Preliminary Study On Expression Profile And Function Of Long Non-coding RNA In Pediatric Acute Leukemia

Objectives: Lnc RNAs play important roles in gene regulation.The contribution of lnc RNAs to the pathogenesis of pediatric acute leukemia is still not unknown. In our study, we describe the expression profile of lnc RNAs in pediatric acute leukemia compared with normal control using microarray analysis and primarily explored their biological functions. We determined AC002454.1 with the greatest difference, and studied its clinical significance. Also to use gene silencing technology for further understanding of its function.To investigate the role and mechanism of lnc RNAs in pediatric acute leukemia, and to lay the foundation for further study of lnc RNAs.Methods: 1. We chose 9 clinical cases of AL（6 ALL cases and 3 AML cases） and 3 normal control cases. There were 3 AL groups: L1（3 AML cases）, L2（3 T-ALL cases）, L3（3 B-ALL cases） andthree control groups: C1, C2, C3（each group includes 3 cases）. Total RNAs were extracted from bone marrow/peripheral blood samples, then transcribed into c DNAs. The original datas from microarray scanner were used for statistical analysis and the differential expression profiles of lnc RNAs/m RNAs between AL and control group were obtained.2. To confirm the microarray data, we selected 97 dysregulated lnc RNAs from the microarray analysis of 43 cases of newly diagnosed pediatric AL（including 21 ALL cases, 22 AML cases） and 21 cases of control. Total RNAs were extracted from bone marrow samples, then transcribed into c DNAs by specific primers. The expression of lnc RNAs were detected by real-time fluorescence quantitative polymerase chain reaction（q RT-PCR） and was analyzed with comparative method in cycle threshold（Ct） value. Statistical analysis was used to the expressions of lnc RNAs in leukemia. We determined AC002454.1 with the greatest difference, and studied the relationship between the expression of AC002454.1 and its clinical indicators.3. Bioinformatic analysis（gene ontology analysis and pathway analysis）was used for further resarch on the differential expression profiles based on NCBI Ref Seq, UCSC, RNAdb,Lnc RNAs databases and we constructed a coding and non-coding gene expression network based on the correlation analysis between differentially expressed lnc RNAs and m RNAs.4. K562 and NB4 cell lines were selected as research objects. They were transfected with specific si RNAs to down-regulate AC002454.1expression. CCK8, flow cytometric and Western-blot analysis were used for evaluating its effect on the biological behaviorof leukemia cells, such as proliferation, apoptosis, cell cycle and CDK6 expression.Results: 1. A total of 2409 differently expressed lnc RNAs were found in pediatric AL. Among them, 1394 lnc RNAs were up-regulated and 1015 lnc RNAs were down-regulated for more than 2 folds in pediatric AL compared to normal controls. 2331 differently expressed m RNAs were found in pediatric AL. Among them, 1073 m RNAs were up-regulated and 1258 m RNAs were down-regulated for more than 2 folds in pediatric AL compared to normal controls. A total of 1884 differently expressed lnc RNAs were found in pediatric ALL. Among them, 1106 lnc RNAs were up-regulated and 778 lnc RNAs were down-regulated for more than 2 folds. 1857 differently expressed m RNAs were found in pediatric ALL. Among them, 905 m RNAs were up-regulated and 952 m RNAs were down-regulated for more than 2 folds. A total of 4289 differently expressed lnc RNAs were found in pediatric AML. Among them, 2215 lnc RNAs were up-regulated and 2074 lnc RNAs were down-regulated for more than 2 folds. 4003 differently expressed m RNAs were found in pediatric AML. Among them, 1769 m RNAs were up-regulated and 2234 m RNAs were down-regulated for more than 2 folds.2. To confirm the microarray datas, we selected 97 dysregulated lnc RNAs from the microarray analysis using q RT-PCR. Our datas revealed that same lnc RNAs expression profile in ALL and AML was significantly different from normal controls. Uc002 ehu.1, AC002454.1, uc001 guz.2, BC035649, uc001 kpt.2, AK123765, NR₀26776, NR₀27182, ENST00000508489 were up-regulated in pediatric ALL. ENST00000415964, uc010 arh.1, ENST00000457799, ENST00000511213, NR₀02712, X61079, ENST00000417930 were down-regulated in pediatric ALL. AC002454.1, uc002 ehu.1, ENST00000509150, uc001 guz.2, ENST00000457457, uc001 kpt.2, NR₀27182, AK123765, BC031319,AK124936, uc003 hhv.1, NR₀26776 were up-regulated in pediatric AML. AK027193, AK024584, BC005232, AK094982, uc003 ebe.1, ENST00000512129, AF088004, uc002 vje.1, uc010 arh.1, ENST00000428188, ENST00000457799, ENST00000415964 were down-regulated in pediatric AML.The results suggested that the expression of these lnc RNAs was consistent with microarray data. The expression of AC002454.1 was no significant difference with the sex, age, WBC count, chromosome, fusion gene, risk stratification andprognosis（P>0.05）. The expression of AC002454.1 was significantly related with the immunophenotype in children with newly diagnosed AL（P<0.05）.3. GO analysis showed that in up-regulated m RNAs: 413 m RNAs involved in biological process, 90 m RNAs involved in cellular component, 94 m RNAs involved in molecular function. Cell cycle phase, intracellular part and catalytic activity were the most enrichments. In down-regulated m RNAs: 665 m RNAs involved in biological process, 59 m RNAs involved in cellular component, 118 m RNAs involved in molecular function. Immune response, plasma membrane and protein binding were the most enrichments.4. Pathway analysis showed that 24 pathways corresponded to up-regulated m RNAs andcell cycle, consisted of 26 target genes, was the most enrichment. 32 pathways corresponded to down-regulated m RNAs and hematopoietic cell lineage, consisted of 23 target genes, was the most enrichment.5. We constructed a coding-noncoding gene co-expression network that included 10 lnc RNAs（ENST00000435695, ENST00000509150, NR₀27182, AK124936, uc003 hhv.1, AK027193, BC005232, ENST00000428188, ENST00000415964, NR₀02712） and 797 targeted genes.6. After AC002454.1 expression was down-regulated, the proliferations of K562 and NB4 cells were inhibited. The apoptosis rates of K562 and NB4 cells were increased. In addition, cell cycle results showed that the NB4 cells in G2/M phase were increased. After AC002454.1 expression was down-regulated, CDK6 expression in K562 and NB4 cells was lower than the control group.Conclusions: 1. Our study was the first to reveal differentially expressed lnc RNAs in pediatric AL by using lnc RNA microarray analysis. Some of the differentially expressed lnc RNAs were validated by q RT-PCR to promote the credibility of results. Lnc RNA microarray analysis is an ideal and effective method for screening differentially expressed genes in pediatricAL.2. The expressions of lnc RNAs were significant difference between AL and normal control. We determined AC002454.1 with the greatest difference. The expression of AC002454.1 was significantly related with the immunophenotype in pediatric AL. It is expected to be a new molecular markers of pediatric AL.3. Preliminary bioinformatics analysis of the microarrayshowed that differential expression lnc RNAs and m RNAs could regulatecell transformation and carcinogenesis through participation in whole processes and pathways of leukemia cells. The gene network map of lnc RNAs with their target genes, suggested that these genes and their pathways may be new targets for the treatment of pediatric AL.4. In vitro studies demonstrated that down-regulation of AC002454.1 can affect proliferation, cell cycle, apoptosis and CDK6 expression of leukemia cells. Our study provides the research ideas and experimental base for further clarifying the biological function of AC002454.1 in the future.