on cell proliferation.Western Blot was used to detect the effect of knockdown or overexpression on caspase-3 of apoptosis-related proteins.The effects of ASF1 B knockdown or overexpression on apoptosis and cell cycle were detected by flow cytometry.3、Profiling technology was used to detect differentially expressed proteins in 4 cell lines before and after ASF1 B knockdown or overexpression.Western blot was used to verify the results of mass spectrometry.IP-mass spectrometry(IP-MS)technolo gy was used to explore the binding between ASF1 B protein and associated proteins.Results:1、TCGA and GTEx database analysis showed that the expression of ASF1 B in LUAD tissues was higher than that in normal lung tissues(p<0.05),and was also higher than that in adjacent normal tissues(p<0.05).The express ion of ASF1 B protein in LUAD tissues was higher than that in adjacent normal tissues detected by IHC(p<0.05).Downloaded LUAD mass spectrum data also supported the IHC results.The expression of ASF1 B in LUAD tissues was positively correlated with clinical stage,T stage,N stage and age(p<0.05).ASF1 B had prognostic and early diagnosis value in patients with LUAD.Correlation analysis of ASF1 B with Tumor-infiltra ting immune cells showed that the expression of ASF1 B was associated with multiple Tumor-infiltrating immune cells.The methylation site CG26259181 of ASF1 B was associated with over survival(OS)in LUAD.The expression level of ASF1 B was not related to CNV,somatic mutation and MSI(p<0.05).2、The expression of ASF1 B protein was low in A549 and H1299 cell lines,and high in H1975 and H1650 cell lines.The constructed ASF1 B sh RNA significantly reduc ed the expression level of ASF1 B protein in H1975 and H1650 cell lines.The expression of this protein was more than twice as high in the overexpressed ASF1 B cell lines as in the control group.CCK-8 and EDU results showed that cell prolifera tion of H1975 and H1650 was reduced in the sh ASF1 B group compared with Scram ble group.Flow cytometry results showed that apoptosis rate of H1975 and H1650 in sh ASF1 B group were increased compared with the scramble group;compared with the scramble group,the caspase-3 expression of H1975 and H1650 were increa sed;Flow cytometry results showed that cell cycle distribution of H1975 and H1650 was influenced.Compared with Vector group,A549 and H1299 cells in ASF1 B OE group showed no difference in cell proliferation,apoptosis and cell cycle.3、Profiling results from four cells with different biological backgrounds were interse cted,resulting in a total of 58 proteins.Among these proteins,a total of 5 proteins(POLE3,CKS1 B,HFR,RPS29 and TMEM230)were expressed with general significance.There was no overlap between the IP-MS results and the profiling results,suggesting that ASF1 B regulates the above five proteins in an indirect way.Conclusion:1、ASF1B is a molecular marker for early diagnosis and prognosis of LUAD.The relationship between ASF1 B and prognosis may be regulated by CG26259181 loc us.The expression level of ASF1 B was correlated with age,clinical stage,T stage and N stage.The expression level of ASF1 B was related to tumor immune infiltra ting cells and their surface markers.2、Knocking down the expression of ASF1 B reduce the proliferation of LUAD cells,induces cell apoptosis,and influence the cell cycle distribution.3、ASF1B can promote the progression of LUAD by indirectly regulating above 5 prot eins.