| Objective:1.To study the expression of P90 ribosomal S6 kinase(RSK1)in patients with acute myeloid leukemia(AML),and to explore the correlation between its expression levels and clinical features and prognosis of patients,so as to provide a theoretical basis for finding new prognostic markers and biomarkers of AML.2.To explore the effect of RSK1 expression on apoptosis and proliferation of AML cells(THP-1,MV4-11)and its mechanism.Methods:1.Patients with AML diagnosed for the first time in the Pediatric and Adult Hematology Unit of the Affiliated Hospital of Zunyi Medical University from December 2017 to December 2020 were set as AML group.The clinical data(including sex,age,blood routine,percentage of primordial and immature cells in bone marrow,risk stratification,FAB classification,prognosis,etc.)were collected,and non-hematological patients in the same period were selected as control group(Control).Bone marrow mononuclear cells were collected from two groups,and the expression levels of RSK1 m RNA and protein were detected.To analyze the relationship between the expression levels of RSK1 and clinical features and prognosis,and to further analyze the expression of RSK1 and its significance with prognosis in the database of cancer genome map(TCGA).2.Human myeloid leukemia cells(THP-1,MV4-11,K562,OCI-AML2,MOLM14)were selected to detect the expression of RSK1 protein by Western Blot,and THP-1 and MV4-11 cells were selected for follow-up study.Lentivirus with knockdown and overexpression of RSK1 was transfected into THP-1 and MV4-11cells to construct THP-1 and MV4-11 cells with stable knockout and overexpression of RSK1.Cell proliferation was detected by CCK-8 assay and soft agar colony formation assay.The expression of apoptosis-related proteins such as Bax,Bcl-2 and Caspase3 was detected by Western Blot.The rate of apoptosis was detected by flow cytometry.Western Blot was used to detect the expression of STAT3,p-STAT3,JAK2 and p-JAK2 proteins related to JAK2/STAT3 signal pathway.3.After adding STAT3 inhibitor to THP-1 and MV4-11 cells with RSK1overexpress,the apoptosis rate was detected by flow cytometry,the cell proliferation was detected by CCK-8,and the expression of RSK1,PCNA and p-STAT3 protein was detected by Western Blot.4.STAT3 agonist was added to THP-1 and MV4-11 cells with RSK1 knockout,and the apoptosis rate was detected by flow cytometry.CCK-8 assay was used to detect cell proliferation.Western Blot was used to detect the expression of RSK1,PCNA and p-STAT3 protein.5.AML subcutaneous tumor model mice were constructed,and 5-week-old female nude mice were selected.5×10~6/100μL of MV4-11 cells,MV4-11 cells of knockout RSK1 and control MV4-11 cells were injected into the axillary back of nude mice,respectively.The growth status of subcutaneously transplanted tumor and the general conditions of nude mice(spirit,diet,body weight,hair)were observed.About5 weeks later,the cervical vertebra was dislocated and the mice were immediately dissected,and the related indexes were detected,the tumor size and weight,liver size and weight,and spleen size and weight were measured,Tumor HE staining was used to understand the transplanted tumor,and Tumor immunohistochemical staining was used to detect the expression of RSK1 and p-STAT3.6.The AML model of NOD/SCID mice was constructed.5×10~6/200μL of MV4-11 cells,MV4-11 cells of knockout RSK1 and MV4-11 cells of the control group were injected through the tail vein and inoculated into NOD/SCID mice aged 6to 8 weeks.The body weight,hair changes and mental state of the mice were detected.About 8 weeks later,the cervical vertebra was dislocated,and the mice were dissected immediately.The weight and size of liver and spleen in each group were measured.Peripheral blood smears and bone marrow smears were stained with Wright’s staining,liver and spleen tissues were stained with HE,flow cytometry was used to detect the expression of CD33 in bone marrow,liver and spleen cells,and immunohistochemistry was used to detect the expression of RSK1 and p-STAT3 in bone marrow.Results:1.A total of 40 patients with AML and 9 controls were included in this study.Of the 40 patients with AML,17 were males and 23 were females,with a median age of12.25 years,18 patients were over 18 years old and 22 patients were less than 18years old.According to FAB classification,there were 15 cases of M2 type,5 cases of M3 type,5 cases of M4 type,12 cases of M5 type and 3 cases of unknown type.Fusion gene was detected in 33 of 40 AML patients,of which 23 cases were positive and 10 cases were negative.Of the 40 patients with AML,32 had chromosome karyotype detection,of which 21 were positive and 11 were negative.Including 19cases in high risk group,8 cases in medium risk group,5 cases in low risk group and8 cases with unknown risk.2.Compared with the control group,the expression of RSK1 m RNA and RSK1protein in the newly diagnosed group was significantly higher than that in the control group(P<0.05).The expression of RSK1 was not related to white blood cell count,percentage of primordial immature cells in bone marrow,hepatosplenomegaly and degree of anemia in newly diagnosed AML patients,and had nothing to do with risk degree(mild to moderate to severe),chromosome abnormality and fusion gene abnormality(P>0.05).Compared with the control group,the expression level of RSK1 in patients with M4 and M5 was significantly higher(P<0.05),while the expression level of RSK1 in M2,M3 and unclassified patients was also higher than that in the control group,but the difference was not statistically significant(P>0.05).3.The results of survival analysis showed that the overall survival rate(OS)of the high expression group was worse than that of the low expression group of RSK1m RNA(P<0.05),and the same result was obtained in the TCGA database analysis.4.After overexpression of RSK1 in THP-1 and MV4-11 cells,the results of CCK-8 and soft agar colony formation assay showed that the ability of cell clone formation and cell proliferation were significantly higher than those of the control group,and the rate of apoptosis was lower than that of the control group(P<0.05).Western Blot detection showed that the expression of Cleaved-Caspase3 and Bax protein decreased(P<0.05),the expression of Bcl-2 protein increased(P<0.05),the expression of Caspase3 protein had no significant difference compared with the control group(P>0.05),the expression of PCNA protein increased(P<0.05),the expression of p-JAK2 and p-STAT3 protein increased(P<0.05),but the expression of JAK2 and STAT3 protein had no significant difference(P>0.05).5.After RSK1 knockout in THP-1 and MV4-11 cells,the results of CCK-8 and soft agar colony formation assay showed that the ability of cell clone formation and cell proliferation were significantly lower than those of the control group,and the apoptosis rate was higher than that of the control group(P<0.05).Western Blot detection showed that the expression of Cleaved-Caspase3 and Bax protein increased(P<0.05),the expression of Bcl-2 protein decreased(P<0.05),the expression of Caspase3 protein had no significant difference compared with the control group(P>0.05),the expression of PCNA protein decreased(P<0.05),the expression of p-JAK2 and p-STAT3 protein decreased(P<0.05),but the expression of JAK2 and STAT3 protein had no significant difference(P>0.05).6.When STAT3 inhibitor was added to THP-1 and MV4-11 cells overexpressing RSK1,flow cytometry showed that apoptosis in overexpression group was significantly lower than that in control group(P<0.05),but after adding STAT3inhibitor,apoptosis in overexpression RSK1 group was less than that in control group,but there was no significant difference(P>0.05).CCK-8 assay showed that the cell proliferation in the overexpression group was significantly higher than that in the control group(P<0.05),but after the addition of STAT3 inhibitor,the proliferation in the overexpression RSK1 group was slightly higher than that in the control group,and the difference was not statistically significant(P>0.05).Western Blot detection showed that the overexpression of RSK1 and STAT3 inhibitor could decrease the expression of RSK1 protein and PCNA protein.7.After RSK1 knockout in THP-1 and MV4-11 cells,adding STAT3 agonist,flow cytometry showed that the apoptosis rate of RSK1 knockout group was significantly higher than that of the control group(P<0.05),but after the addition of STAT3 agonist,the apoptosis rate of RSK1 knockout group was slightly higher than that of the control group,and the difference was not statistically significant(P>0.05).CCK-8 assay showed that the cell proliferation in the knockdown RSK1 group was significantly lower than that in the control group,but after the addition of STAT3agonist,the cell proliferation in the knockdown RSK1 group was slightly lower than that in the control group,and the difference was not statistically significant(P>0.05).Western Blot detection showed that the addition of STAT3 agonist after knockout RSK1 coμLd increase the expression of RSK1 protein and PCNA.8.The subcutaneous tumorigenesis model of nude mice showed that the tumor formation rate,tumor growth rate,tumor size,weight and spleen infiltration in the knockout MV4-11 group were slower than those in the control group,and the results of HE staining showed that,compared with the control group,the HE staining in the RSK1 knockout group showed small vacuoles,nuclear condensation and deeper staining.9.In the NOD/SCID AML model mice:compared with the control group,the general condition of the RSK1 knockout group was better;the hepatosplenomegaly was alleviated;the proportion of primordial cells in peripheral blood and bone marrow smear decreased;the expression of CD33 in bone marrow,spleen and liver cells decreased;the immunohistochemical results showed that the level of STAT3phosphorylation in the RSK1 knockout group was lower than that in the control group.Conclusions:1.There was a high expression of RSK1 in patients with AML,especially in M4and M5.2.The expression level of RSK1 is related to the prognosis of patients with AML,and the prognosis of patients with high RSK1 expression is poor.3.RSK1 participates in the apoptosis and proliferation of THP-1 and MV4-11cells.Knockdown of RSK1 can promote the apoptosis and inhibit the proliferation of THP-1 and MV4-11 cells.Overexpression of RSK1 can inhibit apoptosis and promote cell proliferation.4.It was confirmed in vivo and vitro experiments that RSK1 inhibits apoptosis and promotes cell proliferation by regulating JAK2/STAT3 pathway. |
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