The Functional Role Of SARI In Human MG-63 Osteosarcoma Cells And Its Mechanisms Of Action

Background and Objective:Osteosarcoma,the most common type of primary malignant bone tumor and the peak incidence of the osteosarcoma occurs in the second decade of life during the adolescent growth spurt.Osteosarcoma can be treated with surgery,radiotherapy,and chemotherapy.Current treatment regime of surgery and intensive chemotherapy only cure less OS in advanced stages.There is a clear need for newer effective agents for patients with osteosarcoma,especially for patients who afflicted with metastatic and recurrence tumor.Novel precise and effective OS-targeting treatment approaches are being developed on the condition that the biology of osteosarcoma as well as the molecular mechanisms could constantly be investigated and uncovered.The SARI?suppressor of AP-1,regulated by IFN?gene,also known as BATF2 or ATF-like2,is a putative cancer-suppressor gene that located at chr:11q13.1.The SARI gene possesses three coding exons and encodes a protein?SARI?with molecular mass of 29.4 kDa.Since its discovery,the SARI has been demonstrated to be associated with the occurrence of several types of carcinomas.Of note,decreased expression of SARI is associated with a worse clinical outcome in hepatocellular carcinoma and oral tongue squamous cell carcinoma.All of the above-mentioned studies have in common that SARI may be served as a potential candidate biomarker or target for new diagnosis,prognosis and therapies in cancers.The biological feature of SARI in osteosarcoma?OS?remains unclear.In the current study,we firstly made characteristic analysis of SARI gene and protein,based on which,the pCMV-SARI-Flag eukaryotic expression vector was constructed and tansfected into MG-63osteosarcoma cells,and the effects as well as the underlying mechanisms were evaluated and traced.Our study contains three sections:Part One:Characteristic analysis of SARI gene and protein;Part two:Construction and identification of the pCMV-SARI-Flag eukaryotic expression vector;Part three:Effects of SARI overexpression on the biological characteristics of MG-63 osteosarcoma and its mechanism of action.Part One:Characteristic analysis of SARI gene and proteinMethods:1.The basic information of SARI was obtained from the online UCSC database.The promote region and sequences were predicted by Promoter 2.0 and Neural Network Promoter Prediction;The underlying transcription factors and sequences in the 5000 bp 5'UTR region were predicted by CONSITE;The underlying CpG islands were predicted using the online Database of CpG island Finder,EMBOSS Cpgplot,and CpGFinder programs.2.The secondary structure of SARI RNA were predicted and obtained based on the Vienna RNA Package and the online UCSC database.The expression profiles of SARI RNA in tissues were obtained from the analyses of GTEx and U133plus2Affymetrix microarray;3.I–TASSER was utilized to analyze the protein structure and function predictions of SARI protein,including the secondary structure,the final structure models,ligand binding sites,enzyme active sites and Proteins structurally close to the target in the PDB library.PROSITE was also employed to predict the domains and functional sites of the SARI protein.4.The information of subcellular localization of SARI in cells was got from the GeneCards and UniProtKB databases.The interaction network between SARI and other protein families was analyzed and predicted using the STRING Interaction Network.Results:1.The SARI gene locates at chr:11q13.1 and possesses three coding exons,with a full length cDNA sequence of 825 bp;2.The possible promoter regions of SARI gene may locate at 777⁸27 bp,1579¹629 bp,2542²592 bp and 2800 bp;3.The promoter/upstream 5000 bp region is predicted to have several transcription factor binding sites,which are mainly for the transcription factors of Snail,HFH-1,SQUA,and RREB-1,and so forth.4.The underlying CpG islands may locate at 835¹605 bp of the promoter/upstream 5000 bp region in the SARI gene.5.Analyses of the GTEx and U133plus2 Affymetrix microarray showed that SARI RNA are relatively high expressed in pulmonary alveolar macrophage,cultured endothelial progenitor cells,colon,colonic mucosa,sigmoid colon and mucosa,rectum,spleen,kidney,lung,and bone marrow;the subcellular localization of SARI is mainly in nucleus and golgi apparatus;6.I–TASSER showed that the sencondary structure of SARI protein was characterized by the presence of Helix and Coils;7.The final structure models were obtained using the I–TASSER program;the ligand binding sites comprise amino acid residues of 6 Gly,7 Asn,8 Gly,9 Leu,36Gln,39 Thr,43 Asp,45 Leu,46 His,47 Gln,49 His,76 Thr,Val 79,83 Leu,86 Met,87 Asp,122 Glu,and 129 Ser.No enzyme active sites were predicted.SARI protein shows the highest structural similarity to the 5h64B transferase protein family;8.SARI contains the basic-leucine zipper domain,and the DNA binding sites comprise amino acid residues of23 Lys,24 Lys,26 Lys,27 Asn,28 Arg,30 Ala,31Ala,32 Gln,33 Arg,34 Ser,35R,37 Lys,38 His and 39 Thr.9.The protein network showed that SARI could interact or communicate with JUN,JUNB,CEBPA,CEBPG,DBP,EPAS1 and MAFF protein families.Part two:Construction and identification of the pCMV-SARI-Flag eukaryotic expression vectorMethods:1.The full length SARI cDNA sequence was cloned form the SARI positive cDNA library using PCR,and then connected to the pCMV-C-Flag eukaryotic expression vector by the digestion using Hind III and Xba I,and connecting using the T₄ DNA ligase;2.The identification of the combined pCMV-SARI-Flag vector was enabled using DNA sequencing and double enzyme digestion by Hind III and Xba I;3.The pCMV-SARI-Flag vector was transfected into MG-63 osteosarcoma cells?SARI expression negative?using Lipo6000^TM,and the total RNA and protein from transfected cells were correspondingly extracted.The alteration of SARI in mRNA and protein levels following transfection was respectively examined by RT-PCR and Western blotting.Results:1.DNA sequencing demonstrated that the full length SARI cDNA sequence was successfully connected to the pCMV-C-Flag vector,and no single nucleotide mutaions were found by blasting with the NCBI database;2.RT-PCR and Weatern blotting showed that SARI mRNA and protein levels were up-regulated following the transfection of pCMV-SARI-Flag vector.Part three:Effects of SARI overexpression on the biological characteristics of MG-63 osteosarcoma and its mechanism of actionMethods:1.The pCMV-SARI-Flag vector was transfected into MG-63 osteosarcoma cells?SARI expression negative?using Lipo6000^TM,and light microscope was utilized to observe the morphological changes of MG-63 osteosarcoma cells during transfection;CCK-8 assay was applied to plot the growth curve;Wound healing assay was performed to further assess the effects of SARI restoration on cell migration;2.Effects of SARI restoration on cell apoptosis were respectively evaluated by Caspase-3 activity analysis,active Caspase-3 expression by indirect immunofluo-rescence,and DNA ladder electrophoresis;3.The expression alterations of apoptosis related protein involved in the endogenous mitochondrial apoptosis pathway was examined by Western blotting,and that the interactions between SARI and c-JUN was confirmed by co-immunopre-cipitation.Results:1.Cell growth was prohibited in pCMV-SARI-Flag transfected MG-63 cells at48 h following transfection characterized by the presence of swelling and atrophy in cellular morphology even deaths without adherence in 40%⁵0%cells;2.Up-regulation of SARI in MG-63 cells resulted in an markedly inhibition of the cell growth,proliferation and migration,all with P value less than 0.05;3.Overexpression of SARI in MG-63 cells could promote the Caspase-3 activiy?P<0.05?;indirect immunofluorescence also displayed that cleaved Caspase-3expression was detectable;DNA ladder electrophoresis showed the generation of typical“DNA ladder”in SARI overexpressed MG-63 cells;4.Western blotting showed that overexpression of SARI in MG-63 cells resulted in an down-regulation of Bcl-2,up-regulation of Bax as well as an activation of both Capspase-3 and Caspase-9,all with P<0.05;5.Co-immunoprecipitation and immunoblotting revealed that exogenous SARI could interact with c-JUN in MG-63 cells.Conclusions:1.The genic and proteic characteristics of SARI were summarized and predicted by bioinformatics analysis;2.The pCMV-SARI-Flag vector was successfully constructed;3.Up-regulation of SARI in MG-63 osteosarcoma cells resulted in an markedly inhibition of the cell growth,proliferation and migration;4.SARI behaves as a potent tumor suppressor in MG-63 osteosarcoma cells and the apoptosis effects may due to the activation of the intrinsic apoptotic singling pathway;5.SARI could bind with c-JUN during its anti-cancer effects in MG-63osteosarcoma cells.