ANG Promotes Proliferation And Invasion Of The Cell Of Lung Squamous Carcinoma By Up-regulating HMGA2

Purpose:The lung cancer,as the most common tumor among all the malignant tumors,causes the most cancer deaths worldwide.While it is the fact that the prognosis of lung cancer is poor,despite significant therapeutic improvements have been made in recent years.However,it is studied that preventing the invasion and metastasis of cancerous cells plays an important role in lowering the morbidity and mortality of this disease.Squamous cell carcinoma of the lung currently has ranked as the second most frequent histological subtype of non-small cell lung cancer and accounted for about 40 % of all lung cancers.Angiogenin(ANG),as a angiogenic ribonuclease of the RNase A superfamily,is up-regulated in a variety of human cancers,which has a 33% amino acid identity and an overall homology of 56% to that of RNase-A.Angiogenin(ANG)has been shown to play a permissive role in tumor angiogenesis.Recent research indicates that ANG also directly stimulates cancer cell proliferation by enhancing rRNA transcription,thus has been linked to worse clinical prognosis in one study.NG cleaves tRNA and promotes ribosomal RNA transcription affecting a wide variety of responses including proliferation,cell invasion and invasion and tube formation in the nucleus,and nuclear ANG cannot be detected in normal,non-endothelial cells.High mobility group AT-hook 2(HMGA2)is involved in various biological processes such as the formation of the embryo and the development of malignant cancer.Members of the HMGA protein family are often referred to as ”architectural transcription factors'',because of their ability to regulate expression of a large number of target genes through alteration of chromatin structure.HMGA2 gene has shown to be of prognostic relevance in several other cancer entitiesBased on this knowledge,the current study examines the impact of ANG-HMGA2 pathway on the proliferation and invasion of the squamous cell carcinoma of the lung cell line SK-MES-1 and discusses its possible mechanisms of action,as clarified that our results provide experimental evidence for the inclusion of ANG-based targeted therapies in the clinical treatment of SQCLC.Methods:The present study is divided into three sections.1.Twelve patients' normal tissue and cancerous tissue were collected.ANG expression in the squamous cell carcinoma of the lung was evaluated by qRT-PCR,western-blot and immunohistochemical.2.We transfected SK-MES-1 cells with Ad-ANG and guaranteed the transfection efficiency.The regulation of ANG on proliferation and invasion of SK-MES-1 cells were analyzed by Cell Counting Kit-8,Transwell migration camber.PCR and western-blot was utilized for HMGA2,the screening potential target genes of ANG.3.We transfected SK-MES-1 cells with Ad-ANG/sh HMGA2 and tested the transfection efficiency through qRT-PCR and western-blot.The proliferation and invasion of SK-MES-1 cells transfected by Ad-ANG/sh HMGA2 were analyzed by Cell Counting Kit-8,Transwell migration camber.Chromatin immunoprecipitation(ChIP)assays were adopted for direct DNA-protein bind relationship detection.ResultsQuantitative RT-PCR and western-blot were used to examine the expression levels of ANG and HMGA2 in normal tissue as well as cancerous tissue in SQCLC patients.qRT-PCR results showed that ANG and HMGA2 mRNA levels were significantly higher in cancerous tissue,as compared with the normal adjacent tissue.Similarly,western-blot and immunohistochemical results showed a higher local expression levels of ANG and HMGA2 protein in cancerous tissue in SQCLC patients.We transfected SK-MES-1 cells with Ad-ANG and the transfection efficiency reached ? 80%.Cells transfected with Ad-GFP were used as control.The expression levels of ANG and HMGA2 were examined 48 hours after transfection.The relative mRNA level of ANG and HMGA2 in the Ad-ANG SK-MES-1 cells increased significantly,as compared with the Ad-GFP cells.An increased expression of ANG and HMGA2 protein in the Ad-ANG SK-MES-1 was also observed according to western blot results.Cell proliferation examination was performed after transfection using a CCK8 assay.It was found that Ad-ANG SK-MES-1s manifested increased proliferation level at 24 hours,48 hours and 72 hours after transfection.With regard to SK-MES-1 cells invasion capability evaluation,transwell invasion chamber assay was applied to assess the impact of ANG on this cell property.Results showed that Ad-ANG SK-MES-1s presented significantly increased invasion capability,as compared with the Ad-GFP cells.A rescue experiment was performed via transfection of sh HMGA2 in Ad-ANG SK-MES-1 cells.As expected,the HMGA2 mRNA and protein levels in the Ad-ANG/sh HMGA2 SK-MES-1 cells were effectively decreased,compared with the Ad-ANG/sh Scramble cells.Furthermore,it was found that Ad-ANG/sh HMGA2 SK-MES-1 cells showed decreased proliferation and invasion.Results from ChIP assay showed that ANG directly bound to the promoter of HMGA2.ConclusionIn this study,we examined the expression of ANG in clinical SQCLC tissues by Western blotting and qRT-PCR.The results of qRT-PCR analysis showed that ANG and HMGA2 mRNA level in SQCLC cancerous tissue revealed more increases compared with that in the benign tissues,and the ANG mRNA expression showed a linear correlation of the HMGA2 expression in SQCLC cancerous tissue.It suggested that ANG might play a role in the tumorigenesis of SQCLC.To extend our clinical studies and investigate its biological function,we employed adenovirus to over-express ANG in SQCLC cell line SK-MES-1,which thus suggested that over-expression of ANG could become a positive factor for expression of HMGA2.In addition,over-expression of ANG could enhance the ability of proliferation and invasion of lung squamous carcinoma cells in vitro.But when over-expressed ANG along with silencing HMGA2,the ability of cells could weaken.In conclusion,ANG promotes the invasion and proliferation of SQCLC cells through the mediator HMGA2 in vitro.