Screening And Functional Study Of MicroRNAs And Proteins Related To The Signaling Pathway Of Diffuse Large B-cell Lymphoma

Objective: Diffuse large B-cell lymphoma(DLBCL)is the most common B-cell lymphoma in adults.After R-CHOP standard chemotherapy 30-40% of patients still have poor treatment and relapse resistance or even death in a short period of time.Therefore,it is urgent to find new drug targets to improve the survival of patients with DLBCL.This study aims to screen and verify the possible the key factors and signaling pathways related to the occurrence and development of DLBCL at miRNA,mRNA and protein levels,and in cell level further to study the functions of the key factors regulating the signaling pathways.The mechanism of Homo sapiens(hsa)-miR-193a-3p targeting TCL1 regulating PI3K/AKT signaling pathway affecting the proliferation,apoptosis and cycle of DLBCL cells was preliminally elucidated,providing a theoretical foundation for further understanding the occurrence and development and providing new therapeutic targets of DLBCL.Methods: 1)Fresh frozen human DLBCL tissues were selected as experimental group and human lymph node reactive hyperplasia(LRH)as control group,and differential miRNAs were screened by microarray.Bioinformatics analysis of differential miRNAs was conducted to clarify the target genes,biological functions and enrichment signaling pathways of differential miRNAs.The expression of differentially related miRNAs in important signaling pathways was verified by qRT-PCR;2)Fresh frozen human DLBCL tissue was selected as the experimental group and human LRH as the control group.Proteomics was used to screen differential proteins.Bioinformatics analysis was carried out to elucidate the biological function and enrichment signal pathway of different proteins.Parallel reaction monitoring(PRM)and immunohistochemistry were used to verify the protein expression of differential proteins related to important signaling pathways,and the correlation between protein expression and clinicopathological characteristics and prognosis of DLBCL patients was analyzed.Cross analysis of miRNA microarray and proteomics research results was conducted to determine the mechanism of hsa-miR-193a-3p targeting TCL1 regulating PI3K/AKT signaling pathway affecting the proliferation,apoptosis and cycle of DLBCL cells at the cellular level;3)The interaction between hsa-miR-193a-3p and TCL1 was detected by dual luciferase reporter genes.Human DLBCL cells were selected as the experimental group and normal peripheral blood B lymphocytes as the control group.The gene expressions of hsa--193a-3p and TCL1 were detected by qRT-PCR,and the DLBCL cell lines were screened for subsequent experiments.DLBCL cells were transfected with hsa-miR-193a-3p mimic and inhibitor,siRNA-TCL1,siRNA-TCL1+hsa-miR-193a-3p inhibitor,respectively.The negative and blank controls were set up.The proliferation,apoptosis and cycle of DLBCL cells were observed by CCK8 and flow cytometry.The gene and protein expressions of TCL1,the important regulatory factors AKT and p-AKT of PI3K/AKT signaling pathway,apoptosis-related factors BCL-2 and BAX in DLBCL cells were detected by qRT-PCR and Western-blot.Results: 1)This study screened 204 differential miRNAs,54 up-regulated and 150 down-regulated.7522 target genes were predicted by differential miRNA.The target genes of differential miRNA were enriched in Pathway in cancer,MAPK,Focal adhesion,PI3K/AKT signal pathways.qRT-PCR verified differential miRNAs related to PI3K/AKT signaling pathway in DLBCL and showed that the expression level of hsa-miR-193a-3p,hsa-miR-19b-3p,hsa-miR-370-3p and hsa-miR-490-5p were decreased,but hsa-miR-630 was increased;2)This study screened 335 differential proteins,131 up-regulated and 204 down-regulated.Differential proteins were mainly enriched in Pathways in cancer,PI3K/AKT,Alcoholism and Necroptosis signal pathways.PRM and immunohistochemistry verified the differential proteins related to PI3K/AKT signaling pathway in DLBCL and found that the protein expressions level of TCL1,HSP90,AKT and IKK? were increased.Survival analysis found that high expression of TCL1 was an independent factor for short overall survival and progression-free survival in DLBCL patients;3)TCL1 was confirmed to be the target gene of hsa-miR-193a-3p.In DLBCL cells,the expression of hsa-miR-193a-3p was significantly decreased,while the expression of TCL1 mRNA was significantly increased.Hsa-miR-193a-3p mimics is a tumor suppressor in DLBCL,and the expression of hsa-miR-193a-3p is increased in DLBCL.The high expression of hsa-miR-193a-3p mimics can reduce the proliferation activity of DLBCL cells,increase apoptosis,block cell cycle in S phase,block DNA synthesis,and regulate cell conversion between S phase and subsequent G2/M,G0/G1 phase.Hsa-miR-193a-3p inhibitor reduced the expression of hsa-miR-193a-3p in DLBCL,and its low expression increased the proliferation and apoptosis of DLBCL cells,and could reverse the regulation effect of mimic on cell cycle.TCL1 is an oncogene in DLBCL,and TCL1-siRNA can silence the expression of TCL1 in DLBCL.Silencing the expression of TCL1 can inhibit the proliferation of DLBCL cells,promote apoptosis,arrest the cell cycle in S phase,and inhibit the activation of PI3K/AKT signaling pathway.However,adding hsa-miR-193a-3p inhibitor can reverse the inhibitory effect of siRNA-TCL1 on PI3K/AKT signaling pathway.Conclusion: 204 differential miRNAs were screened by miRNA microarray,54 were up-regulated and 150 were down-regulated.Proteomics was used to screen 335 differential proteins,131 were up-regulated and 204 were down-regulated.The related factors validation of PI3K/AKT signaling pathway,which an important signal pathway that has been screened,found that the expression of hsa-miR-193a-3p in DLBCL was decreased and the protein expression of TCL1 was increased and the survival analysis showed that the high positive expression of TCL1 protein was an independent influencing factor for the short overall survival and progression-free survival of DLBCL patients.Hsa-miR-193a-3p can regulate the proliferation,apoptosis,cycle and other biological behaviors of DLBCL cells through the target gene TCL1,and hsa-miR-193a-3p inhibitor can reverse the inhibitory effect of siRNA-TCL1 on PI3K/AKT signaling pathway,thus affecting the occurrence and progress of DLBCL.