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Knockdown of NUDT21 Inhibits Proliferation and Promotes Apoptosis of Human K562 Leukemia Cells Through ERK Pathway

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Knockdown of NUDT21 Inhibits Proliferation and Promotes Apoptosis of Human K562 Leukemia Cells Through ERK Pathway Journal name: Cancer Management and Research Article Designation: Original Research Year: 2018 Volume: 10 Cancer Management and Research Dovepress Running head verso: Zhang and Zhang Running head recto: NUDT21 promotes growth of K562 leukemia cells open access to scientific and medical research DOI: http://dx.doi.org/10.2147/CMAR.S173496 Open Access Full Text Article ORIGINAL RESEARCH Knockdown of NUDT21 inhibits proliferation and promotes apoptosis of human K562 leukemia cells through ERK pathway Lan Zhang Background: NUDT21 is a mammalian precursor mRNA(pre-mRNA) 3’ end processing factor Weihua Zhang and plays an important role in the selection of poly(A) sites in 3’-untranslated region (3’-UTR). NUDT21 links alternative polyadenylation with regulation of glioblastoma and osteosarcoma Department of Haematology, First Hospital of Shanxi Medical University, progression and is found to be related to drug resistance in childhood acute leukemia. However, Taiyuan 030001, China the effect of NUDT21 on leukemia cells and the underlying mechanism are unknown. Methods: We knocked down NUDT21 in K562 cells and applied qRT-PCR and western blotting to quantitate the mRNA and protein expression. Cell proliferating and apoptosis were investigated subsequently by flow cytometry, BrdU, Caspase3/7. RNA microarray and intracellular signaling array were used to determine the important cell signaling pathways. Results: We clarified that the mRNA expression levels of NUDT21 are higher in primary chronic myelocytic leukemia patients and K562 leukemic cells compared with healthy controls and PBMCs. Downregulation of NUDT21 expression in K562 cells inhibits proliferation and promotes apoptosis. Screening by mRNA chip and intracellular signaling array, we found that MAPK/ERK pathway represented the main molecular mechanism underlying the effects of NUDT21 knockdown in K562 cells. Conclusion: NUDT21 played an important role in promoting proliferation and inhibiting apoptosis in leukemia K562 cells. The underlying mechanisms involved the modulation of PTEN and a set of downstream molecules including ERK1/2. Impact statement: The present work shows that the expression of NUDT21 was upregulated in chronic myelocytic leukemia and K562 cells. Silencing NUDT21 inhibited the proliferation and promoted the apoptosis of K562 cells. Subsequent experiments confirmed that NUDT21 promoted K562 proliferation through regulating the expression of p-ERK. Our findings may provide insights into the molecular mechanism underlying the effects of NUDT21 on leukemia cells and a novel strategy for the treatment of leukemia. Keywords: NUDT21, MAPK/ERK, K562 cell line, leukemia, cell proliferation, cell apoptosis Introduction Leukemia is a heterogeneous group of disorders resulting from the acquisition of chromosomal rearrangements, multiple gene mutations, and abnormal epigenetic regu- lation. Epigenetics indicates heritable changes in gene expression without nucleotide Correspondence: Weihua Zhang sequence variation, including DNA methylation, histone deacetylation, and chromatin Department of Haematology, First remodeling.1 With the noncoding RNAs in the field of epigenetics becoming a new Hospital of Shanxi Medical University, 85 Xinjian South Road, Taiyuan 030001, hot topic of research on abnormal epigenetic regulation in leukemia genesis,2 more China studies have focused on posttranscriptional regulation of genes. Recently, NUDT21, a Tel +86 351 463 9562 Email [email protected] protein capable of regulating precursor messenger RNA (pre-mRNA) 3′-end formation submit your manuscript | www.dovepress.com Cancer Management and Research 2018:10 4311–4323 4311 Dovepress © 2018 Zhang and Zhang. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://dx.doi.org/10.2147/CMAR.S173496 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Zhang and Zhang Dovepress at the posttranscriptional level, has been revealed to be closely cytic leukemia (CML) and K562 cells. Silencing NUDT21 related to disease progression. inhibited the proliferation and promoted the apoptosis of K562 It has become increasingly recognized that pre-mRNA cells. Subsequent experiments confirmed that NUDT21 pro- 3′-end formation is crucial for mRNA maturation in eukary- moted K562 proliferation through regulating the expression otic cells, promoting mRNA stability, efficient nuclear trans- of p-ERK, which is the effector of the MAPK/ERK pathway. port, and translation.3 CFIm is a cleavage and polyadenylation specificity factor, and it was first purified from Hela cell Materials and methods nuclear extracts as four polypeptides of 25, 59, 68, and 72 Patients and cell culture kDa (CFIm25, CFIm59, CFIm68, and CFIm72, respectively). The human leukemia cell lines, K562, Jurkat, and HL-60 cells CFIm is a heterodimer composed of the smallest CFIm25 were purchased from the Cell Resource Center, IBMS, CAMS/ subunit and any one of the three large subunits, while PUMC, and maintained in DMEM supplemented with 10% CFIm59, CFIm68, and CFIm72 are structurally related.4 FBS. Cells were incubated at 37°C and maintained at 5% CO . Although CFIm68 depletion decreases 3′-untranslated region 2 Peripheral blood mononuclear cells (PBMCs) were collected (3′-UTR) length, the most significant primer activation signal from the healthy people’s medical examination specimen at switching was found to occur after knockdown of CFIm25.5 the First Hospital of Shanxi Medical University. The use of CFIm25, also known as NUDT21 (nudix, nucleoside the primary cells was approved by the ethics committee of The diphosphate linked moiety X-type, motif21), has a NUDIX First Hospital of Shanxi Medical University (Taiyuan, China). hydrolase domain that acts like an authentic RNA-binding The bone marrow samples of primary CML patients protein. 3′-UTRs are important for fine-tuning transcript and and healthy control subjects were obtained from The First protein levels because they contain binding sites for regula- Hospital of Shanxi Medical University, China. The median tory molecules such as RNA-binding proteins and miRNAs.6 age of the CML patients was 58 years (range, 49–76 years). NUDT21 regulates 3′-UTR length by binding to the proximal All samples of CML were positive for the BCR/ABL gene. cleavage and polyadenylation site and directing alternative The study was approved by the ethics committee of The polyadenylation (APA).7 The global shortening of mRNAs First Hospital of Shanxi Medical University (Taiyuan, China). through APA that occurs during enhanced cellular proliferation Written informed consent was obtained from all subjects. All represents an important mechanism of regulated gene expres- the bone marrow samples were kept frozen at –70°C until use. sion. The 3′-UTR truncation of growth-promoting mRNA tran- scripts that relieves intrinsic microRNA- and AU-rich element (ARE)-mediated repression has been observed to correlate Quantitative reverse polymerase chain with cellular transformation. Like alternative splicing, usage reaction (qRT-PCR) of APA signals allows a single gene to encode various mRNA Total RNA was isolated from cultured cell lines and bone transcripts.5 For example, use of APA signals often removes marrow samples using TRIzol Reagent (Thermo Fisher large parts of the 3′-UTR, which contain the possible miRNA- Scientific, Waltham, MA, USA). The IQ5 detection system targeting sites and AREs, thus influencing the fate of mRNAs. (Bio-Rad Laboratories Inc., Hercules, CA, USA) and SYBR Specific APA events play important roles in cell growth and Green Real time PCR Master Mix (Thermo Fisher Scientific) differentiation or disease, such as immunoglobulin M (IgM) were used for qRT-PCR analysis. The conditions of qRT-PCR switching,8 spermatogenesis,9 and tumorigenesis.10,11 were as follows: predenaturation for 5 minutes at 95°C, dena- It has been demonstrated that NUDT21 plays pivotal turation for 30 seconds at 90°C, annealing for 40 seconds roles in the regulation of glioblastoma cell proliferation and at 60°C, and extension for 40 seconds at 72°C, for a total of tumorigenicity.12,13 Redis et al14 reported that lncRNA CCAT2 40 cycles. The PCR primers are listed as follows: NUDT21 regulated tumor metabolism through binding different CFIm forward, 5′-CGGCTACCCCATGTGTTACTG-3′; NUDT21 subunits (CFIm25 and CFIm68). NUDT21 was also identified reverse, 5′-TCACCACCAGGTAGTTTGAAGAAA-3′; as a gene associated with resistance to etoposide in children PTEN forward, 5′-CGACGGGAAGACAAGTTCAT-3′; with acute leukemia.15 These findings identified the potential PTEN reverse, 5′-AGGTTTCCTCTGGTCCTGGT-3′; relevance of NUDT21 to tumor progression. However, the GAPDH forward, 5′-TTTGTCAAGCTCATTTCCTG-3′; role of NUDT21 in leukemia cell growth and the underlying and GAPDH reverse, 5′-TGGTCCAGGGTTTCTTACTC-3′. mechanism remain to be fully elucidated. GAPDH was used as an internal control, and fold changes In the present study, it was first confirmed that the were calculated by relative quantification (2−DDCt). Each expression of NUDT21 was upregulated
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