| Objective: 1.the clinical study of pseudogene B cell-specific Moloney murine leukemia virus integration site 1 pseudogene 1(BMI1P1)is a pseudogene of BMI1 stem cell gene.This study was aimed to investigate BMI1P1 expression in de novo AML patients and to analyze its clinical relevance,whether it might serve as a biomarker for predicting disease prognostic.2.the function study of pseudogene POU domain class 5 transcription factor 1B gene(POU5F1B)is a pseudogene of OCT4 stem cell gene.This study was aimed to explore the pathogenic mechanism of POU5F1 B gene in hepatocellular carcinoma(HCC).Methods: 1.The BMI1P1 levels of 144 de novo AML patients and 36 healthy donors were detected by Real-time quantitative PCR.2.POU5F1B-HCC and si-POU5F1B-HCC cells were constructed and the expression of POU5F1 B transfected HCC cells was detected by RQ-PCR.3.The proliferation,survival,migration,anti-apoptosis,self-renewal and drug resistance ability of POU5F1 B transfected cells were observed by cell growth experiment,survival experiment,plate colony formation experiment,apoptosis assay,scratch assay,tumor sphere formation experiment and drug resistance experiment.4.To predict the mi RNAs that target POU5F1 B and the conserved sites bound by the seed region of these mi RNAs,different target prediction programs were used,including Target Scan,Miranda,DIANA and mi RDB.The expression of mi RNAs,OCT4,NANOG and MYC in POU5F1 B transfected cells was detected by RQ-PCR,and the expression level of OCT4 and MYC in POU5F1B transfected cells were also detected by Western blot.Result: 1.BMI1P1 was significantly down-regulated in AML compared with control(P < 0.001),a receiver operating characteristic curve was revealed that BMI1P1 expression could differentiate patients with AML from control subjects(AUC = 0.895,95% CI: 0.835-0.954,P < 0.001).The percentage of blasts in bone marrow(BM)were significantly lower in BMI1P1 high-expressed group versus low-expressed group(P = 0.008).BMI1P1 high-expressed cases had significantly higher complete remission(CR)than those low-expressed cases(P = 0.023).Furthermore,Kaplan–Meier demonstrated that both whole AML cohort and no-M3-AML patients with low BMI1P1 expression showed shorter leukemia free survival(LFS,P = 0.002 and P = 0.01,respectively)and overall survival(OS,P < 0.001 and P = 0.011,respectively)than those with high BMI1P1 expression.Multivariate analysis also showed that BMI1P1 over-expression is an independent favorable prognostic factor for OS in both whole and non-M3 cohort of AML patients(HR = 0.462,95% CI = 0.243-0.879,P = 0.019 and HR = 0.483,95% CI = 0.254-0.919,P = 0.027).To further investigate significance of BMI1P1 expression in the follow-up of AML patients,the BMI1P1 level monitored in 26 de novo AML patients was significantly increased form initial diagnosis to postCR(P < 0.001).These results indicated,for the first time,that BMI1P1 may contribute to diagnosis of AML and assessment of therapeutic effect.2.Study on Pathogenesis of POU5F1 B in HCC:(1)The expression of POU5F1 B in POU5F1 B transfected cells was significantly higher than that in the control group(P<0.01).(2)The growth rate of POU5F1B-Hep3 B cells was significantly faster than that of Mock-Hep3 B cells,and did not sto P the growth trend(P <0.01).The survival rate of POU5F1B-Hep3 B cells was higher than that of the controls cells(P <0.01).The population-dependent and proliferative ability of POU5F1B-Hep3 B cells was significantly higher than that of the control group(P <0.01).(3)The migration ability of POU5F1B-Hep3 B cells was significantly higher than that of the control group(P <0.01).POU5F1B-Hep3 B cells had been cured at 36 h,while the control group did not.(4)Mock-Hep3 B cells were more prone to apoptosis than POU5F1B-Hep3 B cells(P<0.01).(5)POU5F1B-Hep3 B cells could accumulate to form larger tumor sphere.The tumor formation rate of POU5F1 B was significantly higher than that of the control group(P <0.01).(6)The resistance of POU5F1B-Hep3 B cells to chemotherapeutic drugs doxorubicin and 5-fluorouracil was significantly higher than that of the control group(P <0.05),but it could not increase the resistance to cisplatin.(7)The expression levels of OCT4 and MYC in POU5F1 B transfected cells were significantly higher than those in the control group(P <0.01).(8)Target Scan,Miranda,DIANA and mi RDB predictors predict that the co-mi RNA targeting POU5F1 B was hsa-mi R299-3p,and that of POU5F1 B and OCT4 has its binding site on the 3’UTR,and POU5F1 B overexpression may be let OCT4 escape mi R-299-3p and other tumor suppressor mi RNA binding,so that OCT4 expression increased instead.Conclusions: 1.Pseudogene BMI1P1 was down-expressed in AML.Pseudogene BMI1P1 may serve as biomarkers for detection of AML.Interestingly,BMI1P1 may serve as an important prognostic marker for AML and its initiation of treatment;2.Construction of POU5F1B-Hep3 B,Mock-Hep3 B,si-POU5F1B-Hep3 B and siMock-Hep3 B cell lines;3.POU5F1 B can promote the proliferation,migration,colony formation and antiapoptotic effect of Hep3 B cells in vitro,and can promote the formation of tumor globules and participate in drug resistance;4.POU5F1 B may act as a competitive bait to regulate the expression of OCT4 by targeting mi R-299-3p,and can co-amplify with MYC,thus promoting the development of tumor. |
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