| Obejective1.Knock out micro RNA-155 at the genome level by applying CRISPR /Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated gene-editing technology in FLT3-ITD+acute myeloid leukemia MV411 cell line and establish a miR-155 knockout cell line.2.Based on the miR-155 knockout MV411 cell line,study the effects of proliferation and glucose metabolism pathways after knocking out miR-155 in MV411 cell line,and preliminary explore the relationship between cell’s sensitivity change in tyrosine kinase inhibitor(TKI)and glucose metabolism.Methods1.RT-PCR was used to detect the expression of miR-155 in different cells and the change of the expression of miR-155 after applying the chemotherapeutic drugs doxorubicin,Quizartinib,and Midostaurin into MV411 cells.2.Design sg RNA targeting miR-155 and construct a vector,package the lentivirus,infect MV411 cells,and test lentivirus transfection efficiency(knockout efficiency).The experimental group was MV411/KO25,and MV411/KO26,and the control group(Scramble,Scr)was MV411 / Scr.Construct the miR-155over-expression,lentiviral vector,package the lentivirus,and infect MV411 cells.The experimental group was MV411/OE,and the control group/was MV411/Con.3.Knock out micro RNA-155 at the genome level by applying CRISPR /Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated gene-editing technology in FLT3-ITD+acute myeloid leukemia MV411 cell line and establish a miR-155 knockout cell line.4.Draw cell growth curves and perform cell clone formation experiments to examine the effects of cell growth and proliferation capacity in miR-155 knockout and overexpression cell lines.5.Detect the drug(doxorubicin,Quizartinib,Midostaurin)sensitivity(IC50)of MV411 cells after miR-155 knockout or overexpression by the MTT method.Explore the effect of miR-155 on cell drug sensitivity and its mechanism.6.Detect cell glucose metabolism by detecting glucose uptake,lactic acid production,ATP production.Detect the expression of platelet 6-phosphofructokinase(PFKP),a key enzyme for glycolysis,Pyruvate kinase(PK),lactate dehydrogenase(LDH)and hexokinase(HK)by western blot.Explore the possible mechanism of miR-155 regulation of glycolysis.Results1.Compared with THP-1 and U937 cells,the expression level of miR-155 in MV411 cells with FLT3-ITD mutation is higher;The expression level of miR-155 in MV411 cells was negatively correlated with the drug concentration dependence of the ADM and the FLT3-ITD inhibitors(Quizartinib and Midostaurin),a kind of chemotherapeutic drugs.2.Successfully constructed the miR-155 knockout lentiviral vector(LV-sg Cas9-P2A-puro PCA06825-1/LV-sg Cas9-P2A-puro PCA06826-1)and the overexpression vector(LV-miR-155-OE).The degrees of the virus solution were 1E+9TU/ml,6E + 8TU/ml,and 4E+8TU/ml respectively.After successful infection of MV411 cells,MV411/KO25 and MV411/KO26 cells detected by Surveyor mutation detection was approximately 12.2%,and 18.9% respectively;the relative expression of miR-155 in MV411/OE cells was 2.181 ± 0.12 times that of control cells.3.Cell proliferation experiments and cell clone formation experiments indicate that the proliferative capacity of MV411 cells was significantly inhibited after the miR-155 gene was knocked out.The clone formation rates of MV411 / Scr,MV411 / KO25,MV411 / KO26,were: 30% ± 2.48%,14.7% ± 1.02%.In contrast,the over-expression of miR-155 promoted cell proliferation.The clone formation rates of MV411 / Con and MV411 / OE cells were: 25.8% ± 1.36%,47.8% ± 2.79%(P﹤0.05).4.The change of miR-155 gene expression in MV411 cells was significantly negatively correlated with the drug sensitivity of the cells to ADM,Quizartinib,and Midostaurin.After miR-155 was knocked out,the IC50 of MV411/Scr,MV411/KO25 and MV411/KO26 to ADM were 0.066 ± 0.022 μg / ml,0.041 ±0.012 μg/ml,0.031 ± 0.009 μg/ml(P﹤0.05).After miR-155 was over-expressed,the IC50 of MV411/Con and MV411/OE cells to ADM were 0.0166 ± 0.004μg/ml and0.0353 ± 0.007μg/ml,respectively(P﹤0.05).After miR-155 was knocked out,the IC50 of MV411 / Scr,MV411 / KO25,MV411 / KO26 cells to Quizartinib were0.825 ± 0.109 n M / ml,0.590 ± 0.092 n M /ml,0.303 ± 0.053 n M/ml(P﹤0.05).The IC50 of MV411/Con and MV411/OE cells to Quizartinib were 0.264 ± 0.087 n M /ml,0.813 ± 0.298 n M / ml,respectively(P﹤0.05).After miR-155 overexpression,the IC50 of MV411 / Con and MV411/OE cells to Midostaurin were 0.0203 ±0.007 n M/ml,0.035 ± 0.010 n M/ml,respectively(P﹤0.05).5.After miR-155 was knocked out,the values of glucose uptake,lactic acid production,and ATP production of MV411/KO25 and MV411/KO26 cells in the same time period were significantly reduced compared with the MV411/Scr group(P﹤0.05).After miR-155 overexpression,the value of the glucose uptake of MV411/Con cells in the same time period was significantly increased compared with the MV411/OE group(P﹤0.05).6.After miR-155 knockout,the expressions of PFKP,PKM1/2,PKM2,and HK1 in the cells of each group of MV411/KO25 and MV411/KO26 were down-regulated compared with the control group(P <0.05);Among the MV411/OE cells,the expressions of PFKP,PKM1/2,PKM2,and HK1 were up-regulated compared with the control cells(P <0.05).ConclusionsKnock out of micro RNA-155 could inhibit the proliferation and clone formation of MV411 cells,improve the drug sensitivity of MV411 cells to chemotherapy drugs and FLT3 inhibitors,and inhibit the glucose uptake,lactic acid and ATP production of MV411 cells,which may be related to the decreased expression and activity of key glycolytic enzymes in MV411 cells after mir-155 knockout. |
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