The Study Of BCL11A-siRNA Targeted On SUDHL6Cells And Its Molecular Mechanism

Objective： To investigate whether B-cell chronic lymphocytic leukemia(CLL)/lymphoma11A gene (BCL11A) small interference RNA(siRNA) canenhance apoptosis induced by vincrinstine (VCR) of diffuse large B-lymphoma cellsSUDHL6and further analyze the gene expression profile of SUDHL6cells afterBCL11A knockdown and B malignantcells..Methods:1.Chemically synthesized BCL11A-siRNA and scrambled siRNA controlwere transfected into in SUDHL6cells using Hiperfect transfection reagent,respectively.Then the BCL11A-siRNA was combined with VCR, blank control group,scrambled siRNA group, BCL11A-siRNA group, VCR group, scrambled siRNA+VCR group were used as control. The tansfection efficiency was detected by Flowcytometry(FCM) at6hours after transfection with siRNA. Cell proliferation wasassayed by count kit-8method at48h and72h. Cell morphology was deteced byHoechchst33258staining. The apoptosis rate was detected by FCM (AnnexinV-FITC/PI double staining).2. The global gene expression profile was identified usingthe Affymetrix HG-U133Plus2.0array.after BCL11A-siRNA were transfected intoSUDHL6cells. Differentially expressed genes were analyzed by DAVID software.Twenty-one differentially expressed genes were further validated and analyzed fromthe BCL11A siRNA-treated SUDHL6cells by real-time quantitative reversetranscript-polymerase chain reaction (qRT-PCR).The PCR products was observed byagarose gel electrophoresis and DNA sequencing was used to verify the PCRproducts.Results：1. After the siRNA were transfected to SUDHL6cells, the tansfectionefficiency was (44.27±1.66)%by FCM. CCK8showed that the SUDHL6cells wereobviously inhibited in BCL11A-siRNA+VCR group, which had statisticallydifference compared with blank control group, scrambled siRNA group, BCL11A-siRNA group, VCR group, scrambled siRNA+VCR group（p<0.05）.Hoechchst33258staining showed a large number of apoptotic cells, Anexxin V/PIdouble dying assays by FCM indicated that the cell apoptotic rates inBCL11A-siRNA585+VCR group were(71.46±2.53)%,which had statisticallydifference compared with blank control group, scrambled siRNA group,BCL11A-siRNA group, VCR group, scrambled siRNA+BCL11A-siRNA group,the cell apoptosis rates of the latter five group were（3.10±0.30)%,(4.53±0.23)%,(39.64±5.17)%, and (49.73±6.74)%,(50.32±6.18)%respectively by FCM（p<0.05）.2.Gene chip result showed that：there were4,874genes differentiallyexpressed between the BCL11A siRNA-and negative control-transfected cells, inwhich2140genes were upregulated and2734genes were downregulated at least2-fold. The differentially expression genes are involved in various signaling pathways,which mainly closely related to cancer,apoptosis,cell proliferation,B cellsurvival/receptor and TGF-et al pathways. Among the twenty-one verified genes,PTEN、TERT、BCL2L11、TGF2、MYC、TNFAIP3、JUN、JUND were upregulated;BCL2、BAX、TNFSF10、FOXP1、MDM2、COL3A1、ING1、BCL6、RUNX1、RUNX1T1、EBF1、EP300、BMPR2were downregulated. qRT-PCR validation of theselected differentially expressed genes demonstrated agreement the microarrayanalysis with the exception of three genes: ING1and BCL6. Among the verifiedgenes, there is no statistically difference in the expression level of TNFAIP3,RUNX1between BCL11A-siRNA and scrambled siRNA group(P>0.05). But there isstatistically difference in the expression level of the others genes (P<0.05). qRT-PCRconfirmed that BCL2L11was upregulated7.24-fold, and BCL-2was downregulated3.23-fold after the transfection of BCL11A siRNA. The sizes predicted for the PCRproducts of the selected differentially expressed genes were confirmed by2%agarosegel electrophoresis and gene sequencing.Conclusion：1. BCL11A-siRNA can enhance apoptosis induced by vincrinstine of SUDHL6cells.2. BCL11A siRNA-induced apoptosis in SUDHL6cells might occurr via the upregulated BCL2L11and downregulated BCL-2.3. Our results indicate that in malignant B cells, BCL11A might closely associate withvarious related-tumor gens.