MiR-143 Induces Apoptosis In MDS Cells And Enhances Sensitivity To Chemotherapeutic Drugs

PART ONE:MIR-143 INHIBITED THE GROWTH AND INDUCED APOPTOSIS OF SKM-1 CELLS XENOGRAFT TUMOR IN MICEObjective:By detecting miR-143 expression level in MDS/AML patients' bone marrow and myelodysplastic syndromes(MDS)cell line SKM-1 and MUTZ-1 cells,to explore the effects of miR-143 on proliferation and apoptosis of SKM-1 cells,and further explore the effects of miR-143 on growth and apoptosis of SKM-1 cell xenograft in NOD/SCID mice under the condition of complex micro environment.Methods:Bone marrow samples from thirty patients with MDS/AML were collected(18 patients with MDS,12 patients with sAML,8 patients with low-risk MDS,10 patients with high-risk MDS,2 patients with MDS 5q-syndrome),and 10 healthy human as healthy control.The total RNA was extracted from Mononuclear cells-seperated from fresh bone marrow,SKM-1 cells and MUTZ-1 cells by kits.The relative expression of miR-143 was detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR).Recombinant lentiviral vectors LV-hsa-miR-143,LV-hsa-miR-143-inhibitors and their negative control empty vector LV-control were stably transfected into SKM-1 cells.The transfection efficiency of the cells was measured using inverted fluorescence microscopy and flow cytometry.The expression of miRNA-143 in each group after transfection was detected by qRT-PCR.The effect of the changes of the expression level of miR-143 in the cells on the proliferation of SKM-1 cells was detected by CCK-8 method.The cell cycle and apoptosis rate of SKM-1 cells were detected by flow cytometry after the expression level of miR-143 was changed.The LV-hsa-miR-143 group,LV-control group and CON group cell suspension(2×107/200?l)were injected to each group of NOD/SCID mice.Three weeks after tumor formation,the xenograft subcutaneous tumor expression levels were measured by fluorescence in vivo fluorescence imaging instrument.After the mice were sacrificed by cervical dislocation method,subcutaneous tumor was separated and measured its weight(g)and volume(mm3).A complete part of each tumor was then fixed in 4%polyformaldehyde,and paraffin embedded sections were stained with H&E and immunohistochemistry respectively.Finally,we detected the protein expression of cleaved-caspase-3,-8,-9,FasL and AKT in tumor tissues by using Western blot method.Results:Compared with healthy controls,the relative expression levels of miR-143 in the bone marrow samples of MDS/AML patients and SKM-1 cells and MUTZ-1 cells were significantly lower;the high risk MDS group(RAEB-2)was lower than the low risk MDS group(RAEB-1).The expression level of miR-143 in patients with a chromosome 5 deletion(del(5q-))was significantly lower than in patients without chromosomal deletion.The proliferation rate of over-expression miR-143 group was much lower than that of LV-control group and CON group,while the proliferation rate of LV-hsa-miR-143-inhibitors group was significantly higher than that of the two control groups.The apoptotic rate of LV-hsa-miR-143 group was significantly increased by more than one time compared with the two control groups,while the apoptosis rate of LV-hsa-miR-143-inhibitors group was reduced to about a quarter of the two control groups.In addition,the detection of cell cycle in each group showed that compared with the two control groups,the number of cells in LV-hsa-miR-143 group in G1 phase increased by more than 20%,while the number of cells in LV-hsa-miR-143-inhibitors group decreased by 20%.The results of the in vivo living fluorescent imager showed that the fluorescence intensity of the subcutaneous tumor of LV-hsa-miR-143 group mice was significantly lower than that of LV-control group and CON group.The weight and volume of subcutaneous tumor in LV-hsa-miR-143 group was significantly smaller than that of LV-control group and CON group.Compared with LV-control group and CON group,chromatin accumulation,fragmentation and nuclear pyknosis increased significantly and the number of apoptotic bodies increased in LV-hsa-miR-143 group,suggesting that over-expression of miR-143 can promote the apoptosis of mouse subcutaneous tumor tissue cells.Immunohistochemical staining showed that FasL staining was strongly positive in LV-hsa-miR-143 group,while AKT staining was negative,suggesting that miR-143 probably promoted apoptosis in mice tumor tissues through Fas/FasL pathway.The protein expression of FasL and AKT in subcutaneous tumor tissues was detected by Western blot.Compared with two control groups,the expression of apoptosis-related factors caspase-3,-8,-9 and FasL in LV-hsa-miR-143 group were significantly increased at the protein level,while those in LV-hsa-miR-143-inhibitors group were just the opposite.The detection of AKT-a FasL antagonist protein-showed that the protein expression decreased at the time of miR-143 over-expression,but increased in the knockdown of miR-143.Conclusion:miR-143 expression levels in bone marrow samples of MDS/AML patients and SKM-1 cells and MUTZ-1 cells were lower than those of healthy controls.miR-143 increased cell arrest by increasing G1 phase and increasing cell apoptosis by Fas/FasL pathway.It inhibited the proliferation of SKM-1 cells.In NOD/SCID mouse xenograft tissues,over-expression of miR-143 also increased apoptosis through the Fas/FasL pathway and inhibited the growth of subcutaneous tumors.PART TWO:MIR-143 ENHANCES DRUG SENSITIVITY OF CYTARABINE IN SKM-1 CELLS.Objective:To explore the role of miR-143 in cytosine arabinoside(Ara-C)in the chemosensitivity of human myelodysplastic syndrome cell line SKM-1 and the mechanism.Methods:The recombinant lentiviral vectors LV-hsa-miR-143 and LV-control were stably transfected into SKM-1 cells.The experiment was divided into three groups:LV-hsa-miR-143 group,LV-control group,and SKM-1 group.The growth inhibition rate of SKM-1 cells on different concentrations of chemotherapeutic drugs cytosine arabinoside was detected by CCK-8 assay before and after miR-143 transfection.Flow cytometry was used to detect the cycle and apoptotic rate of the three groups of cells.Western blot was used to explore the mechanism of miR-143 enhancing cytotoxicity of cytarabine on SKM-1 cells.Results:The percentage of growth inhibition rate of SKM-1 cells over-expressing miR-143 was significantly different from that of LV-control group and SKM-1 group(P<0.05)at the dosage of Ara-C concentration of 2.5 ?M,5 ?M,10 ?and 25 ?M.The apoptotic rate of SKM-1 cells over-expressing miR-143 group was 20%higher than that of the two control groups when the Ara-C concentration was 5?M.In addition,by detecting the cell cycle of each group,compared with the two control groups,the number of SKM-1 cells in the over-expressing miR-143 group in the G1 phase increased by more than 15%when the concentration of Ara-C was 5?M.The protein expression of apoptosis related factor Akt and pAkt detected by Western blot method showed that the protein signal of pAkt in LV-hsa-miR-143+Ara-C(5?M)group was obviously lower than the group of LV-control+Ara-C(5?M)and SKM-1+Ara-C(5?M)group.Conclusion:Over-expressing miR-143 could increase the sensitivity of SKM-1 cells to Ara-C,which showed that the apoptosis rate increased by reducing pAkt and increased G1 phase block in cell cycle.