The Study Of Long Non-coding RNA H19 Imprinting State And Function In Acute Leukemia

Objective:(1)Understanding the expression of long-noncoding RNA H19 in patients with acute leukemia, and analyzes its relationship with prognosis and survival.(2)Discuss the relationship between CBS6 methylation and LOI.(3)Understanding the influence of H19 in NB4.Provides a new targets for the treatment of acute leukemia.Methods:(1)Real-time qPCR is applied to detect the expression of H19 acute leukemia patients and normal level.Comparing the differences of H19 expression level between patients and normals.(2)Using pyrosequencing, detect CBS6 methylation in patients and normals.(3)SNP sequencing is applied to screening heterozygote in patients according Rsa I. In heterozygotes,the CBS6 methylation were detected and contrasted in LOI and MOI,which were speculated the LOI’s relationship with the methylation.(4)Real-time qPCR is used to screen the high expression of H19 in acute leukemia cell lines. Using RNA interference, constructs the H19 interfering lentivirus, interfere the high expression of H19 leukemia cell lines.(5)Flow cytometry is used to detect the cells’ cycle after infection. CCK-8 method、clone formation inhibition test were used to detect the cells’ prolifer/ation.(6)Benzimidazole dye and Flow cytometry were used to observe cell’s apoptosis.Results :(1)the H19 expression in patients is significantly higher than normals(P =0.0031).(2)through the detection of DNMT3 A and DNMT3 B and DNMT1,the expression of DNMT3 A and DNMT3 B in patients is higher than normals(P=0.0004, P<0.0001), while the DNMT1 is opposite(P=0.0142). IGF2 shows low expression(P =0.048), but this result needs to be further explored.(3)the survival analysis indicated that patients with high expression of H19 have worse OS and RFS than the lower expression of H19 patients,and the difference is statistically significant(P= 0.0351, P= 0.0351).(4)In patients with acute leukemia,CBS6 methylation is lower than normals, but not obvious(P=0.0163). Combine the methylation of CBS6 and LOI、 MOI, further analysis shows that the methylation of CBS6 in LOI are significantly lower than the MOI(P=0.033).(5)By constructing vector of RNA interference andlentivirus packaging build stable H19 interference’s NB4 cells.(6)By the method of CCK-8,clone formation inhibition experiment, flow cytometry can be found that H19 inhibition of NB4cells’ proliferation than the control group.also the apoptosis is increased than the control group.Conclusion:1、Long non-coding RNAH19 in in patients with acute leukemia have abnormal high expression, and it affects the patients’ survival and prognosis. It may can be a biomarker for acute leukemia treatment as a oncogene.2、Patients with acute leukemia have much CBS6 methylation than normal, especially in AML. According further analysis of between LOI and MOI, LOI patients CBS6 methylation were significantly lower than MOI.Methylation of CBS6 may be one of mechanism of LOI.3、NB4 cells’ proliferation was significantly affect by H19, and the apoptosis rate increased significantly.It show that H19 may be a oncogene in acute leukemia,and participated in the acute leukemia cell proliferation and apoptosis.