Expression And Molecular Mechanism Of JAK/STAT Signaling Related Genes In Acute Leukemia

Objective: To investigate the expression and clinical significance of erythropoietin(EPO)and erythropoietin receptor(EPOR)in patients with acute leukemia(AL).methods: We collected 30 patients with acute lymphoblastic leukemia(ALL)and 30 patients with acute myeloid leukemia(AML)who were newly diagnosed in our hospital from June 2016 to June 2017 as the case group,19 patients with non-hematological diseases hospitalized in our hospital in the same period and volunteers as Control group.Divided into ALL and AML according to the latest WHO diagnostic classification criteria in 2008.The risk of ALL and AML was divided into high,medium and low risk according to the Chinese Journal of Pediatrics Blood Group "2006 Chinese Children’s Acute Leukemia Treatment Proposal" and the Chinese Medical Association Hematology Branch "Legal Leukemia Treatment Guidelines 2011".According to immunophenotyping,ALL is divided into B-cell leukemia and T-cell leukemia,AML is divided into M0-M7.Everyone was extracted 2ml bone marrow.(1)The levels of EPO and EPOR in plasma were determined by ELISA kit.(2)The EPO,EPOR m RNA expression levels were determined by RT-RCR.(3)The EPO and EPOR protein levels were detected by Western Blotting.Final,statistical analysis.Results:(1)ELISA results showed: The levels of EPO protein in marrow plasma of ALL and AML groups were significantly higher than those in the control group,the difference was statistically significant(P < 0.05),EPOR protein levels in patients with ALL and AML groups were significantly lower than those in the control group,the difference was statistically significant(P < 0.05).(2)The results of RT-RCR showed that the levels of EPO and EPOR m RNA in ALL and AML groups were significantly higher than those in the control group(P < 0.05),there were no significant difference between ALL and AML groups(P > 0.05).The levels of EPO m RNA in ALL high risk group were significantly higher than those in the medium,low risk and the control groups(P < 0.05),the difference was statistically significant(P < 0.05),the EPO m RNA levels in the medium and low risk group respective compared with the control group were no significant difference(P > 0.05).The EPOR m RNA level in the high risk group was significantly higher than those in the medium,low and the control groups,the difference was statistically significant(P < 0.05).There was no significant difference in the level of EPOR m RNA between the medium,low and the control groups(P > 0.05).The levels of EPO and EPOR m RNA in the AML high risk group were significantly higher than those in the medium,low risk group and the control group,the difference was statistically significant(P<0.05),there was no significant difference in the medium,low group and the control groups(P>0.05).The levels of EPO m RNA in M5 group were significantly higher than those in M2 group and control groups(P<0.05),there was no statistical difference between M2 group and Control group(P>0.05).The level of EPOR m RNA in M5 group was significantly higher than those in the control group(P<0.05).The level of EPOR m RNA in M2 group respective compared with M5 group and control group was no significant difference(P>0.05).(3)The results of Western Blotting showed that the EPO and EPOR expression levels in ALL and AML groups were significantly higher than that control group(P < 0.05).(4)The levels of EPO and EPOR m RNA in ALL and AML groups were not correlated with hemoglobin level and erythrocyte count(P>0.05).Conclusion:1.There were high expressions of EPO and EPOR gene in ALL and AML patients.2.The EPO and EPOR are associated with the risk of ALL and AML.High risk patients have higher expression Levels of EPO and EPOR.3.There was no correlation between the expression of EPO and EPOR and the amount of hemoglobin and erythrocyte count in peripheral blood.Objective: To investigate the expression and clinical significance of LNK,STAT3 m RNA in acute myeloid leukemia(AML)patients.methods: A total of 35 cases of patients with newly diagnosed AML in our hospital from June 2016 to August 2017 were collected,the control group of 17 specimens from the same period in our hospital in patients with non-hematological diseases and volunteers.AML was included according to the latest WHO diagnostic classification in 2008.The risk of AML was divided into high,medium and low risk according to the Chinese Journal of Pediatrics Blood Group "2006 Chinese Children’s Acute Leukemia Treatment Proposal" and the Chinese Medical Association Hematology Branch "Legal Leukemia Treatment Guidelines 2011".According to immunophenotyping,AML is divided into M0-M7.Bone marrow 2ml each,total RNA was extracted and reverse transcribed into c DNA,the expression of LNK and STAT3 m RNA in bone marrow was detected by RT-PCR.Finally,statistical analysis.Results: The results of RT-PCR showed that:(1)The levels of LNK and STAT3 m RNA in bone marrow of AML group were significantly higher than those in control group,the difference was statistically significant(P < 0.05).(2)The levels of LNK and STAT3 m RNA in high risk group were significantly higher than those in the control group,medium and low risk groups,the difference was statistically significant(P < 0.05).there was no statistical difference in the medium,low risk and the control groups(P>0.05).(3)The levels of LNK and STAT3 m RNA in M5 group were significantly higher than those in M2 group and Control group,the difference was statistically significant(P < 0.05),there was no statistical difference between M2 group and Control group(P>0.05).(4)There was a positive correlation between LNK and STAT3 m RNA in AML group(R=0.5,P=0.01).Conclusion:1.There were high expressions of LNK and STAT3 gene in the bone marrow of AML patients.2.The LNK and STAT3 expression levels and the risk of AML patients are related,and high risk patients have higher expression levels.3.There was a positive correlation between the expression of LNK and STAT3 in AML patients.Objective: To investigate the effect of LNK gene on the molecular mechanism of EPO,EPOR and STAT3 genes in JAK/STAT signaling pathway in acute myeloid leukemia cells(THP-1).methods:(1)THP-1 cells were cultivated to logarithmic growth phase.(2)The lentivirus was used as a vector to silence and overexpress the LNK gene stably.(3)After 72 hours infection,the GFP expression levels was observed under a fluorescence Inverted microscope.(4)Puromycin was used to screen stably transfected THP-1 cell lines.(5)Detect lentiviral Infection efficiencies by flow cytometry.(6)The LNK silencing and overexpression effect was confirmed by RT-PCR.(7)The cck8 method was used to detect the proliferation of THP-1 cells and the apoptotic rate of THP-1 cells was detected by flow cytometry.(8)The expression of STAT3,EPO and EPOR m RNA was detected by RT-PCR.(9)The protein levels of LNK,STAT3,P-STAT3,EPO and EPOR were detected by Western Blotting.Final statistical analysis.Results:(1)The best MOI for infecting THP-1 cells with lentivirus as a vector was100.(2)The optimal concentration of puromycin for screening stably transfected THP-1 cell lines is about 2.5 μg/ml.(3)72 hours after lentivirus infection,the expression level of GFP was above 85% under fluorescence inverted microscope.(4)RT-PCR test results showed that: LNK and STAT3 m RNA expressions of LNK silencing group(Si RNA)were signifycantly lower than the empty virus group and Control group,the difference was statistically significant(P < 0.05),The LNK and STAT3 m RNA expressions of LNK overexpression group(SH2B3)were significantly higher than the empty virus group and Control group,the difference was statistically significant(P < 0.05).however,there was no significant difference between the control group and the empty virus group(P > 0.05).In addition,the level of EPO m RNA: Si RNA group was significantly lower than the control group,the difference was statistically significant(P < 0.05).LNK overexpression group was significantly higher than the control group(P < 0.05).The LNK overexpression group compared with the empty virus group and the control group was no significant difference(P> 0.05).There were no significant difference in EPOR m RNA levels of different groups(P> 0.05).(5)Using cck8 detection of THP-1 cells proliferation showed: the OD450 nm value of THP-1 cells in Si RNA group at 24 h,48h and 72 h was significantly lower than the Control group,the difference was statistically significant(P < 0.05),there was no significant difference between LNK overexpression group and the Control group(P>0.05),there was no significant difference between the empty virus group and the control group(P> 0.05).(6)Using flow cytometry test results show: the apoptosis rate of THP-1 cells in the Si RNA group was significantly higher than the control group,the difference was statistically significant(P < 0.05),LNK overexpression group compared with the control group,the difference was not statistically significant(P>0.05),there was no significant difference between the empty virus group and the control group(P> 0.05).(7)Western Blotting test results show: the level of LNK and STAT3 protein in LNK overexpression group was significantly higher than that in Control group,the difference was statistically significant(P < 0.05),but its P-STAT3,EPO,EPOR protein levels compared with the control group,the difference was not statistically significant(P > 0.05).The protein levels of LNK,STAT3,P-STAT3,EPO and EPOR in Si RNA group were significantly lower than those in Control group(P < 0.05),there was no significant difference between the control group and the empty virus group(P > 0.05).Conclusion: 1.Successfully established the LNK gene silencing and overexpression of THP-1 cell line;2.Silencing LNK gene could significantly inhibit the proliferation of THP-1 cells and promote its apoptosis.3.LNK gene silenced,STAT3 expression decreased,LNK gene overexpression,STAT3 expression increased,indicating that LNK participates in the regulation of STAT3.