| Part one The effect of Tanshinone IIA on the proliferation of OCI-AML3 cells and the screening of miRNA-497Objective:To study the effect of Tanshinone IIA(TIIA)on the proliferation of human acute myeloid leukemia cells,and screen the key miRNAs of tiia after it was used in human acute myeloid leukemia cells by transcriptional sequencing,which will lay a foundation for further research.Methods:the TIIA concentration was obtained by CCK-8 method.The best concentration of TIIA was used to extract RNA from 10 primary acute myeloid leukemia patients and then to get the candidate miRNA.Result:The growth inhibition rate of OCI-AML3 cells was the best when the concentration of TIIA was 15ng/ml for 36h.The miRNA expression was detected by using the 15ng/ml TIIA on the primary leukemia cells and after 0,4,8 and 12 hours.The expression of miR-497 was the most obvious,so miR-497 was selected as the research object.Summary:TIIA has an inhibitory effect on the proliferation of AML OCI-AML3 cells,and the expression of miR-497 was significantly increased after TIIA acted on primary leukemia cells of AML patients.Part two Effects of Tanshinone IIA on miR-497 and MAPK pathways in OCI-AML3 cells and primary leukemia cells of acute myeloid leukemiaObjective:To investigate the effects of TIIA on miR-497 and MAPK pathways in OCI AML3 cells and 10 primary leukemia cells,and to lay a foundation for further research.Methods:The expression of mir-497 in OCI-AML3 cells and 10 primary leukemia cells were detected by qPCR.The phosphorylation levels of ERK1/2,JNK and p38 in OCI-AML3 cells and patients with bone marrow mononuclear cells were detected by Western blot.Results:The expression of miR-497 in OCI-AML3 cells increased with the increase of TIIA(0h,4h,8h,12h)and the expression of miR-497 in OCI-AML3 cells increased gradually(P<0.05);At 8 hours,12 hours,there was no significant difference in miR-497 expression(P>0.05).At the same time,miR-497 was not significantly different in OCI-AML3 cells and primary leukemia cells(P>0.05);With the increase of time,the phosphorylation level of ERK1/2 decreased gradually(P<0.05),but JNK and p38 had no significant change(P>0.05).Summary:TIIA increased the expression of miR-497 and decreased the phosphorylation level of ERK1/2 in both acute myeloid leukemia OCI-AML3 cells and primary leukemia cells.Part three Effects of Tanshinone IIA on MAPK pathway in OCI-AML3 cells after miR-497 inhibitionObjective:OCI-AML3 cell lines were selected as the research object to investigate the effect of TIIA on MAPK pathway in acute myeloid leukemia cells after miR-497 inhibition.Methods:miR-497 inhibitor vector was constructed and transfected into OCI-AML3 cells.The green fluorescence was observed under fluorescence microscope to verify the transfection efficiency.The expression of miR-497 was detected by qPCR.After the best dose of tiia was applied to OCI-AML3 miR-497 inhibitor cells,the expression of mirna-497 was detected by qPCR.The proliferation of cells was obtained by CCK-8 method.The phosphorylation levels of ERK1/2,JNK and p38 were detected by Western blot.Results:The OCI-AML3 cells transfected with miR-497 inhibitor vector for 48 hours were observed under fluorescence microscope,and many cells were green fluorescent.miR-497 was significantly lower in miR-497 inhibitor cells than in the control group(P<0.05).The expression of miR-497 in OCI-AML3 miR-497 inhibitor cells was not significantly changed with time(P>0.05),and the bands of p-ERK1/2,p-JNK,p-p38,ERK1/2,JNK and p38 did not change significantly.After 15ng/ml TIIA was applied to OCI-AML3 miR-497 inhibitor cells,the proliferation of miR-497 inhibitor+TIIA,blank group and miR-497 inhibitor group was faster and significantly higher than that of tiia group.The proliferation of miR-497 inhibitor+TIIA and miR-497 inhibitor group was faster than that of the blank group.Summary:After miR-497 inhibited,TIIA had no effect on the proliferation and MAPK pathway of AML3 cells in AML.Part four Effects of Tanshinone IIA on miR-497 and MAPK pathway in OCI-AML3 cells after ERK1/2 inhibitionObjective:The effects of TIIA on miR-497 and MAPK pathways in acute myeloid leukemia cells after ERK1/2 inhibition were investigated in OCI-AML3 cells.Methods:The best concentration of MAPK kinase inhibitor(U0126)on OCI-AML3 cells was obtained by CCK-8 method.The phosphorylation of ERK1/2 was detected by Western blot.The expression of miR-497 in OCI-AML3 cells was detected by PCR.The phosphorylation levels of ERK1/2,JNK and p38 in OCI-AML3 cells were detected by Western blot method.The effect of TIIA on the proliferation of OCI-AML3 cells was obtained by CCK-8 method.Results:For OCI-AML3 cells,the best concentration was U0126 of 40ng/ml.After ERK1/2 inhibition(adding 40ng/ml U0126),with the increase of time(0h,4h,8h,12h),the expression of miR-497 in OCI-AML3 cells was up regulated,and the mRNA expression of miR-497 was not significantly different from ERK1/2 inhibition group and tiia group,but significantly higher than that of the control group,ERK1/2 with the increase of time There was no significant change in phosphorylation level of JNK and p38(P>0.05),and ERK1/2 was very low.After ERK1/2 inhibition,15 ng/ml TIIA was used on OCI-AML3 cells,and the proliferation of the cells was detected at 12h,24h,36h and 48h,respectively.The results showed that the proliferation of ERK1/2 inhibited+TIIA,blank group and ERK1/2 inhibited group was inhibited,and the growth rate of cells decreased with the increase of the time of action,and was significantly lower than that of the blank group.Summary:When ERK1/2 was inhibited,the proliferation rate of OCI-AML3 cells was decreased with or without the involvement of TIIA,while the expression of miR-497 was increased under the action of TIIA,irrespective of whether ERK1/2 was inhibited or not.Conclusions:1.TIIA has a significant inhibitory effect on the proliferation of AML OCI-AML3 cells,and the expression of miR-497 was significantly increased after TIIA acted on primary leukemia cells of AML patients.2.TIIA increased the expression of miR-497 and decreased the phosphorylation level of ERK1/2 in both acute myeloid leukemia OCI-AML3 cells and primary leukemia cells.3.After miR-497 inhibited,TIIA had no effect on the proliferation and MAPK pathway of AML3 cells in AML.4.When ERK1/2 was inhibited,the proliferation rate of OCI-AML3 cells was decreased with or without the involvement of TIIA,while the expression of miR-497 was increased under the action of TIIA,irrespective of whether ERK1/2 was inhibited or not.5.The inhibition effect of TIIA on the proliferation of OCI-AML3 cell line may be realized through miRNA-497/ERK1/2 pathway.First,it elevates miRNA-497,and then inhibitions the phosphorylation level of ERK1/2,and finally realizes the inhibition effect on the proliferation of OCI-AML3 cell line.Moreover,TIIA can inhibit the proliferation of primary cells of acute myeloid leukemia,which provides a new idea for the treatment of acute myeloid leukemia. |
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