Hsa_circ₀001947 Suppresses Acute Myeloid Leukemia Progression By Sponging Hsa-miR-329-5p

BackgroundAcute myeloid leukemia（AML）is characterized by blocked accumulation of clonal myeloid progenitor cells and exhibits complex clinical and biological heterogeneity[1].Although great efforts have made in AML,the prognosis of AML is still poor and its 5-year survival rate is only 25%[2].It is of clinical significance to explore the pathogenesis and identify new targets for AML.Circular RNAs（circRNAs）are a widely expressed class of non-coding RNAs generated in a diverse set of eukaryotic organisms,including animals,plants,yeasts and protists[3-5].Recently it has been considered as the latest research hotspot in the field of RNAs[6].Unlike the traditional linear RNAs（containing 5’and 3’ ends）,circRNAs have a closed continuous loops,which are difficult to be degraded by RNA exonuclease and are more stable in expression[6-8].The most common method of circRNAs biogenesis is through backsplicing,in which the 5’ splice site（5’ss）of a downstream exon is paired with the 3’ss of an upstream exon[3].One model believes that the partial folding of RNA during the transcription of pre-RNA closes the original non-adjacent exons,resulting in exon skipping,which makes the spanned region form a ring.The RNA intermediate is further ligated by lasso to form a circRNA consisting of an exon.Another model suggests that a reverse complement sequence located in an intron region results in intron region pairing and further mediates reverse splicing to form a circular RNA[9,10].CircRNAs were reported to be cell-specific,which are relatively abundant in the brain and exist in human body fluids such as blood[11].The relationship between circRNAs and linear RNA isoforms was complicated.There are reports that circRNAs are produced in competition with linear RNAs[12].However,it was also reported that there is no definite correlation between circRNAs and liner RNAs[13].CircRNAs contain plenty of microRNAs（miRNAs）binding sites,which offer itself an important role as the miRNAs sponge in cells.Through the function of binding certain miRNAs,circRNA could eliminate the inhibitory effect of miRNAs on its target genes,thereby increasing the expression level of the target genes[7].This mechanism is called competitive endogenous RNA（ceRNA）[7,8].Moreover,circRNAs are suggested to carry out diverse functions including sequestration and trafficking of proteins,regulation of transcription and generation of short proteins[14-16].By interacting with disease-associated miRNAs,circRNAs play a significant regulatory role in various diseases,from which the tumor-related research caught our great attention.It has been reported that circTADA2As serve as miR-203a-3p sponge to suppress breast cancer progression and metastasis[17],and circPSMC3 is a sponge of miR-296-5p to suppress the proliferation and metastasis of gastric cancer by acting as a ceRNA[18].In addition,circOMAl mediated the miR-145-5p to suppress the growth of tumor in nonfunctioning pituitary adenomas[19].These findings indicate that circRNAs could be a promising new diagnostic markers and treatment targets in solid tumors.However,there are rare data about the role of circRNAs in hematological diseases,especially in the AML until now.It was reported that silencing of circ₀009910 inhibits acute myeloid leukemia cell growth through increasing miR-20a-5p[20].Moreover,we have proven that hsa_circ₀004277 could be characterized as a new biomarker and treatment target for AML via circRNAs profile and bioinformatics analysis.In particular,we found chemotherapy could significantly restore the expression of hsa_circ₀004277,indicating the increasing level of hsa_circ₀004277 was associated with successful treatment[21].However,the detailed mechanism of circRNAs in the pathogenesis of AML is still unclear.In this study,we investigated the role and possible mechanism of hsa circ₀001947 in the development of AML,and found that hsa_circ₀001947 exerts a tumor suppressor effect in AML via hsa_circ₀001947-hsa-miR-329-5p-CREBRF network,which may become a potential therapeutic target for AML.ObjectiveThis study was designed to further validate the differential expression of hsa_circ₀001947 in bone marrow of AML patients and healthy controls;to further elucidate the antitumor effect of hsa_circ₀001947-hsa-miR-329-5p-CREBRF network in AML,and may provide new treatment target for AML.Materials and methodsWe used circRNAs microarray to determine the differential expression profile.Quantitative reverse transcription polymerase chain reaction（qRT-PCR）analyzed the expression of hsa_circ₀001947.The siRNA was used to assess the function of hsa_circ₀001947 in vitro and in vivo.A dual-luciferase and mimics/inhibitor were to determine the target gene relationship between hsa-miR-329-5p and CREBRF.Results1.Profile of circRNA expression in AML patients:Based on the principle of log2（fold changes）≥1,we selected two circRNAs,upregulated hsa_circ₀058058 and downregulated hsa_circ₀001947,as the candidate for the following study.2.Expression of hsa_circ₀001947 in AML patients:The expression level of hsa_circ₀001947 in newly diagnosed（ND）or relapsed-refractory（RE）AML patients was much lower compared to that in healthy controls.Meanwhile,the expression level of hsa_circ₀001947 was elevated in patients achieved complete remission（CR）compared to that in the ND group.3.Characterization of hsa_circ₀001947 in AML:Hsa_circ₀001947 was derived from the exon of AFF2 and it was resistant to be digested by RNase R.4.Hsa_circ₀001947 is clinically favorable in AML patients:4.1 The expression level of hsa_circ₀001947 was negatively correlated with white blood cell（WBC）,the percentage of primitive cells in bone marrow（BM%）and the percentage of primitive cells in peripheral blood（PB%）or positively linked to hemoglobin（HGB）in AML patients.4.2 QRT-PCR showed the restoration of dys-regulated hsa_circ₀001947 expression after chemotherapy.5.Receiver-operating characteristic（ROC）curve analysis of hsa_circ₀001947 in AML patients:Results indicate that the expression difference of hsa_circ₀001947 makes itself of significant diagnostic value.6.Hsa_circ₀001947 manifests inhibitory effect for AML cells in vitro and in vivo6.1 In vitro:Cell Counting Kit-8（CCK-8）analyzed THP-1 cells in the down-regulated group and the negative control group.The results showed that down-regulation of hsa_circ₀001947 promoted the proliferation of THP-1 cells and decreased the sensitivity to Cytosine arabinoside（Ara-c）induced drugs compared with the negative control.The results of cytometry showed that the apoptotic rate decreased,indicating that hsa_circ₀001947 has an inhibitory effect on AML cells.6.2 In vivo:The tumor volume in sicirc xenografts was bigger significantly than negative control xenografts and tumor weight were heavier in the sicirc group.We stained tumor cells with antibody Ki67 to compare the proliferation of the two groups.Results revealed that the proliferation ratio of the sicirc group was significantly higher than that of the negative control group.The percentage of apoptotic cells from the sicirc xenografts was lower than the cells from negative control xenografts determined with Annexin V and PI.Conclusion:1.The microarray show the circRNA expression profile in AML patients,from which two circRNAs were selected.2.Hsa_circ₀001947 has specific expression in AML and can be used as a potential diagnostic indicator and therapeutic target for AML.3.SiRNA down-regulation of hsa_circ₀001947 promotes proliferation of THP-1 cells and inhibits apoptosis,reducing drug sensitivity induced by Ara-c.4.No difference in proliferation between transfected mimics group and negative control.But,the drug sensitivity to Ara-c was decreased;transfected with the inhibitor group,hsa-miR-329-5p inhibited the proliferation of THP-1 cells and increased drug sensitivity to Ara-c compared with the negative control.5.The hsa_circ₀001947-hsa-miR-329-5p-CREBRF network exerts a tumor suppressor effect in AML.