The Expression Of Circular RNAs As Valuable Predictor For Pediatric B-Cell Acute Lymphoblastic Leukemia Diagnosis

Objective: With the development of molecular technology,molecular biomarkers play an important role in the diagnosis and prognosis of pediatric acute B-cell lymphoblastic leukemia?B-ALL?,however,20%-30% of pediatric B-ALL could not be classified into any of the established molecular subtypes.In this study,we explore circular RNAs?circ RNAs?as the new biomarker of diagnosis and prognosis in pediatric B-ALL.Methods: Target circ RNAs?hsa_circ₀004123,hsa_circ₀001857,hsa_circ₀001708?were screened by literature review.Total RNA isolated from B lymphoblast cell line HMy2.CIR was reversely transcribed into c DNA.Using c DNA as template,PCR was used to amplify the characteristic fragments with the back-splice junction of hsa_circ₀004123,hsa_circ₀001857 and hsa_circ₀001708.T-A cloning method was used to construct the recombinant plasmid.Gradient dilution recombinant plasmid was used as the standard substance.SYBR fluorescent dye method was used to establish the real-time PCR standard curve.Finally,the absolute quantitative PCR method for the detection of circ RNAs was established and evaluated.To explore the diagnostic and prognostic value of circ RNAs in pediatric B-ALL,the expression of circ RNAs?hsa_circ₀004123,hsa_circ₀001857,hsa_circ₀001708?in 193 children with B-ALL and 20 control patients with non-hematologic tumors were detected by this method.Results: A fluorescence quantitative PCR method for detecting circ RNAs was successfully established and evaluated with a wide linear range,high sensitivity and specificity and good repeatability.The expression of hsa_circ₀004123,hsa_circ₀001857,hsa_circ₀001708 and their corresponding parent genes were quantitatively detected by this method.Hsa_circ₀004123?p=0.0057?,hsa_circ₀001857?p<0.0001?and their parent genes ETV6?p=0.038?and PAX5?p<0.0001?were significantly upregulated in pediatric B-ALL compared with controls.Hsa_circ₀001708?p=0.1796?and its parent gene IKZF1?p=0.7294?showed an increased expression trend in pediatric B-ALL,but no statistical significance.Receiver operating characteristic curve?ROC curve?was used to examine the potential diagnostic value in distinguishing individuals with pediatric B-ALL from healthy individuals.The area under the curve?AUC?respectively was 0.6937,0.8544 and 0.5968 of hsa_circ₀004123,hsa_circ₀001857 and hsa_circ₀001708,indicating hsa_circ₀001857and hsa_circ₀004123 had promising potential as biomarker.To validate the prognostic value of circ RNAs,the survival curves were analyzed.Pediatric B-ALL with overexpression or low expression of hsa_circ₀004123,hsa_circ₀001857,hsa_circ₀001708 and their parent genes ETV6,PAX5,IKZF1 could not indicate a poor or better prognosis.A positive correlation between hsa_circ₀004123,hsa_circ₀001857,hsa_circ₀001708 and their respective parent genes ETV6,PAX5 and IKZF1?R2 was 0.6357,0.7635 and 0.8381,respectively?was observed inpediatric B-ALL.Conclusion: The absolute fluorescence quantitative PCR method could be used for detecting the expression of corresponding circ RNAs in patient samples.Hsa_circ₀004123 and hsa_circ₀001857 were potential diagnostic markers in pediatric B-ALL.