The Effect And Mechanism Of Mir-149 Antisense Oligonucleo Tide On The Proliferation And Apoptosis Of Jurkat Cell Line

Objective Acute lymphoblastic leukemia(Acute lymphoblastic leukemia ALL)is a type of malignant,clonal disease characterized by uncontrolled proliferation of hematopoietic stem and progenitor cells of the bone marrow,differentiation disorders,and suppressed apoptosis.In recent years,the incidence of the disease has increased.The trend,although at this stage,after drug chemotherapy,the remission rate of the disease has increased significantly,but due to the existence of multidrug resistance problems,the recurrence rate is also higher than before.miRNA is an endogenous non-coding RNA that binds to the non-coding sequence at the 3’ end of its corresponding target gene,resulting in degradation of the mRNA corresponding to the target gene or inhibition of the translation process to reach the post-transcriptional level The purpose of a series of regulation and control of the relative expression level of the target gene.Among many miRNAs,MiR-149 has been studied and found to be abnormally expressed in a variety of malignant diseases.Among them,JunB mRNA is considered to be one of the direct targets of miR-149 in leukemia cells,and miR-149 is abnormal in leukemia cells.Expression,through the targeted regulation of JunB mRNA expression,and then participate in the occurrence and development of a variety of leukemia.JunB protein is the main component of the transcription factor of the active protein and participates in many processes such as cell mitosis,cell cycle and apoptosis pathway regulation,signal molecule transmission and so on.It plays a role in the invasion and metastasis of malignant tumors.At present,there are few studies on the expression level of miR-149 in leukemia Jurkat cell lines,and there is no study on the expression level and effect of JunB mRNA.In order to investigate the effect of miR-149 on proliferation and apoptosis of Jurkat cells and its possible mechanism of action,this study provides a basis for the clinical treatment of leukemia.Therefore,this experiment was designed.Methods Antisense oligonucleotide sequence of artificially synthesized miRNA-149,negative control sequence NC with no homology to human gene sequence,and liposome transfection method both mediated by Lipofectamine 2000,The miRNA-149 antisense oligonucleotide sequence and NC sequence were transfected into Jurkat cells,and the expression levels of miR-149 and JunB mRNA in Jurkat cells after transfection were detected by Real-time PCR;Flow cytometry was used to detect the effect of transfected miRNA-149 antisense oligodeoxynucleotides on the proliferation and apoptosis of Jurkat cells.The Western-Blot method was used to detect the antisense oligonucleotides of miRNA-149.The effect of JunB protein expression.Results1.The designed miR-149 antisense oligonucleotide sequence can effectively silence the expression of miR-149 in Jurkat cells.Compared with the blank control group,the expression level of miR-149 in experimental group was significantly decreased(0.401 ±0.065 vs 1.021 ±0.145),JunB mRNA expression level was significantly increased(1.003 ±0.072 vs.0.164±0.013),the two experimental groups exist significant difference(P<0.05);transfection negative sequence group Jurkat cell miR-149 and JunB mRNA expression levels No significant changes were found(0.998±0.234 vs 1.02±0.1453,0.153±0.014 vs 0.164±0.013).The two experimental groups exist no significant difference(P>0.05).It was suggested that this experiment successfully combined the antisense oligonucleotide sequence that can effectively silence the expression of miR-149 gene and up-regulate the expression of JunB mRNA.The synthesized NC sequence has no homology with human genome and can be used in the next experiment.2.Flow cytometry and MTT assay detected the transfection of miR-149 antisense oligonucleotide sequence,compared with the blank control group,Jurkat cell apoptosis rate was significantly increased,the survival rate was significantly reduced,the difference between the two Statistically significant,there was no significant change in the apoptosis rate of the NC sequence group,and the difference was not statistically significant.It is suggested that the miR-149 antisense oligonucleotide sequence can inhibit the proliferation of Jurkat cells.3.Western Blot assay showed that the expression of JunB protein in each experimental group was changed.Compared with the blank control group,the expression of JunB protein was increased after transfection of miR-149 antisense oligonucleotide sequence(0.798±0.013 vs 0.116± 0.003),the difference was statistically significant(P<0.001).The expression of JunB protein in the NC group was(0.152 ±0.004 vs 0.116 ±0.003),P>0.05,and the difference was not statistically significant.The experimental results are consistent with the results of Real-time PCR,suggesting that the designed miR-149 antisense oligonucleotide sequence can specifically and effectively up-regulate the expression of JunB mRNA in Jurkat cells,which in turn increases the expression of JunB protein.Conclusions1.The miR-149 antisense oligonucleotides synthesized in this experiment can inhibit the expression of miR-149 in Jurkat cells and up-regulate the expression of JunB mRNA.2.Transfection of miR-149 antisense oligonucleotides increased the expression of JunB protein.3.Transfection of miR-149 antisense oligonucleotides can significantly inhibit Jurkat cell proliferation and promote apoptosis,which may be achieved through the activation of multiple apoptosis-related signaling pathways in cells.