The Mechanisms And Effects Of GAS5,A Long Non-coding RNA,on The Proliferation Of Leukemia Cells

Leukemia is a disease caused by the malignant clone of hematopoietic stem cells.When leukemiacellsin bone marrow and other hematopoietic tissues multiply and accumulate,eventually infiltrating other tissues and organs,it will block normal hematopoiesis.So far,the reason and mechanism of leukemia arestill unclear and require the further study.LncRNA refers to a class of ncRNAs whose transcript length is greater than 200 nt.It is transcribed in eukaryotic cells without or little coding protein.There is growing evidence that LncRNA plays an important rolein the development of the different tumors.The long-chain non-coding RNA 5(GAS5)whose entire length is about 630 nt is located on the chromosome 1q25.1 of the non-coding protein,and it mainly accumulates in the growth-suppressing cells.Recent years,it was found that the aberrant expression of GAS5 in variety of cancer models,such as breast cancer,lung cancer,bladder cancer,liver cancer,prostate cancer,etc.The effects of GAS5 on tumor growth,invasion,metastasis,apoptosis,and cell sensitivity to chemotherapeutic agents have been reported.HLelievre’s group found that the expression of GAS5 was up-regulation in myeloproliferative diseases.In terms of leukemia,only a piece of literature reported that the patients with rs55829688 CC genotype had a high expression of Lnc RNA GAS5 in peripheral blood mononuclear cells.The high expression of Lnc RNA GAS5 may worsen the myelosuppression,and in turn may lead tothe poor prognosis of AML.However,the expression way of LncRNA GAS5 in other leukemia cells have not been reported.Insulin-like growth factor 1 receptor(IGF1R)is one of the four cell surface receptors of IGF family.Locating at the surface of cells,IGF1 R is the tetrameric tyrosine receptor,with proto-oncogene activity.The abnormal expression of IGF1 R in most solid tumors and hematologic malignancies may promote the development of tumor.In different solid tumors and hematologic malignancies,by inhibiting IGF1 R or stimulating IGF1 Channel,IGF1 R pathway can regulate the function and phosphorylation of some important kinase pathways at its transcription and post-transcriptional level,such as PI3 K / AKT pathway,Raf-ERK1 / 2,Src and FAK(focal adhesion kinase).Therefore,we assume that whether the abnormal expression of Lnc RNA GAS5 exists in leukemia cells;can it regulate the proliferation and apoptosis of leukemia cell;and is the specific mechanism involved in IGF1 R pathways?ObjectiveSelecting leukemia cell line as the research object,we compared the expression of Lnc RNA GAS5 in mononuclear cells of peripheral blood from healthy adults and that in different leukemia cells.And we explore the mechanisms of influences of LncRNA GAS5 on proliferation of human leukemia cell line,which can provide guidance for the diagnosis and treatment of leukemia.MethodsHuman leukemia cell lines(KG1a、HL60、K562)were cultured in vitro.Mononuclear cells were isolated from peripheral blood of healthy adults.The plasmid of interference LncRNA GAS5 slow virus infectious KG1 a cells was cultured,and the steady turn strains was screened.The expression of GAS5 in AML cell line was assessed by RT-PCR.The effects of KG1 a cells proliferation in vitro were detected by CCK-8 and typan blue assay.After down-regulated LncRNA GAS5,the cell cycle and apoptosis of KG1 a,mock(KG1a)and siGAS5(KG1a)cells were measured by FCM,and the morphological changes of apoptotic cells were observed by fluorescence microscope after Hochest staining.The expression of apoptotic related proteins p53,Bax,Bcl-2 and Cleaved caspase-3,cell-cycle related proteins cyclinD1 and CDK4,the expression change of IGF1 R,AKT and ERK were examined by Western Blot.KG1 a and siGAS5(KG1a)cells were treated with 0.05μmol/LPPP,and then analyzed the levels of IGF1 R,AKT and ERK protein.Results1.The results of semi-quantitative RT-PCR showed that GAS5 was highly expressed in AML cell lines(KG1a,HL-60 and K562 cells)compared to mononuclear cells.2.Trypan blue stain and CCK-8 assay showed that the down-regulation of GAS5 significantly inhibited proliferation and promoted apoptosis of KG1 a cells.3.After the interference of LncRNA GAS5,the life cycle of KG1 a cells was blacked in G0/G1 phase.The portion of cycles in G0/G1 phase was increased from(30.27±2.12)% to(44.26±6.98)%(P<0.05).4.The rate of apoptosis in siGAS5(KG1a cells)group was up-regulated from(5.63±1.90)% to(17.40±3.44)% compared to control group(P<0.05).5.Hoechst staining revealed that the down-regulation of GAS5 could induce compaction of nuclear chromatin,and the typical change of cell apoptosis occurred.6.Western Blot suggested that the expression of p53,Bax and Cleaved caspase-3 increased sharply,but the expression of Bcl-2,cyclinD1 and CDK4 proteins significantly decreased.7.Western Blot suggested that GAS5 down-regulation could inhibited the expression of p-IGF1 R,p-AKT and p-ERK significantly.And the downward trend was more obvious after treatment with PPP.Conclusion1.LncRNA GAS5 was overexpression in leukemia cell lines,the down-regulation of GAS5 inhibited the proliferation of leukemia cells KG1 a.2.The down-regulationof GAS5 caused the activation of P53 signal pathway,down-regulation of the expression of CyclinD1 protein,and the imbalance of Bcl-2/Bax,and ultimately activated caspase-3.All these inhibited the proliferation and induced apoptosis of KG1 a cells.3.The down-regulation of GAS5 inhibited the proliferation of leukemia cells KG1 a mainly through down-regulating the expression of p-IGF1 R,the activation of AKT and ERK signaling pathway.