Research On The Inhibitory Effect And The Underling Mechanisms Of AS-miR-21 In Human Non-small Cell Lung Cancer A549 Cells

BACKGROUNDmicroRNAs(miRNAs)are a class of endogenous non-coding small RNAs of approximately 22 nucleotides in length,which act as post-transcriptional gene modulators and play important roles in growth,differentiation,proliferation and apoptosis of cells.In recent years,many reports have showed the miRNAs are closely related to the pathogenesis of tumors,miR-21 was also confirmed highly expressed in many kinds of tumor tissue.miR-21 antisense oligonucleotide named AS-miR-21 was designed and synthetized to observe its effect on target genes of miR-21 and biological:characteristics of NSCLC A549 cells and to clarify the mechanism of AS-miR-21 in pathogenesis of NSCLC.This research may provide new targets of biological therapy for patients with NSCLC.Part ?.The Effct of AS-miR-21 on Biological Behaviour in NSCLC A549 CellsObjective To observe the effects of AS-miR-21 on biological behavior,such as proliferation,apoptosis,invasion,migration,cell cycle and Morphologicalcharacteristics of NSLCL A549 cells in vitro.Methods Selected NSCLC A549 cells,which were staying logarithmic phase for cell experiments.The experiments were divided into AS-miR-21 group,miR-negative control(miR-NC)group and blank group.Firstly,the qRT-PCR was used to measure the expression of miR-21 in A549 cells after As-miR-21 transfection.Subsequently,the influence on the proliferation,colony formation,apoptosis,invasion,migration,cell cycle and morphological characteristics in A549 cells were evaluated by methyl thiazolyl tetrazolium(MTT),colony formation assay,flow cytometry(FCM),transwell invasion test,wound healing assay and light microscope,respectively.Results Compared with miR-NC group and Blank grpup,the expression level of miR-21 significantly decreased(p<0.05)after transfected with AS-miR-21.MTT assay showed that As-miR-21produced an obvious inhibitive effect on the proliferation of A549 cells(p<0.05);the colony formation experiment showed that the ability colony formation was decreased by As-miR-21 notely(p<0.05).FCM showed that As-miR-21 promoted apoptosis(p<0.05),increased G0/G1 stage(p<0.05),declined S stage of cell line A549(p<0.05).Transwell invasion test showed that the ability of cell invasion were significantly decreased(p<0.05).Also,wound healing assay showed that the ability of cell migration significantly decreased(p<0.05).Under the inverted microscope,A549 cells of miR-NC group and Blank group proliferated and spread quickly on the culture bottle.They appeared as triangle,and arranged compactly.Cells of As-miR-21 group demonstrated cell shrinkage and appeared roundness.Conclusion As-miR-21 may regulate the biological behavior through down-regulation miR-21,such as suppressing proliferation,invasion,migration,promoting apoptosis.Part ?.The Mechanism Research of AS-miR-21 Regulatiing NSCLC A549 Cells Proliferation,Apoptosis and Invasion.Objective To explore the mechanism of AS-miR-21 regulating NSCLCA549 cells proliferation,apoptosis and invasion.Methods To validate whether miR-21 directly recognizes the 3'-UTR of PTEN mRNA,luciferase reporter assay was employed.qRT-PCR,Western blot,and immunohistochemistry were used to measure the expression of PTEN mRNA and PTEN protein respectively.To investigate the expression of PI3K and Akt,Wester blot was employed.Result PTEN was identified as one of target genes of miR-21 in A549 cells by dual luciferase report gene assay.The AS-miR-21 group PTEN mRNA expression had no significant difference on A549 cells(p>0.05)by qRT-PCR.AS-miR-21 could lead to the increment of PTEN protein,the downregulation of PI3K,Akt,by Western blot,and Immunohistochemistry.Conclussion As-miR-21 might regulate NSCLC A549 cell proliferation,apoptosis and invasion by PI3K/PTEN/AKT signal pathway.Part ?.Antineoplastic and Antiangiogenic Effects of AS-miR-21 onNSCLC A549 Cell Xenograft AssessObjective To explore antineoplastic and antiangiogenic effects of AS-miR-21 on human NSCLC A549 cell xenograft.Methods Xenograft models were produced.in 20 nude mice by subcutaneously implantation of human NSCLC A549 cells.when the tumor grew to about 70mm3 on 15d,a total of 15 nude mice with confirmed tumor lesions closely were randomized into 3 groups:Blank group,miR-NC group,AS-miR-21 group.Animal body weight and tumor volume were monitored regularly throughout the study.The nude mice were sacrificed 28d post-inoculation.The tumor weight,volum and tumor growth inhibition rate were recorded.Histopathological analysis for xenograft tissues were observed by HE staining.The immunohistochemical method were adopted to detect CD34 expression which was to analyze the microvessel density.Result NSCLC A549 cell xenograft models were established in nude mice,Mice in AS-miR-21 group were found a significant lessening of tumor volume and tumor weight,compared with Blank group and miR-NC group(p<0.01).By HE staining,binucleate cells could be observed in Blank group and negative control group,while cell debris and necrotic area could be observed in AS-miR-21 group.Microvascular density measurement:the number of vessels in the tumor filed of AS-miR-21 group,Blank group and miR-NC group was 32+9,102+23,108+22,respectively.Compared to Blank group and miR-NC,it was decreased significantly in AS-miR-21group(p<0.01).Conclussion As-miR-21 could inhibite the growth and angiogenesis of NSCLC A549 cell xenografts.Part IV.The Mechanism Research of AS-miR-21 Antineoplastic andAntiangiogenic Effects on Human NSCLC A549 Cell XenograftObjective To explore the mechanism of AS-miR-21 antineoplastic and antiangiogenic effects on NSCLC A549 cell xenograft.Methods Xenograft models were established by subcutaneously implantation of NSCLC A549 cells.The model nude mice were randomized into 3 groups:Blank group,miR-NC group,AS-miR-21 group.The nude mice were sacrificed 3d after last AS-miR-21 treatment.qRT-PCR was performed to detect the expressions of PTEN,PI3K,Akt,VEGF,VEGFR2,MMP-2 and MM-9.The expressions of p53,bax,bcl-2,VEGF,MMP-2,MMP-9 in xenograft were determined by immunohistoehemical staining and western-blot.Result The mRNA level PI3K,Akt,VEGF,VEGFR2,MMP-2 and MM-9 increased,while there were no statistical difference of PTEN mRNA in all groups.The result of immunocytochemistry staining showed that the protein expressions of PTEN,bax,p53 in AS-miR-21 group increased,while the expression of bcl-2,PI3K,Akt VEGF,MMP-2 and MMP-9 decreased.CoLtclussion The mechanism of AS-miR-21 antineoplastic and antiangiogenic effects might be through PTEN/PI3K/AKT signal pathway.