Expression And Biological Function Of MiR-199a-3p In Diffuse Large B-cell Lymphoma

Background Diffuse large B-cell lymphoma(DLBCL)is the most common aggressive nonHodgkin’s lymphoma,which has great differences in cytology,gene expression,and immunophenotype.At present,the first-line treatment of DLBCL is mainly chemotherapy combined with rituximab and other biological targeted therapies.This treatment method enables 40%-60% of patients to achieve complete remission,but there are still some patients who relapse or are refractory after standard first-line treatment.For patients with relapsed and refractory DLBCL,exploring new molecular targets will have extremely important significance for the diagnosis,treatment and prognosis of DLBCL.Studies have found that the onset and progression of DLBCL is closely related to micro RNA(miRNA),and can become a molecular biomarker for DLBCL diagnosis,treatment and prognosis monitoring.Studies at home and abroad have found that miR-199a-3p is highly expressed in some malignant tumors and can affect the biological functions of cells.However,there are currently few studies on miR-199a-3p in DLBCL.expression and biological function in DLBCL to explore the role of miR-199a-3p in the pathogenesis and progression of DLBCL.Objective1.To study the expression of miR-199a-3p in diffuse large B-cell lymphoma cell lines and normal human lymphocytes,and to explore the significance of the abnormal expression of miR-199a-3p in diffuse large B-cell lymphoma cell lines.2.To study the effect of miR-199a-3p on the cytological functions of diffuse large Bcell lymphoma cell lines LY8,U2932 and EJ-1,such as proliferation,apoptosis,migration,invasion and colony formation.Methods1.The qRT-PCR method was used to determine the expression of miR-199a-3p in DLBCL cell lines LY8,U2932,EJ-1,normal human lymphocytes were used as a control,and the differentially expressed miR-199a-3p was statistically analyzed.2.After successfully transfecting the DLBCL cell line with the lentiviral vector,qRTPCR showed that the expression of miR-199a-3p in the miR-199a-3p inhibitor group was significantly reduced(P﹤0.05),while the expression of miR-199a-3p inhibitor NC in the miR-199a-3p inhibitor NC group was significantly reduced.The expression of miR-199a-3p did not change significantly(P﹥0.05).3.The CCK-8 cell proliferation experiment was used to detect the effect of miR-199a-3p on the proliferation of DLBCL cell lines,and flow cytometry was used to determine the effect of miR-199a-3p on the apoptosis of DLBCL cell lines.4.Flow cytometry detected the apoptotic rate of miR-199a-3p inhibitor group,miR-199a-3p inhibitor NC group and control group.It was found that the levels of apoptosis of the three groups were statistically different(P﹤0.05).5.The target genes of miR-199a-3p were searched through the Starbase database,and the proteins interacting with Bcl-2 and c-MYC were searched using the protein action database STRING;the Western Blot experiment was used to detect Bcl-2 and c-before and after the lentivirus transfection of the DLBCL cell line The expression of MYC protein,so as to speculate the possible mechanism of miR-199a-3p affecting the occurrence and development of DLBCL.Results1.The qRT-PCR results showed that the expression of miR-199a-3p in DLBCL cell lines EJ-1,LY8,U2932 was significantly higher than that of normal human lymphocytes,and the difference was statistically significant(P﹤0.05).2.After lentivirus transfection,the cell line RNA was extracted for qRT-PCR detection.The results showed that the expression level of miR-199a-3p in the miR-199a-3p inhibitor group was significantly down-regulated(P ﹤ 0.05),while miR-199a-3p inhibitor NC The expression of miR-199a-3p in the group did not change significantly(P﹥0.05).3.The CCK-8 method cell proliferation experiment showed that the OD value of the miR-199a-3p inhibitor group decreased significantly with the extension of the culture time.Compared with the control group and the miR-199a-3p inhibitor NC group,the difference was statistically significant(P ﹤ 0.05),but there was no statistically significant difference between the control group and the miR-199a-3p inhibitor NC group(P﹥0.05).4.Flow cytometry showed that the levels of cell apoptosis in the miR-199a-3p inhibitor group,miR-199a-3p inhibitor NC group and control group were statistically different(P﹤0.05).5.The Transwell migration test showed that the number of cells in the lower chamber of the miR-199a-3p inhibitor group was significantly lower than that of the miR-199a-3p inhibitor NC group and the control group,and the difference was statistically significant(P﹤0.05),while miR-199a-3p There was no significant difference in the number of cells in the lower chamber between the inhibitor NC group and the control group(P﹥0.05).6.Clone formation experiments showed that the clonality of miR-199a-3p inhibitor NC group and control group were not significantly different(P﹥0.05),but compared with miR-199a-3p inhibitor group,the clonality of miR-199a-3p inhibitor group Significantly weakened(P﹤0.05).7.A search of the Starbase database found that the target genes for miR-199a-3p were BCL2L11,BID,PMAIP1 and MAX.The proteins found in the STRING database that interact with BCL-2 and c-MYC also included BCL2L11,BID,PMAIP1,and MAX,among which BCL2L11 and Bcl-2 has the highest correlation;Western Blot experiments showed that the expression of Bcl-2 protein in the miR-199a-3p inhibitor group decreased.Compared with the miR-199a-3p inhibitor NC group and the control group,the difference was statistically significant(P﹤0.05),while the expression of cMYC protein did not change significantly before and after transfection(P﹥0.05).Conclusion1.miR-199a-3p is highly expressed in DLBCL cell lines.Inhibition of miR-199a-3p expression can reduce tumor cell proliferation,migration and cloning ability,and promote its apoptosis.2.In diffuse large B-cell lymphoma cell lines,Bcl-2 may be regulated by the miR-199a-3p/BCL2L11 pathway.