The Role And Mechanism Of AFAP1-AS1 In Germinal Center B-cell-like Diffuse Large B-cell Lymphoma

Objective:Diffuse large B-Cell lymphoma?DLBCL?is a group of aggressive B-cell lymphoma with significant heterogeneity.It is the most common subtype of non-Hodgkin's lymphoma,accounting for 35-40%,and is mainly divided into germinal center subtype?GCB?and non-germinal center subtype.Among them,GCB subtype is thought to originate from germinal center blast cells,and its pathogenesis is complex.Although genetic abnormalities are important initiators for the development of GCB-DLBCL,the role of epigenetic alterations in tumor progression is getting more and more attention.Long noncoding RNAs?lncRNAs?are hot topics in recent years.Abnormal expression of lncRNAs is closely related to many diseases including tumors.However,the function and mechanism of lncRNAs in GCB-DLBCL remain unclear.In this paper,we focused on the differentially expressed lncRNAs in GCB-DLBCL,selected a specific lncRNA based on fold change and explored its biological function and possible mechanism.Methods:Part I:Microarray detection and bioinformatics analysis of long noncoding RNAs in germinal center B-cell-like diffuse large B-cell lymphoma and normal CD19+B cells1.CD19-positive cells were isolated from the buffy coats of healthy donor blood with CD19 microbeads.LncRNA/mRNA microarrays were used to screen differentially expressed lncRNA/mRNA in OCI-Ly1 and OCI-Ly19 cell lines compared with CD19-positive cells.2.Collect specimens,including 10 cases of GCB-DLBCL tissue samples and 10 cases of lymph node reactive hyperplasia?RLN?tissue samples.We detected the expression level of ENST00000425358,ENST00000455011,ENST00000451368,NR₀26892?AFAP1-AS1?,ENST00000464929,ENST00000475089,ENST00000558952 and ENST00000424690 in OCI-Ly1 and OCI-Ly19 cell lines to verify the results of microarray.Besides,we detected the expression level of those confirmed lncRNAs mentioned above by qRT-PCR in 10 GCB-DLBCL tissue samples and 10 RLN tissue samples again.3.A lncRNA-mRNA co-expression network was constructed based on the lncRNAs verified by qRT-PCR and mRNA expression profiles.The function of differentially expressed lncRNAs was predicted by bioinformatics analysis.Part II:Effect of AFAP1-AS1 knockdown on cell proliferation,cell cycle and apoptosis in germinal center B-cell-like diffuse large B-cell lymphoma1.We selected AFAP1-AS1 for further study.Firstly,we constructed and packaged adenovirus that could silence AFAP1-AS1 expression and then infected logarithmic growth phase GCB-DLBCL cells with adenovirus.2.Three groups were established:experimental group?sh-AFAP1-AS1 interfering sequence adenovirus infected cell group?,unrelated sequence control group?sh-NC unrelated sequence adenovirus infected cell group?and blank group?uninfected adenovirus cell group?.3.CCK-8 method was used to detect the proliferation of three groups.4.Flow cytometry?Annexin V/7-AAD?was used to detect the apoptosis rate of three groups.5.Flow Cytometry?PI?was used to detect the cell cycle progression of three groups.6.Western blot was used to detect the expression of apoptosis and cell cycle-related proteins in three groups.Part III:The mechanism of AFAP1-AS1 in germinal center B-cell-like diffuse large B-cell lymphoma1.Two groups were established:experimental group?sh-AFAP1-AS1 interfering sequence adenovirus infected cell group?and unrelated sequence control group?sh-NC unrelated sequence adenovirus infected cell group?2.Label-free protein mass spectrum was used to detect differentially expressed proteins in two groups.3.The differentially expressed proteins were subjected to KEGG Pathway to analyze enriched signaling pathways and determine downstream molecules of AFAP1-AS1.4.We predicted the microRNA that could bind complementarily with AFAP1-AS1and its downstream molecules via bioinformatics analysis.5.The reporter gene vectors H-AFAP1-AS1-WT/H-AFAP1-AS1-MUT and H-BTK-3'UTR-WT/H-BTK-3'UTR-MUT were constructed.Dual luciferase reporter assay was used to verify that mi R-670-3p could bind with AFAP1-AS1 and BTK mRNA complementarily and negatively regulated their expression.Results:Part I:Microarray detection and bioinformatics analysis of long non-coding RNAs in germinal center B-cell-like diffuse large B-cell lymphoma and normal CD19+B cells1.Microarray results revealed that 1,648 lncRNAs displayed significant upregulation,and 2,671 lncRNAs displayed significant downregulation in the two cell lines?Fold Change?2.0,P<0.05?.2.The results of qRT-PCR showed that the expression of ENST00000424690,ENST00000425358 and NR₀26892?AFAP1-AS1?were upregulated in OCI-Ly1and OCI-Ly19 cell lines,as well as in 10 GCB-DLBCL tissue samples,while the expression of ENST00000464929 and ENST00000475089 were downregulated?Fold Chang?2.0,P<0.05?.The results of microarray and qRT-PCR were basically the same,verifying the reliability of our microarray results.3.The lncRNA-mRNA co-expression network showed that the expression of three cell cycle related genes?GTSE1,CDK4 and CDK1?and four ubiquitin-proteasome related genes?PSMA7,PSMD14,UBE2K and UBE2C?were negatively correlated with the expression of ENST00000464929 and ENST00000475089.Additionally,ENST00000425358 and NR₀26892?AFAP1-AS1?were significantly associated with the expression of several proto-oncogenes or tumor suppressor genes?MYB,RABL3 and XAF1?.Part II:Effect of AFAP1-AS1 knockdown on cell proliferation,cell cycle and apoptosis in germinal center B-cell-like diffuse large B-cell lymphoma1.The titers of adenovirus AFAP1-AS1 shRNA1³ were 1.58×10¹⁰ PFU/ml.AFAP1-AS1 expression was significantly downregulated in AFAP1-AS1 shRNA1³adenovirus-infected cells compared with blank group and unrelated sequence control group?P<0.05?.2.The results of CCK8 assay showed that the absorbance values of sh-AFAP1-AS1group were significantly decreased compared with blank group and unrelated sequence control group,and the differences became greater with the time going.3.The results of flow cytometry?Annexin V/7-AAD?showed that the rates of early and late apoptotic cells in sh-AFAP1-AS1 group were significantly higher than those of blank group and unrelated sequence control group in OCI-Ly1 and OCI-Ly19 cells,with statistical difference?P<0.05?.4.Flow cytometry?PI?results showed that the proportion of cells in G0/G1 phase in sh-AFAP1-AS1 group was significantly higher than that in blank group and unrelated sequence control group,while the proportion of cells in S phase was significantly reduced,with statistical difference?P<0.05?.5.Western blot results showed that the expression of Cleaved Caspase-3 and pro-apoptosis protein Bax were upregulated in sh-AFAP1-AS1 group compared with blank group and unrelated sequence control group in OCI-Ly1 and OCI-Ly19 cells,while the expressions of Cyclin D1,Cyclin E and Bcl-2 were downregulated?P<0.05?.Part III:The mechanism of AFAP1-AS1 in germinal center B-cell-like diffuse large B-cell lymphoma1.After silencing AFAP1-AS1 in OCI-Ly1 cells,the results of protein mass spectrum showed that 122 proteins were upregulated and 124 proteins were downregulated.Among them,4 DLBCL-driven genes?POU2F2,BTK,PTPN6 and XPO1?were downregulated.2.Our KEGG Pathway analysis showed that the differential proteins detected by protein mass spectrum were enriched in several tumor-related pathways including ubiquitin-proteasome pathway,B cell receptor pathway?BCR?and TNF pathway.3.Western blot results showed that the expression level of BTK kinase in the BCR pathway and its phosphorylated form was decreased in sh-AFAP1-AS1 group compared with unrelated sequence control group in OCI-Ly1 and OCI-Ly19 cells,consistent with the results of protein mass spectrum?P<0.05?.4.Bioinformatics analysis predicts the complementary binding of AFAP1-AS1,BTK and miR-670-3p and their binding sites.5.Dual luciferase reporter assay confirmed that miR-670-3p could bind complementarily with AFAP1-AS1 and BTK,respectively,and downregulate the expression of mi R-670-3p.Conclusions:1.Compared with normal CD19+B cells,1,648 lncRNA transcripts were upregulated in OCI-Ly1 and OCI-Ly19 cell lines,with 2,671 lncRNA transcripts downregulated,and AFAP1-AS1 was one of the lncRNAs with significant fold change.2.Silencing AFAP1-AS1 could inhibit the proliferation of GCB-DLBCL cells,promote apoptosis and arrest the cell cycle at G0/G1 phase by affecting the expression level of Bcl-2,Bax,Cyclin D1 and Cyclin E1.3.After silencing AFAP1-AS1,the differentially expressed proteins were enriched in several tumor-associated pathways,including the ubiquitin-proteasome pathway,BCR signaling pathway and TNF signaling pathway.4.The overexpressed AFAP1-AS1 in GCB-DLBCL may inhibit downregulation of BTK by miR-670-3p through competitive binding with miR-670-3p,leading to the upregulation of BTK expression.