Effects Of MicroRNA-132 On Proliferation And Apoptosis Of Leukemia Cells And Related Mechanisms

Objective:Leukemia is a kind of malignant tumor originating from hematopoietic bone marrow stem cells.With the gradual improvement of chemotherapy regimen,the remission rate is also obviously increased or even cured.However,due to the emergence of drug resistance,the recurrence rate is higher.There is also a clear upward trend before.As an endogenous non-coding single-stranded RNA,miRNA is 21 to 25 nucleotides in length and binds to the 3'non-coding end of the corresponding target gene to regulate cell behavior,ie,post-transcriptional level the expression of the target gene is regulated.At present,there are few studies on the expression level and mechanism of action of miR-132 in K562 and Jurkat cell lines.In order to explore the effect of miR-132 on proliferation and apoptosis of leukemia cells and related mechanisms,and provide more basis for targeted therapy of leukemia,this experiment was designed.Methods:1.Identification of overexpression of miRNA-132The biological company(Shanghai Jima Co,Ltd.)synthesized the miRNA-132 mimics sequence and the negative control sequence NC sequence which has no homology with the human gene sequence.miRNA-132 mimics and NC sequences were transfected into K562 and Jurkat cells by Lipofectamine 2000-mediated lipofection.The transfected K562 was detected by real-time quantitative PCR(qRT-PCR).The expression level of miR-132 in Jurkat cells was identified by Lipofectamine 2000 as a mediator of lipofection efficiency.2.Changes in cell proliferation and apoptosis after transfection of K562 and Jurkat with miRNA-132 mimicsThe growth curve of K562 and Jurkat cells after overexpression of miRNA-132 was determined by CCK-8 method.The proliferation and apoptosis of each cell line after transfection were detected by flow cytometry.3.Detection of miR-132 transfected K562 and Jurkat cell malignant cell behavior-related factors after transfectionThe expression of sirtuins family gene SIRT1 and tumor suppressor gene P53 was detected by qRT-PCR.The expression of SRIT1 and acetylated P53 protein was detected by Western-Blot method.4.Statistical analysisStatistical analysis was performed using SPSS 22.0 statistical software.The difference was statistically significant at P<0.05,and the measurement data was expressed as mean± standard deviation.Differences between groups were measured by LSD-t test.One-way ANOVA analysis was used for comparison between groups.Results:1.The results of real-time PCR showed that the expression of miR-132 was significantly increased in K562 and Jurkat cells transfected with miR-132 mimics group,and the expression level of SIRT1 mRNA was significantly decreased.Significance(P<0.05),but the expression level of P53 mRNA was not significantly changed,the results were not statistically significant(P>0.05).The expression of miR-132,SIRT1 and P53 mRNA were detected in K562 and Jurkat cells transfected with negative sequence.There was no significant change in the mean water,and the difference was not statistically significant(P>0.05).This indicated that the expression of miRNA-132 in K562 and Jurkat cells was increased by transfection,and the expression of SIRT1 mRNA was down-regulated,while the high expression of miRNA-132 had no significant effect on the expression of tumor suppressor gene P53.2.The results of CCK-8 showed that the proliferation of K562 and Jurkat cells in miR-132 mimics group was significantly inhibited compared with the negative control group and the blank control group.The difference was statistically significant(P<0.05)3.Flow cytometry results showed that the apoptosis rate of K562 and Jurkat cells transfected with miR-132 mimics group was significantly higher than that of the blank control group,and the difference was statistically significant(P<0.05)The apoptosis rate of the negative sequence group was not significantly different from that of the blank control group,and the difference was not statistically significant(P>0.05).It is indicated that high expression of miR-132 can inhibit the proliferation of K562 and Jurkat cells4.The expression of SRIT1 and acetylated P53 protein in each experimental group was detected by Western Blot.In K562 and Jurkat cells,the expression of SIRT1 protein was decreased and decreased in the transfected group(miR-132 mimics group)compared with the blank control group.About 61.0%and 47.3%,the difference of each group was statistically significant(P<0.05),while the transfection group compared with the blank control group,the expression of acetylated P53 protein increased,about 53.6%and 62.6%,The difference was statistically significant(P<0.05),the expression of SIRT1 protein in the negative control group(miR-132-NC group)was not significantly different from the blank control group(P>0.05),which was not statistically significant.The results of this experiment are consistent with the results of Real-time PCR,indicating that high expression of miR-132 can effectively inhibit the expression of SIRT1 protein in K562 and Jurkat cells.The increase of acetylated P53 protein expression indicates that the low expression of SIRT1 protein is beneficial to P53.Acetylation enhances its anti-cancer effect.Conclusions:High expression of miRNA-132 can inhibit the proliferation of leukemia K562 and Jurkat cell lines and promote its apoptosis,which may be related to the activation of SRIT1/P53 pathway.