| Background and Objective: Circular RNA(circRNA)has been shown to play an important role in the pathogenesis of many tumors.However,researches about circRNA in acute myeloid leukemia(AML)were still rare.In this study,the circRNA with the most potentially clinical value was screened out by the circRNA microarray.The expression levels of the circRNA and its host gene were detected,its clinical significances were analyzed,its function and molecular mechanism were preliminarily researched.Method: 1.3 normal samples,3 acute lymphoblastic leukemia(ALL)ones and 5 AML ones were collected and then the circRNA microarray was used to detect the circRNA with the most obvious difference between AML and normal control;hsa＿circRNA＿100199 was selected to perform preliminary research as well as its host gene RNF220.2.We verified the results of the circRNA microarray and also detected the expression of hsa＿circRNA＿100199 and RNF220 in enlarged AML samples by qRT-PCR.3.The relationships between their expression levels and clinical laboratory indicators and survivals were retrospectively analyzed.4.Specific sh RNA lentiviral vectors was used to targeted knockdown of RNF220 in AML cell lines including THP-1 and MV4-11 cells.The knockdown efficiencies were verified by qRT-PCR and its effects on cell growth,apoptosis and cell cycle were observed.5.In MV4-11 cells with RNF220 knockdown,Western Blot was used to verify the expression changes of Wnt classical pathway gene-related proteins(such as Wnt2b,Wnt5a/b).6.In MV4-11 cells,the effect of RNF220 knockdown on hsa＿circRNA＿100199 or of hsa＿circRNA＿100199 knockdown on RNF220 was detected by qRT-PCR to explore the possible regulatory relationship between them.Results: 1.The circRNA microarray results suggested that hsa＿circRNA＿100199 was the circRNA with the greatest expression difference between AML and the normal control(Fold change=18.49,P <0.05).2.The specificities of this circRNA primers and qRT-PCR used in hsa＿circRNA＿100199 detection was identified by the RNase R digestion assay and sequencing.3.In these samples used in circRNA microarray analyze,the expression level of hsa＿circRNA＿100199 in AML was significantly higher than that in ALL and normal controls detected by qRT-PCR(21.81 ± 5.713 vs.0.3427 ± 0.3287,P=0.015),which was consistent with circRNA microarray results.Increased RNF220 expression was also observed in AML(2.798 ±0.706 vs.1.15 ± 0.109,P=0.040).4.In enlarged samples including 94 AML patients,the median expression level of hsa＿circRNA＿100199 was 0.7369(0.0018-4.4880),which was significantly higher than that in normal people(median expression level was 0.0097,range 0.0049-0.0142,P=0.0002).The median expression level of RNF220 in AML patients was 0.9725(0.2290-14.4708),which was also significantly higher than that in normal people(median expression level was0.3587,range 0.1508-0.5147,P=0.0005).And there was a positive correlation between the expression levels of the hsa＿circRNA＿100199 and RNF220(P=0.0002,R=0.377).5.In AML,the percentage of blasts in the hsa＿circRNA＿100199 high expression group was higher than that in the hsa＿circRNA＿100199 low expression group(71.70 ± 17.57 vs.61.03 ± 21.07,P <0.05).The percentage of blasts in the RNF220 high expression group was also higher than that in the RNF220 low expression group(71.60 ± 1.46 vs.62.53 ± 20.32,P <0.05).There were no significant differences between the expression levels of these two genes and indicators such as age,gender,white blood cell count,hemoglobin,platelet,risk stratification,and complete remission rate.6.Overall survival(OS)of the hsa＿circRNA＿100199 high-expression group(median: 12.5 months,95% CI: 5.75 months to 19.25 months)was marginally(but non-significantly)shorter than that of the low-expression group(median: 33.4 months,95% CI: 9.61 months to 57.19 months,P=0.070).OS in the RNF220 high expression group(median: 12.5 months,95% CI: 5.96 months to 19.05months)was significantly shorter than that in the low expression group(median: 46.7 months,95%CI: 20.46 months to 72.94 months,P=0.0014).Multivariate Cox regression analysis suggested that higher white blood cell count(P=0.019)and the higher expression level of RNF220(P=0.023)were independent adverse factors in AML.7.After RNF220 was knocked down in THP-1cells,the growth rates in the negative control(NC)group at 0,24,48,72,and 96 hours were 1.00 ±0.05,1.64 ± 0.13,1.91 ±0.02,2.59 ± 0.12,and 3.53 ± 0.22,respectively,those in sh RNF220-1group were 1.00 ± 0.10,1.50 ± 0.12,1.34 ± 0.07,1.30 ± 0.17,1.35 ± 0.07 respectively,those in sh RNF220-2 group were 1.00 ± 0.08,1.03 ± 0.05,1.10 ± 0.11,1.12 ± 0.04,1.13 ± 0.04 respectively and those in sh RNF220-3 group were 1.00 ± 0.06,1.11 ± 0.08,1.01 ± 0.09,1.06 ±0.02,1.19 ± 0.06 respectively.There were significant differences between NC group and 3RNF220 knockdown groups(sh RNF220-1,sh RNF220-2 and sh RNF220-3)(all P<0.001).After RNF220 was knocked down in MV4-11 cells,the growth rate in the NC group in MV4-11 cells at0,24,48,72,and 96 hours were 1.00 ± 0.14,2.98 ± 0.26,7.08 ± 0.43,13.36 ± 0.35,23.01 ± 0.86;significantly higher than those of the sh RNF220-1 group(1.00 ± 0.02,1.36 ± 0.12,1.76 ± 0.19,2.56 ± 0.62,4.99 ± 0.27,P <0.001).8.After RNF220 was knocked down in MV4-11 cells,compared with the negative control group,apoptosis in the sh RNF220-1 group increased significantly(23.63% ± 4.55% vs.10.00% ± 1.85 %,P=0.008).The expression of apoptosisrelated proteins such as Cleaved-Caspase3 and Cleaved-Caspase7 also increased.9.After RNF220 was knocked down in MV4-11 cells,the proportion of G1 phase increased significantly(36.98% ±0.54% vs.26.06% ± 0.57%,P <0.001),and the expression of CDK6 decreased.10.After RNF220 was knocked down in MV4-11 cells,the expression levels of Wnt5 a / b and Wnt2 b decreased.11.After hsa＿circRNA＿100199 was knocked down in MV4-11 cells,the decreased expression level of RNF220 m RNA was observed by qRT-PCR(0.66 ± 0.10 vs.1.00 ± 0.04,P=0.004),but RNF220 knockdown couldn’t affect the expression level of hsa＿circRNA＿100199.Conclusion: The expression of hsa＿circRNA＿100199 and RNF220 was abnormally increased in AML,and the expression levels of them were positively correlated.High expression of circRNA＿100199 has a tendency to poor prognosis in AML patients.High expression of RNF220 is an independent adverse prognostic factor in AML patients.RNF220 may promote the growth of AML cells through Wnt signaling mediated apoptosis and cell cycle. |
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