Study On The Mechanism And Drug Sensitivity Of Hsa-miR-28-5p/YWHAZ Axis In Diffuse Large B Cell Lymphoma

Objective: Micro RNAs(miRNAs)participate in the comprehensive biological process of several cancer types.In the previous study of this group,it was found that hsa-miR-28-5p was down-regulated,but tyrosine 3-monooxygenase/tryptophan5-monooxygenase activating protein zeta(14-3-3ζ or YWHAZ)was upregulated in diffuse large B-cell lymphoma(DLBCL)tissues.It was predicted that YWHAZ was a target gene for hsa-micro RNA(miR)-28-5p using bioinformatics analysis.The purpose of this study is to reveal the mechanism and drug sensitivity of hsa-miR-28-5p/YWHAZ axis in DLBCL.Methods: 1)In this study,the expression level of hsa-miR-28-5p,YWHAZ was examined using quantitative real-time polymerase chain reaction(q RT-PCR)in 15 fresh and frozen DLBCL tissues,respectively.YWHAZ and other differential proteins related to PI3K/AKT signaling pathway were tested by immunohistochemistry in formalin-fixed paraffin-embedded(FFPE)tissues of DLBCL patients.The epidemiological and clinical features of DLBCL were analyzed retrospectively,and then by observing the morphologic features of DLBCL,combining with the results of immunohistochemistry,the clinicopathological features of DLBCL in different immunotyping were analyzed.To analyze the protein expression levels of YWHAZ,AKT,p-AKT,BAD,BAX,and BCL-2,which are related to PI3K/AKT signaling pathway in DLBCL tissues,the correlation between YWHAZ and AKT,p-AKT,BAD,BAX,and BCL-2,and the correlation between the clinicopathological features of DLBCL patients and the expression levels of PI3K/AKT signal pathway-related differential proteins in DLBCL tissues.Survival analysis was used to investigate the correlation between the expression levels of the above-mentioned differential proteins and the prognosis of DLBCL patients.2)A dual-luciferase reporter assay was employed to explore the relationship between YWHAZ and hsa-miR-28-5p.q RT-PCR was used to detect the expression of hsa-miR-28-5p and YWHAZ in DLBCL cells and normal peripheral blood B lymphocytes in order to determine the final cell line.Transient transfection of hsa-miR-28-5p mimic(inhibitor)was performed to overexpress hsa-miR-28-5p(inhibit hsa-miR-28-5p)in DLBCL cells.Transient transfection of siRNA-YWHAZ was performed to suppress the expression of YWHAZ in DLBCL cells.Transient transfection of siRNA-YWHAZ + hsa-miR-28-5p inhibitor was performed to suppress the expression of YWHAZ in DLBCL cells,and simultaneously,the expression level of hsa-miR-28-5p was suppressed in DLBCL cells.To observe the effect of hsa-miR-28-5p inhibitor,hsa-miR-28-5p mimic,siRNA-YWHAZ,siRNA-YWHAZ + hsa-miR-28-5p inhibitor on the biological behavior of DLBCL cells,and set up the corresponding control,respectively.A Cell Counting Kit-8 assay was conducted to evaluate cell proliferation,and flow cytometry was showed to examine cell apoptosis and cell cycle progression.The gene and protein expression levels of YWHAZ,BAX,BAD and BCL-2 were detected by q RT-PCR and Western blot in different modes of expression,respectively.3)Different adriamycin(Doxorubicin)concentrations were used to interfere DLBCL cells.CCK-8 assay was used to detect the proliferation of DLBCL cells in order to find the optimal concentration of Doxorubicin.Transfection of hsa-miR-28-5p mimic NC and hsa-miR-28-5p mimic to Doxorubicin-mediated DLBCL cells,and then set up blank control groups.The effects of hsa-miR-28-5p on Doxorubicin sensitivity in DLBCL cells were observed at different intervention time after DLBCL cells were cultured,or after 24 hours of transfection,each group added different concentration of Doxorubicin,and then DLBCL cells were cultured for 24 hours to observe the effects of hsa-miR-28-5p on Doxorubicin sensitivity.The proliferation,early apoptosis and late apoptosis of DLBCL cells were tested by soft Agar colony forming assay,Mitochondrial Membrane Potential Assay,and Caspase-3 activity assay,respectively.The apoptosis and cell cycle of DLBCL cells were detected by Annexin V-PE/7-AAD,and PI/RNase Staining Buffer,respectively.Results: 1)It was found that hsa-miR-28-5p was significantly downregulated,and YWHAZ was upregulated in DLBCL tissues.Using correlation analysis,it was found that hsa-miR-28-5p was negatively correlated with YWHAZ(r=-0.377,P = 0.040).YWHAZ was negatively correlated with BAD(r =-0.177,P = 0.036),YWHAZ was positively correlated with BCL-2(r = 0.180,P = 0.033).Survival analysis suggested that patients with combined therapy had longer overall survival rate(OS)than those with surgery or chemotherapy alone,and the risk of death was lower than those with surgery or chemotherapy alone.Aa IPI score > 2 and concomitant systemic disease were independently prognostic risk factors for OS in patients with DLBCL.The positive expression of LDH and YWHAZ was independently prognostic risk factors of OS in DLBCL patients,respectively,and the positive expression of BAX was an independently prognostic and protective factor of OS in DLBCL patients.2)It was proved that YWHAZ was the target gene of hsa-miR-28-5p at the cellular level in this study.YWHAZ is an oncogene in DLBCL,silencing YWHAZ expression can inhibit DLBCL cell reproduction,encourage cell apoptosis,cause cell cycle to halt in S phase,and inhibit DNA synthesis of DLBCL cell,the transformation of DLBCL cells between S phase and G2/M phase,G0/G1 phase was regulated.hsa-miR-28-5p is a tumor suppressor in DLBCL,over-expression of hsa-miR-28-5p can inhibit the proliferation of DLBCL cells,promote the apoptosis of DLBCL cells,and lead the cell cycle to arrest in S phase.On the contrary,low expression of hsa-miR-28-5p can promote the proliferation,inhibit the apoptosis of DLBCL cells,and reverse the role of hsa-miR-28-5p mimic on DLBCL cell cycle.hsa-miR-28-5p inhibitor could partially reverse the inhibitory effect of siRNA-YWHAZ on DLBCL cell proliferation,the promoted effect of siRNA-YWHAZ on DLBCL cell apoptosis,and the stopped effect of siRNA-YWHAZ on the DLBCL cell cycle.hsa-miR-28-5p can affect the proliferation,apoptosis and cell cycle of DLBCL cells by targeting YWHAZ.3)The optimal concentration of Doxorubicin was 3.028 μmol/l.The effects of Doxorubicin on DLBCL cells were time-and concentration-dependent.hsa-miR-28-5p mimic + Doxorubicin significantly decreased the proliferation of DLBCL.The apoptosis rate of DLBCL cells was the highest in hsa-miR-28-5p mimic +Doxorubicin group again.It was further confirmed that hsa-miR-28-5p mimic combined with Doxorubicin had the best effect on promoting apoptosis of DLBCL cells.After the intervention of hsa-miR-28-5p mimic + Doxorubicin on DLBCL cells,cell cycle was arrested in S phase,and DNA synthesis was blocked.hsa-miR-28-5p mimic +Doxorubicin could regulate cell cycle of DLBCL cells.These results suggest that overexpression of hsa-miR-28-5p combined with Doxorubicin may be involved in the development of DLBCL by affecting the proliferation,apoptosis,and cycle of DLBCL.Conclusion: It was confirmed that hsa-miR-28-5p was significantly downregulated,and YWHAZ was upregulated in DLBCL tissues by q RT-PCR.Survival analysis showed that patients with combined therapy had longer OS than those with surgery or chemotherapy alone.The positive expression of YWHAZ was an independent prognostic risk factor of OS in DLBCL patients.YWHAZ was the target gene of hsa-miR-28-5p in DLBCL.hsa-miR-28-5p may be a tumor suppressor in DLBCL,and YWHAZ may be a cancer gene in DLBCL.hsa-miR-28-5p can affect the proliferation,apoptosis,and cell cycle of DLBCL cells by targeting YWHAZ.The combination of hsa-miR-28-5p mimic and Doxorubicin may be more effective than Doxorubicin alone in inhibiting DLBCL cell proliferation,promoting apoptosis,and blocking cell cycle.The effects of Doxorubicin on DLBCL cells were time-and concentration-dependent.Over-expression of hsa-miR-28-5p may enhance the sensitivity of DLBCL cells to Doxorubicin,and thus improve the therapeutic effect of DLBCL..