Targeted Suppression Of MiRNA-21 Inhibit K562 Cell Growth Through PTEN-PI3K/AKT Signaling Pathway

Objective:Micro RNA(mi RNA) are about 19-25 nucleotide endogenous small non-coding RNA that play an important role in regulation of tumor occurrence,which has a obviously tissue specificity.Data shows that mi RNA-21 can regulate the hematopoietic cells happening and is involved in cell differentiation,proliferation and apoptosis.Mi RNA-21 is an important member of the mi RNAs family,which regulate the tumor occurrence by binding to target genes and downstream signaling pathways.Tensin homologue(PTEN) is known as a target gene of mi RNA-21, which can restrain angiogenesis,lower proliferation and the invasion of tumor cells by regulating the activity of PI3K/AKT.It is known that PTEN-PI3K/AKT play an important role in the development of leukemia.Leukemia was clonal malignant hematopoietic disorders that result from pluripotent bone marrow stem cells.It mainly displays in malignant cell proliferation,differentiation disorder, apoptosis blocked etc.It can affect the normal hematopoiesis by making tumor cells accumulate in the bone marrow or other hematopoietic system and infiltrate other tissues or organs.With the development of leukemia pathogenesis in recent years,more and more tumor signaling were be found.The PI3K/AKT mediated signal pathway has become a research hotspot in the pathogenesis and treatment in leukemia.The aim of the study was to investigate the expression changes on the genes related to PTEN-PI3K/AKT pathway in transfected cells and observe the cell proliferation and apoptosis by using MTT and follow cytometry after mi RNA-21 inhibitor transfection,in order to further explore the correlation between mi RNA-21 and PTEN protein by using Western-blot.Methods:The K562 cell was cultured in vitro.Cells can be divided into four groups:blank group,transfection group,random sequence control group and fluorescein control group.The mi RNA-21 inhibitor was transfected into K562 cells by using electroporation.Cell morphology and transfection efficiency assayed by using the cell in logarithmic growth phase.Cell proliferation and apoptosis assayed by using MTT and flow cytometry.In the same time,Western-blot and RT-PCR were performed to evaluate the expression of PTEN-PI3k/AKT pathway.Results:1 The transfection efficiency of mi RNA-21 inhibitor in K562 cell assayed by RT-PCRRT-PCR shows that the relative expression levels of mi RNA-21 in transfection group,random sequence control group,fluorescein control group were(8.070±5.138)%,(91.600±2.452)%,(92.247±2.053)% from blank group respectively by transfecting mi RNA-21 inhibitor to K562 cell.The expression of mi RNA-21 in K562 cell was significantly decreased(P<0.05).2 Effects of the K562 cell proliferation after mi RNA-21 inhibitor block the mi RNA-21 expressionCell inhibition rate assayed by using MTT after 12 h,24h,36 h,48h,60 h transfection.Compared to blank group cells,the transfection group cells proliferation were(2.6±0.4)%,(8.1±1.0)%,(15.5±1.4)%,(29.1±2.2)%,(43.1± 3.1)%,random sequence control group cells proliferation were(3.6±0.5)%,(7.6±0.9)%,(4.6±0.7)%,(4.3±0.6)%,(3±0.5)%,fluorescein control group cell proliferation were(2.9±0.3)%,(2.8±0.4)%,(5.6±1.0)%,(4.1±0.6)%,(5.7± 0.9)% respectively.The cell proliferation were significantly slow down in transfection group(P<0.05).3 Effects of the K562 cell apoptosis after mi RNA-21 inhibitor block the mi RNA-21 expressionFlow cytometry assays showed that the apoptosis of blank group,transfection group,random sequence control group and fluorescein control group were(2.334±0.263)%,(13.370±0.250)%,(3.990±0.436)%,(2.894±0.352)% respectively.The apoptosis value of transfection group after 48 hours transfection was significantly decreased(P<0.05).4 The expression changes of PTEN by inhibiting mi RNA-21 in K562 cellsThe relative expression levels of PTEN protein in blank group,transfection group,random sequence control group and fluorescein control group were(4.911±0.450),(14.701±0.810),(4.733±0.524) and(4.271±0.682)respectively. The expression of PTEN protein in transfection group was significantly increased(P<0.05).5 The expression changes of PI3 K by inhibiting mi RNA-21 in K562 cellsThe relative expression levels of PI3 K protein in blank group,transfection group,random sequence control group,fluorescein control group were(3.343±0.201),(1.015±0.122),(3.220±0.111) and(3.439±0.098) respectively.The expression of PI3 K protein in transfection group was significantly decreased(P<0.05).6 The expression changes of phosphorylation of AKT(p-AKT) by inhibiting mi RNA-21 in K562 cellsThe relative expression levels of p-AKT protein in blank group,transfection group,random sequence control group and fluorescein control group were(3.040±0.094),(0.463±0.080),(3.628±0.103) and(3.447±0.139) respectively.The expression of p-AKT protein in transfection group was significantly decreased(P<0.05).Conclusions:1 Inhibit mi RNA-21 expression could promote the K562 cells early apoptosis and inhibit the proliferation of K562 cells.2 Inhibit mi RNA-21 expression could suppressed the PI3K/AKT pathway by up-regulation of PTEN expression and promote its anti cell proliferation and the apoptosis effect.