| Objective:In our previous study,we used microarray technology to analyze the miRNA expression profile of DLBCL paraffin-embedded specimens and reactive hyperplasia lymph nodes as control,aiming to find differentially expressed miRNAs.In the first part of this study,the differentially expressed miRNAs in DLBCL were screened,and then further validated in a larger sample size.miR-106a-5p was found to be highly expressed in DLBCL,and the correlation between the expression of miR-106a-5p and clinical prognostic indicators of DLBCL patients and its prognostic value were further explored.In addition,the biological function of miR-106a-5p in the occurrence and development of diffuse large B cell tumor was investigated at the cellular level by cell transfection technology.In the second part of this study,GEPIA website was used to predict the expression level of AID in DLBCL,and tissue microarray technology and immunohistochemical staining were used to verify its protein expression level in DLBCL tissues.Combined with clinical information,the clinical significance and prognostic value of AID expression were further explored.Methods:1.Expression and clinical significance of miR-106a-5p in diffuse large B-cell lymphoma screened by gene chipA total of 105 paraffin samples of diffuse large B-cell lymphoma patients from 2016 to 2019 were selected as the observation group,and 10 samples of reactive hyperplasia lymph nodes in the same period were selected as the control group.Total RNA was extracted from the samples.The volcano plot of gene chip(Flod change up-regulation ≥1.5 or down-regulation < 0.5 and P value <0.05)were used to screen the differentially expressed miRNAs,and the results were reverified in a larger sample.Real-time quantitative PCR was used to detect the levels of differentially expressed miRNAs in the two groups of samples,and miR-106a-5p was finally determined as a research indicator for further discussion.The clinical data of the patients were retrospectively collected,and the relationship between the expression level of miR-106a-5p and the clinicopathological characteristics of diffuse large B-cell lymphoma patients and its effect on prognosis and survival were analyzed.2.Preliminary study on the biological function of miR-106a-5p in diffuse large B-cell lymphoma cellsThe expression level of miR-106a-5p was interfered by transient transfection of miR-106a-5p inhibitor by lipofectamine 3000(invetrogen).q PCR was used to detect the transfection efficiency.CCK8 cell proliferation assay,flow cytometry and cell cycle assay were used to verify the regulatory effect of miR-106a-5p on the biological function of diffuse large B-cell lymphoma cells at the cellular level.3.Bioinformatics was used to predict AID expression and verify its clinical significance in DLBCLThe GEPIA website was used to observe the expression of AID in DLBCL and normal controls.The expression of AID in DLBCL was analyzed at the protein level by immunohistochemical staining on the paraffin-embedded tissue microarray,and the AID was semi quantitatively analyzed by histochemical score(H-score).Patients were followed up regularly.Kaplan-Meier method was used to analyze the effect of AID expression on the overall survival of DLBCL patients.The correlation between miR-106a-5p and AID expression was observed by bioinformatics website Star Base co-expression analysis,and verified in tissues,that in order to provide a basis for subsequent specific molecular mechanism research.Results:1.A total of 13 miRNAs were selected for further validation,in which 6 miRNAs were significantly up-regulated,which was consistent with the microarray results.The other 7 miRNAs were down-regulated in the microarray results,but up-regulated in the sample expansion validation.The up-regulated miR-106a-5p,which showed consistency between the results of extended validation and the differential expression profile of gene chip,was used as the research indicator to further explore.The study showed that there was no correlation between the high expression of miR-106a-5p and clinicopathological parameters,but Kaplan-Meier survival analysis showed that the overall survival of patients with low expression of miR-106a-5p was better than that of patients with high expression of miR-106a-5p,and the difference was statistically significant(P=0.0038).2.miR-106a-5p was up-regulated in diffuse large B cell lymphoma cells such as SU-DHL-4,SU-DHL-8,OCI-LY3,OCI-LY19,WSU-NHL and RC.Transient transfection of miR-106a-5p inhibitor effectively reduced the level of miR-106a-5p in SU-DHL-4 and OCI-LY3 cells.After transfection,the proliferation activity of diffuse large B-cell lymphoma cells was decreased,the apoptosis was increased,and the cell cycle was arrested at G0/G1 phase.3.According to GEPIA website,AID was significantly overexpressed in DLBCL compared with normal controls.The results of immunohistochemical staining based on tissue microarray showed that the AID expression in tumor tissues of DLBCL patients was significantly heterogeneous.The H-score of non-GCB subtype patients was significantly higher than that of GCB subtype patients,and the difference was statistically significant(P=0.046).The survival rate of patients with H-score≥10 was significantly worse than that of patients with H-score<10.Patients with score 10(P=0.0209);Taking 10% as the cut-off value,the survival of patients with positive cell count ≥10% was significantly worse than that of positive cell count<10% of patients(P=0.0409).The co-expression analysis of bioinformatics website Star Base showed that the expression of miR-106a-5p was positively correlated with AID in DLBCL,but there was no significant correlation between them in tissue verification.Conclusion:The differential expression profile of gene chip and qPCR verified the abnormal expression of miRNAs in diffuse large B-cell lymphoma.The expression level of miR-106a-5p is up-regulated in DLBCL,and patients with high expression of miR-106a-5p show poor prognosis,which can be used to predict the survival of patients.In addition,interference of miR-106a-5p level in DLBCL cells significantly inhibited cell proliferation and induced cell apoptosis and S phase arrest.The expression of AID in DLBCL patients has obvious heterogeneity,and DLBCL patients with high expression of AID have poor OS.AID can be used as a poor prognostic factor to predict survival. |
