The Anti-tumor Effect Of MiR-28-5p Targeting BECN1 In DLBCL Cells And The Anti-tumor Mechanism Of Curcumin

Lymphoma is the most common malignant tumor in children,and it ranks the third among malignant diseases in children.Childhood lymphoma can be divided into non-Hodgkin’s lymphoma(NHL)and Hodgkin’s lymphoma(HL),in which NHL accounts for about 80% of childhood malignant lymphoma and HL about 20%.With the development of molecular biology research,the importance of autophagy and apoptosis in the genesis and development of many kinds of tumors has been widely concerned,and many key targets of gene regulation have been found,so as to provide theoretical basis for blocking the occurrence and development of tumors.Autophagy is a process in which cells use lysosomes to degrade their damaged organelles and macromolecules so as to realize the metabolic needs of cells and the renewal of some organelles.The introduction of autophagy death into the clinical treatment of cancer is a significant advance in the medical field.beclin1,a mammalian autophagy regulatory gene,is a candidate for tumor suppressor gene,which can induce autophagy,apoptosis and inhibit the proliferation of tumor cells.There are many factors affecting autophagy,among which the anti-tumor effect of curcumin in inducing autophagy has been concered.Curcumin is a lipophilic polyphenolic compound extracted from the rhizome of curcuma chinensis,has many biological activities and pharmacological actions,such as anti-inflammatory,antioxidant,free radical scavenging,anticancer,anti-atherosclerosis,promoting wound repair and immune regulation,etc.In previous research on miRNA expression profiles during human B-cell lymphomagenesis,miR-28 was identified as an intragenic miRNA located on chromosome 3q28,which was specifically induced by the germinal center(GC)reaction.Evidence has revealed the potential involvement of miR-28 in the development of B-cell lymphoma,overexpression leads to the inhibition of lymphoma cell proliferation.In the present study,we hypothesized that curcumin exerts anti-proliferative effects on DLBCL cells by upregulating miR-28 expression.However,the role and mechanism in the pathogenesis and Progression of Diffuse Large B-cell Lymphoma are still unclear.Part one Expression and clinical significance of beclin1 and Bcl-2 in non Hodgkin’s lymphomaObjective: To study the expression and correlation of beclin1 and Bcl-2in non Hodgkin’s lymphoma(NHL)tissues in children,and explore the value of beclin1 and Bcl-2 in diagnosis,targeted therapy and prognosis.Methods: The data of 86 children with NHL diagnosed in the fourth hospital of Hebei Medical University were collected from June 2010 to may2020,and the expression of beclin1 and Bcl-2 was detected by immunohistochemistry.Using sas9.1 statistical software,chisquare test and Spearman test were used for statistical analysis.Results:1.beclin1 expression was lower in mature B-lymphocytic and precursor lymphoblastic NHL tissues,while Bcl-2 protein expression was higher than that in normal lymphoid tissues(P<0.05).2.In the experimental group,the expression level of Bcl-2 was positively correlated with the stage of IPNHLSS.3.In NHL children with clinical complete remission and low risk of IPI,the high expression of beclin1 was relatively high,and the low expression of Bcl-2 was relatively high;In the group with no clinical remission and high risk of IPI,beclin1 expression was relatively low,and Bcl-2 expression was relatively high.4.beclin1 and bcl-2 protein expression in children with NHL.Conclusion:1.beclin1 and Bcl-2 protein expression were negatively correlated in children’s NHL tissues.2.The expression level of Bcl-2 protein in children’s NHL was positively correlated with the stage level of IPNHLSS.3.beclin1 expression was correlated with clinical complete remission,IPI risk and LDH level in children with NHL;The expression level of Bcl-2protein was correlated with the clinical complete remission and the risk of IPI.4.beclin1 was unexpressed and Bcl-2 gene was overexpressed in children’s NHL,and both of them may promote tumor invasion,metastasis and progression,which may be a factor for poor prognosis.Part two The expression and significance of BECN1 in DLBCL were analyzed based on public databaseObjective: To predict the importance of BECN1 in the occurrence and development of DLBCL,biological function analysis and possible related cell signal transduction pathways.Methods: TCGA database and GTEX database were respectively used to download the high-throughput sequencing data of DLBCL patients and healthy population tissues.Rstudio software was used to screen m RNAs differentially expressed between carcinoma and normal tissues.The heatmap of gene expression was drawn by R package Pheat Map.Use the R package GGanatogram package to draw anatomical map;Through the R package cluster Profiler/enrichplot of differentially expressed genes on the GO,KEGG enrichment and enrichment of GSEA analysis function analysis.Results:1.Compared with the normal tissue,the expression of BECN1 in DLBCL tissue was significantly decreased(P<0.01),and there were differences in expression in different organs of male and female.2.GO and KEEG enrichment analysis showed that:Differential gene functions were mainly concentrated in 183 metabolic pathways involving neutrophils activation in immune response,ribonucleoprotein complex synthesis,autophagy and apoptosis.These include the MAPK signaling pathway,human T-cell leukemia associated complex retrovirus(HTLV-1)infection,fluid shear stress and atherosclerosis,hematopoietic cell line and cell cycle signaling pathways.3.There was no statistical difference in the expression level of BECN1 in the group of DLBCL patients aged ≥60 years and the group of patients aged< 60 years.The expression level of BECN1 in DLBCL patients showed no statistical difference between male and female.There was no statistical difference in BECN1 expression level between the T1-2 group and the T3-4group of DLBCL patients.Conclusion:1.BECN1 expression in DLBCL tissues was significantly lower than that in normal tissues.2.BECN1 is mediated by PI3K/ Akt /m TORC1 pathway during the occurrence and development of DLBCL,MAPK signaling pathway plays a role in autophagy and apoptosis of cells too.Part three The role and mechanism of miR-28-5p mediating Curcumin in the proliferation of OCI-LY7 cellsObjective: To investigate the role and mechanism of Curcumin in the proliferation of Human Lymphoma Cell Line OCI-LY7.Methods: OCI-LY7 cell line was established and transfected with Lipofectamine-2000.According to Curcumin concentration gradient: 2.5μM,5μM,10μM,20μM,40μM,the experimental components were divided into 5groups.The expression of miR-28-5p in OCI-LY7 cells was detected by q RT-PCR Assay,and the proliferation of OCI-LY7 cells was detected by MTT assay.Results:1.The effect of curcumin on miR-28-5p levels was detected by q RT-PCR,miR-28-5p expression was significantly upregulated in the curcumin group at24(40μM,P<0.05),48(20μM: P<0.05;40μM: P<0.01),and 72 hours(10μM:P<0.05;20μM: P<0.01;40μM: P<0.01)compared with its levels in the control group.2.The OCI-LY7 cells were treated with curcumin(2.5,5,10,20,and40μM),cell viability was significantly decreased in the curcumin group(40μM)at 24 hours compared with the findings in the control group(P<0.05).In addition,curcumin(10-40μM)significantly decreased cell viability at 48 and72 hours(both P<0.05).3.Compared with the findings for curcumin alone,cell viability was significantly increased in the presence of curcumin and the miR-28-5p inhibitor(P<0.01).The NC failed to influence cell viability.In addition,the efficiency of transfection was verified by the decreased miR-28-5p levels following treatment with miR-28-5p inhibitor(P<0.01).Conclusion:1.Overexpression of miR-28-5p can inhibit the proliferation of OCI-LY7 cells,and curcumin affect the expression of the miR-28-5p.2.The miR-28-5p inhibition is able to inhibit the anti-tumor activity of curcumin,and the miR-28-5p may mediate the anti-tumor effort of curcumin.Part four The regulation effect of miR-28-5p targeting BECN1 on apoptosis and autophagy of OCI-LY7 cellsObjective: To investigate the role and mechanism of Curcumin and MIR-28-5P in OCI-LY7 cell apoptosis and autophagy.Methods: The effects of curcumin and miR-28-5p on apoptosis and autophagy of OCI-LY7 cells were determined by Tunel fluorescence staining and Western blot,and the targeting relationship between miR-28-5p and BECN1 was determined by on-line prediction and dual-luciferase reporter gene assay.Results:1.Results of apoptosis-associated protein fluorescence staining and detection: compared with the control group,curcumin significantly increased cleaved caspase-3 expression(P<0.05).Compared with the curcumin group,cleaved caspase-3 expression was decreased in the curcumin+miR-28-5p inhibitor group(P<0.05).2.The results demonstrated that curcumin significantly decreased the protein levels of BECN1 and the LC3B-II/LC3B-I ratio as well as increased P62 expression(P<0.05)in OCI-LY7 cells.Moreover,the miR-28-5p inhibitor significantly prevented these curcumin-induced changes(P<0.05),whereas the NC had no effects.3.Prediction of targeting relationship between miR-28-5p and BECN1 based on online database.The dual-luciferase reporter assay revealed that the miR-28-5p mimic significantly reduced luciferase activity in the wt BECN1 group compared with the findings in the NC group(P<0.01);however,the miR-28-5p mimic did not affect luciferase activity in the mut BECN1 group.Conclusion:1.The miR-28-5p mediate Curcumin promotes OCI-LY7 cell apoptosis.2.BECN1 is the target gene of miR-28-5p,and the overexpression of miR-28-5p can directly target BECN1 to inhibit autophagy.3.In DLBCL,miR-28-5p targets BECN1 to inhibit autophagy and prevent tumor growth.Summary:1.beclin1 and Bcl-2 protein expressions were negatively correlated in children’s NHL tissues.beclin1 and Bcl-2 protein expression levels were correlated with clinical complete response and IPI risk levels in NHL children.2 BECN1 mediates autophagy and apoptosis through PI3K/ Akt/m TORC1 during the occurrence and development of DLBCL,and may also play a role through MAPK signaling pathway.3.The overexpression of miR-28-5p can inhibit the proliferation of OCI-LY7 cells,and miR-28-5p mediates the anti-tumor effect of curcumin.4.miR-28-5p mediated curcumin induced OCI-LY7 cells apoptosis.BECN1 is the target gene of miR-28-5p,and curcumin induced the overexpression of miR-28-5p,which directly target BECN1 to inhibit autophagy.