| ObjectiveThis research aims to study the expression of hsa-miR-181 a in ALL and the relationship between hsa-miR-181 a and its apoptosis related target gene, and to explore the function of hsa-miR-181 a in different ALL clinical stage in order to provide a new experimental basis on the treatment of ALL.MethodsThe expression of hsa-miR-181 a were detected by Taqman probe fluorescent quantitative Real-time PCR in 4 kinds of acute lymphoblastic leukemia cell lines, 36 cases of ALL patients and 23 cases of non-hematologic malignancy controls in clinical marrow samples, analysis the expression in different clinical stages(initial treatment,remission, relapse) and clinical significance of difference. We collected ALL patients’ the clinical symptoms and signs, laboratory examination results, clinical diagnosis,treatment and prognosis, survival to perform statistical analysis. The hsa-miR-181 a lentiviral vectors were constructed and packaging plasmids were transfected together into 293 T cells to produce high titer virus particles. After the infection of Nalm-6 cells with lentiviral vector, we set up stable hsa-miR-181 a expression cell model to culture.We observed the cell proliferation of Nalm-6 cells using CCK-8 method. The expression of apoptosis related target gene Bcl-2 were detected in over-expression before and after by quantitative Real-time PCR. The protein expression of Bcl-2,caspase-3, caspase-9 were detected in over-expression before and after by western-blot.Results1 Among the three groups of clinical stages, the recurrence group was the highest relative expression of hsa-miR-181 a group with the expression(5.353 ± 4.491),followed with initial diagnosis group(2.332 ± 2.706) and complete remission group(0.461±0.629). The expression of hsa-mi R-181 a in the recurrence group was higher than that in the initial diagnosis group and the remission group with statistically significant difference(P<0.05). The relative expression of hsa-miR-181 a group was in inverse proportion to the amount of hemoglobin by the standard coefficient-0.434 and determination coefficient 0.188 with statistically significant difference(P<0.05).2 pGLV3/H1/GFP-hsa-miR-181a-GFP and Puro lentiviral expression vectors were constructed. GFP was observed by fluorescence microscope and TaqMan probe Real-time PCR were used to confirm that they could highly express in Nalm-6 cell lines,and could produce mature hsa-miR-181 a to play a biological role for further functional study.3 The effect of hsa-miR-181 a on cell proliferation was detected by CCK-8 test and the results showed that hsa-miR-181 a significantly reduced the proliferation of Nalm-6cells(P<0.05). The Bcl-2 expression was detected by Real-time PCR in Nalm-6 cells over-expressed hsa-miR-181 a before and after and Bcl-2 expression in OE group was significantly reduced, that means hsa-miR-181 a may target the expression of Bcl-2 and then promote apoptosis of Nalm-6 cells. The Bcl-2, caspase-3 and caspase-9 protein expression were detected by western blot in Nalm-6 cells over-expressed hsa-miR-181 a before and after. After hsa-miR-181 a over-expressed, Bcl-2 expression decreased,cleave-caspase-3 and cleave-caspase-9 protein increased, that means hsa-miR-181 a may promote the activation of caspase-3 and caspase-9, resulting in the activation of the caspase system of Nalm-6 cells.ConclusionIn ALL, the expression of hsa-miR-181 a was higher in the recurrence group and the remission group. Over-expressed hsa-miR-181 a could inhibit the proliferation of ALL cells. We inferred the high expression of hsa-miR-181 a may have something to do with the compensatory mechanism.The research may provide a novel thought for ALL therapy. |
PDF Full Text Request