Preliminary Study On The Role And Mechanism Of CircHIPK3 In The Pathogenesis Of Acute Myeloid Leukemia

Objective: Acute myeloid leukemia(AML)is a malignant disease of bone marrow hematopoietic stem cells.The pathological process is manifested as malignant clonal proliferation of myeloid stem cells,which inhibits the production and growth of normal hematopoietic cells.The main clinical symptoms are fever,anemia,bleeding.At present,the etiology of AML is not clear,and may be related to genetic factors,environmental factors,physical and chemical stimuli and so on.With the development of new chemotherapeutic drugs,targeted drugs and bone marrow transplantation technology,the treatment and prognosis of AML have been significantly improved.However,most patients still have primary drug resistance,relapse and poor prognosis.Therefore,the pathogenesis of AML to explore,finding new therapeutic targets and prognostic-related molecular markers is imminent.Circ RNA is a special type of non-coding RNA with a closed loop structure.Because of its special structure with a closed loop structure,it is not easy to be degraded by RNase.It is highly conserved in different species and biological evolution processes,and has tissue specificity and development.In recent years,the research on circ RNA has been increasing.A number of studies have shown that it plays an important role in a variety of pathophysiological processes.Especially in various malignant tumors,it has been confirmed that circ RNA plays an oncogene or tumor suppressor gene to participate in the occurrence and development of tumors.At present,there are not many reports on the correlation between AML and circ RNA.The expression of circ RNA in AML and its mechanism of action need to be further studied.A number of studies have shown that circ HIPK3 is significantly differentially expressed in a variety of solid malignant tumors.It plays an oncogene or tumor suppressor gene to participate in tumorigenesis and progression.It can be used as a specific biomarker in some tumors to evaluate curative effects and evaluate prognosis.However,the expression,biological function and mechanism of circ HIPK3 in acute myeloid leukemia is not clear yet.In this study,Realtime-PCR was used to detect the expression of circ HIPK3 in patients with acute myeloid leukemia,and the correlation between the expression level and FAB typing,chromosomal abnormalities,gene mutations and prognosis was analyzed;The aiming circ HIPK3 interference si RNA were transfected into AML cell line,through CCK8,flow cytometry to detect cell proliferation,apoptosis and cell cycle changes.Then we use western blot to detect changes in cell apoptosis and cycle-related proteins,to explore the effect of circ HIPK3 on the biological functions of AML in vitro;Letter analysis predicts the mi RNAs and target genes that bind to circ HIPK3,and dual luciferase experiment verifies targeted results,then cell rescue experiments verify that circ HIPK3 exerts a molecular sponge mechanism to affect the biological behavior of AML.Method: 1.In this experiment,60 AML patients and 30 normal controls were collected.Realtime-PCR method was used to detect the expression of circ HIPK3,and its correlation with FAB typing,chromosomal abnormalities,gene mutations,efficacy and clinical prognosis were analyzed.2.To verify the expression of circ HIPK3 in AML cell lines,we select cell lines with high gene expression in HL-60 and U937.Synthesized circ HIPK3 si RNA transiently transfected into the cells.CCK8 was used to detect cell proliferation,flow cytometry to detect cell apoptosis and cycle,and Western Blot to detect cell cycle and apoptosis-related proteins to verify the effect of circ HIPK3 on the biological behavior of AML cell lines in vitro.3.RNA nucleoplasmic isolation experiments verify the subcellular localization of circ HIPK3;We use starbase,mi RDB,microrna and other databases to predict the interaction between circ HIPK3 and mi R-193a-3p,mi R-193a-3p and the target gene c-Kit;The dual luciferase experiments verified its targeted binding;Realtime-PCR was used to detect the expression of mi R-193a-3p and c-Kit in bone marrow mononuclear cells and cell lines of AML patients and analyze the correlation with circ HIPK3;Realtime-PCR and western blot detect the effect of circ HIPK3 expression change on the expression of mi R-193a-3p and c-Kit;detect the effect of mi R-193a-3p expression change on the expression of c-Kit;Cell rescue experiment verifies that circ HIPK3 plays a molecular sponge role and adsorbs mi R-193a-3p,through the circ HIPK3/mi R-193a-3p/c-Kit axis to participate in the biological behavior of AML.4.Statistical analysis.The experimental data was statistically processed using SPSS22.0 and Graph Pad Prism 7.0 software.Each set of cell experiment data was repeated at least three times.Categorical variables in patient samples are expressed as rates or constituent ratios,and chi-square test is used for difference analysis;Kolmogorov-Smirnov method is used to test the normal distribution of continuous variables,and those that conform to the normal distribution are expressed as mean ± standard deviation,and independent difference analysis is used by T test;continuous data that does not conform to the normal distribution are represented by the median and interquartile range,then we use the non-parametric test to analysis difference;the Kaplan-Meier survival curve is used for prognostic analysis,and the log-rank test is used difference analysis;single factor and COX multivariate analysis to assess prognostic risk factors;P<0.05 was considered as statistically significant.Result: 1.The expression and clinical significance of circ HIPK3 in AML.1.1 The expression level of circ HIPK3 in newly diagnosed AML patients was significantly up-regulated compared with the normal control group,1.2 The expression level of circ HIPK3 differs in the FAB,chromosomal typing and characteristic gene mutation types of AML;among newly diagnosed AML patients,the expression level of circ HIPK3 is positively correlated with the proportion of bone marrow blasts and FLT3 mutation;the results of survival analysis show that the overall AML patients and the normal karyotype patients with low expression of circ HIPK3 have better overall survival time and recurrence-free survival time,and the difference is statistically significant.Univariate and multivariate COX regression analysis suggested that circ HIPK3 is an independent risk factor for evaluating the prognosis of AML patients.2.The biological effect of circ HIPK3 on the cell function of acute myeloid leukemia cell lines 2.1 circ HIPK3 is highly expressed in acute myeloid leukemia cell lines 2.2 The expression level of circ HIPK3 in HL-60 and U937 cells is relatively higher.These two cell lines were selected for verificating si RNA knockdown efficiency.Among them,the knockdown efficiency of circ HIPK3 si RNA1 fragment was higher,and this fragment was selected for subsequent experiments.2.3 CCK8 experimental results showed that compared with the blank group and the negative control group,the proliferation ability of the si-circ HIPK3 group was significantly reduced.2.4 The results of flow cytometry showed that compared with the blank group and the negative control group,the proportion of AML cell apoptosis in the si-circ HIPK3 group was significantly increased.At the same time,Western blot results indicated that the expression of apoptosis-related protein cleaved Caspase-3 was significantly up-regulated and the expression of anti-apototic protein Bcl-2 decreased significantly.2.5 The results of flow cytometry showed that compared with the blank group and the negative control group,the AML cells in the si-circ HIPK3 group were more blocked in the G1 phase,and the proportion of entering the S phase was significantly reduced.At the same time,the Western blot results indicated the expression of cyclin D1 Significantly down.3.Research on the molecular mechanism of circ HIPK3 on AML 3.1 Nuclear and cytoplasmic separation results suggest that circ HIPK3 is mainly located in the cytoplasm.3.2 RNase R experiment confirmed the existence of the ring structure of circ HIPK3.3.3 Knockdown of circ HIPK3 by si RNA does not affect the expression of its linear transcript HIPK3.3.4 Mi R-193a-3p is a downstream target gene of circ HIPK3: bioinformatics predicts that circ HIPK3 can target mi R-193a-3p.mi R-193a-3p is low expression in AML patients and cell lines,and is negative relation with circ HIPK3 expression in AML patients;dual luciferase experiments further confirms the targeted combination of circ HIPK3 and mi R-193a-3p;knocking down of circ HIPK3 can up-regulate the expression of mi R-193a-3p.3.5 c-Kit is a downstream target gene regulated by mi R-193a-3p: bioinformatics predicts that mi R-193a-3p and c-Kit will bind together,and have a common binding site with circ HIPK3,thus circ HIPK3 and c-Kit can bind mi R-193a-3p competitively;c-Kit genes are significantly highly expressed in AML cell lines and patients,and the expression of c-Kit in AML patients is significantly negatively correlated with mi R-193a-3p.The dual luciferase experiment further confirms the targeted relationship,;overexpression of mi R-193a-3p can inhibit the expression of c-Kit.3.6 circ HIPK3 affects the expression of c-Kit to regulate the biological behavior of AML by targeting mi R-193a-3p: In AML patients,the expression level of circ HIPK3 is significantly positively correlated with c-Kit.Knockdown of circ HIPK3 can inhibit the expression of c-Kit.Co-transfection of mi R-193a-3p inhibitor can partially restore the effect of circ HIPK3 knockdown on c-Kit expression,while co-transfection of mi R-193a-3p inhibitor can partially restore circ HIPK3 knockdown on reducing the proliferation,apoptosis and proliferation of HL-60 and U937 cells.Meanwhile,western blot results suggest that the expression levels of apoptosis and cycle-related proteins cleaved Caspase-3,Bcl-2 and cyclin D1 partially recovered.conclusion: 1.circ HIPK3 is significantly highly expressed in AML patients,and is closely related to some indicators that reflect the tumor load of AML.AML patients with high expression of circ HIPK3 have a poor prognosis.The expression of circ HIPK3 is an independent risk factor for assessing the prognosis of AML.2.circ HIPK3 is highly expressed in acute myeloid leukemia cell lines,promotes cell proliferation,inhibits cell apoptosis,and plays the role of oncogene.3.circ HIPK3 acts as a molecular sponge which can competitively inhibits mi R-193a-3p,then regulates the expression of c-Kit.In all,circ HIPK3 regulates the biological behavior of AML through the circ HIPK3/mi R-193a-3p/c-Kit axis.