| Osteosarcoma is the most common primary bone tumor, formed by mesenchymal cellsproducing osteoid and bone matrix. In spite of their relatively low incidence,<10%of alltumors, these sarcomas represent a considerable burden because they are one of the mostfrequent pediatric bone tumors and remain prominent among both teenagers and youngadults Although the prognosis of osteosarcoma has been significantly improved by theadoption of new therapies that combine high-dose chemotherapy with local control of thetumor, the survival rate has now reached a plateau, and further improvements may onlyderive from a better understanding of molecular pathology, particularly mechanismsunderlying bone growth and remodeling. Thus, identification of new targeted therapies is acrucial step forward in the drive towards personalized medicine.Mediator complex, an essential component of the RNA polymerase II (Pol II)-mediatedtranscription machinery, is conserved from yeast to mammals. This component plays a keyrole both in the stimulation of basal transcription and in the regulation of eukaryotic mRNAsynthesis Mediator is a crescent-shaped complex consisting of head, middle and tailsubcomplexes. This physical partition of mediator is consistent with genetic and biochemicaldata indicating that the head complex interacts directly with Pol II and plays a general role inPol II transcription; the middle complex associates with Pol II carboxy-terminal domain; andthe tail complex recognizes and binds activators and is crucial for mediator's coactivatorfunction. Med19is a member of the head complex of mediator. Gene expression microarraystudies of a Med19deletion strain and a Med19truncation strain have shown that theexpressions of broad sets of genes are both up-and down-regulated. Med19mutants havebeen shown to be defective for activation of Gal4and Gcn4genes. On the other hand,Med19mutants also lead to derepression of heat shock, gluco-repressed, and HO genes. HOis transcribed only in haploid mother cells at Start, the G1-to-S phase transition. Theseresults show that Med19may play an important role in regulation cell proliferation and cellcycle. Deregulation of cell cycle progression is a hallmark of cancer. Thus, Med19may bean attractive candidate for the therapeutic targeting of cancer. However, there is littleknowledge about the role of Med19in the prevention and/or treatment of proliferative disease such as cancer.RNA interference (RNAi) is a kind of endogenous gene-silencing mechanism which hasemerged as a powerful post-transcriptional gene silencing new technique. This technologyhas become a powerful tool for studies on gene function, cancer and viral disease therapy. Inthe present study, we employed the RNAi technique to downregulate Med19expression inSaOS-2and U2OS human osteosarcoma cells and investigated the effect of Med19knockdown on the cell proliferation and cell cycle progression.1Materials and methods1.1Cell strainThe human osteosarcoma cell lines SaOS-2and U2OS (obtained from the ATCC)1.2Cell cultureThe human osteosarcoma cell lines SaOS-2and U2OS (obtained from the ATCC) werecultured in DMEM supplemented with10%FBS and100units/ml penicillin plus100μg/mlstreptomycin, and maintained at37°C in5%CO2humidified atmosphere.1.3Design and subcloning of shRNA into Lentiviral VectorThe cDNA sequence of Med19was obtained from GenBank (accession numberNM153450). The oligonucleotides were cloned into the pLVTHM lentivirus vectorcontaining a CMV-driven GFP reporter to produce Med19shRNA lentivirus vector andcontrol lentivirus vector, respectively. The recombinant vectors were confirmed by thedigestion analysis of restriction endonuclease and all inserted sequences were verified byDNA sequencing.1.4Lentiviral packing and transduction in SaOS-2and U2OS cellsTo produce infective lentiviral particles, human embryonic kidney293T cells wereco-transfected with the virion-packaging elements (psPAX2and MD2.G) and theshRNA-expressing vector using Lipofectamine2000. After two days, culture medium wascollected, filtered through a0.45-μm filter (Millipore, Bedford, MA) and concentrated byultro-centrifugation. SaOS-2and U2OS cells were seeded at4×104cells/ml in6-well platesand infected with recombinant lentivirus encoding for shRNA against Med19at amultiplicity of infection (MOI) of10. Five days post-infection, GFP expression wasexamined using fluorescent microscopy (CKX41, Olympus).1.5Real-time quantitative PCR analysis of Med19mRNA expressionTotal cellular RNA was isolated using Trizol Reagent according to the manufacturer'sprotocol. cDNA preparation was performed according to standard procedure using M-MLV Reverse Transcriptase and oligo-dT primer. Real-time quantitative PCR (qPCR) wasconducted with TAKARA TP800-Thermal Cycler DiceTMReal-Time System. The20μl ofPCR mixture includes SYBR green Master Mixture, primer pair, and template. Relativeexpression levels of Med19were determined using the comparative threshold cycle (Ct)methods.1.6Western blot analysis of Med19protein expressionCells were lysed in50μl lysis buffer (2%Mercaptoethanol,20%Glycerol,4%SDS in100mM Tris-HCl buffer, pH6.8) on ice for15min. Cell extracts were prepared on ice byultrasonic disruptionfollowed by centrifugation. The protein concentrations were determinedusing BCA Protein assay. Equal amounts (30μg of protein) of lysate were subjected to12%SDS-PAGE. After electrophoresis, protein blots were transferred onto PVDF membrane(Schleicher&Schuell Co.,USA) using an electro-blotting apparatus (Tanon, Shanghai, China).The membrane was blocked with5%nonfat milk in TBST solution, and incubated overnightwith the corresponding primary antibodies in the blocking solution at4°C. After washedwith TBST solution thrice, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody diluted with TBST solution (1:5000) at room temperaturefor2h. The signals of detected proteins were visualized on ECL plus Western blottingdetection system (Amersham). GAPDH protein levels were used as a control to verify equalprotein loading.1.7Measurement of cell viabilityThe MTT assay was performed to assess the viability of the infected cells. Both ofnon-infected and infected SaOS-2or U2OS cells (3×104cells/ml) were plated in96-wellplates. Then for five days, every24h a batch of cells were added with10μl MTT (5mg/ml)and incubated at37°C for4h. Then culture medium was removed and100μl of DMSO wasadded to each well. The dark-blue crystals of MTT-formazan were dissolved by shaking theplates at room temperature for10min. Spectrometric absorbance was measured on amicroplate reader (Bio-Rad680) at the wavelength of570nm. Each group was done intriplicate.1.8Plate colony formation assayCell growth and survival ability was also determined by the plate-colony-formationassay. Log phase cells were trypsinized into single cell suspension and seeded onto6-wellplates at a density of200cells/well. Cells were cultured for14days at37°C under5%CO2.Culture medium was changed at3-day intervals. Afterward, cells were fixed with4% paraformaldehyde for30min at room temperature. The colonies were stained with Giemsafor15min, then washed with water and air-dried. Cell colonies were counted using a lightmicroscopy. The experiment was performed in triplicate.1.9Flow cytometric analysis of cell cycleCells infected with Med19shRNA lentivirus or control lentivirus for7days andnon-infected cells were harvested by trypsinization, washed twice in cold PBS, fixed with70%ice-cold ethanol and stored at4°C overnight. After fixation, cells were washed twicewith cold PBS and incubated with100μg/ml RNase A and40μg PI for30min in the dark.Cells were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA). The fractions of the cells in G0/G1, S, and G2/M phases wereanalyzed using dedicated software (Becton-Dickinson, San Jose, CA). The experiments wererepeated in triplicate.1.10BrdU incorporationThe proportion of cells in S-phase was measured by using the BrdU cell proliferationassay kit according to the manufacturer's protocol. briefly, cells infected with Med19shRNAlentivirus or control lentivirus and non-infected cells were seeded in96-well plate at3×104cells/ml and incubated for2and4days. After that, BrdU was added and the cells wereincubated for4h. After removing the culture medium, cells were fixed and the DNAdenatured. Then, peroxidase-labelled anti-BrdU was added to bind to the BrdU. The immunecomplexes were detected by the3,3',5,5'-tetramethyl-benzidine substrate reaction, and theresultant color was read at490nm in a microplate reader (Bio-Rad680). The absorbancevalues correlated directly to the amount of DNA synthesis and thereby to the number ofS-phase cells in culture.1.11Statistical analysisData were expressed as means±standard deviations (S.D.). Statistical analysis wasperformed using Student's t-test. Differences were considered to be statistically significantwhen the P value was <0.05.2Results2.1Transduction efficiencyTo determine the lentiviral transduction efficiency in SaOS-2and U2OS cells, GFPexpression was examined by microscopy at MOI of10on5days after infection (Fig.1A).The efficiency of lentiviral vector transduction in SaOS-2and U2OS cells wasapproximately90%at an MOI of10. 2.2Stable knockdown of Med19mRNA and protein expression in SaOS-2and U2OScellsThe mRNA and protein expression of Med19in SaOS-2cells treated by lentivirus-mediated shRNA for5days were analyzed by real-time PCR and Western blotting. Asshown in Fig.1B, the level of Med19mRNA in SaOS-2cells infected with Med19shRNAlentivirus (Med19-shRNA-Lv) was significantly decreased by70%compared with thoseinfected with control lentivirus (Con)(**P<0.01). Additionally, Western blotting alsoshowed that the Med19protein expression levels were significantly reduced inMed19-shRNA-Lv infected SaOS-2cells compared with control cells (Fig.1C). However,these were no significant difference in Med19mRNA and protein expression betweencontrol cells and un-infected cells. The above results indicated that the expression of Med19in SaOS-2cells could be downregulated effectively by shRNA-targeted Med19.2.3Med19knockdown inhibited cell growth and proliferation in SaOS-2and U2OScellsTo investigate the effect of Med19knockdown on proliferation, vaibility of cells wasfirstly assessed by an MTT assay. As shown in Fig.2, down-regulation of Med19expressiondecreased the viability of SaOS-2and U2OS cells in a time-dependent manner. After fivedays of infection, the percentages of viable cells in SaOS-2and U2OS cells infectedMed19-shRNA-Lv decreased about61%and52%, respectively, as compared to the cellsinfected with control lentivirus (Con)(*P<0.01).Furthermore, the results of colony formation assay showed that lentivirus-mediatedshRNA against Med19caused a significant decreased (about95.3%and96.8%, respectively)in the number of colonies in SaOS-2and U2OS cells, as compared to the cells infected withcontrol lentivirus (Con) in which the number of colonies was not affected (**P<0.01, Fig.3).All these results indicated that shRNA-mediated down-regulation of Med19expressionsignificantly inhibited cell growth and proliferation in SaOS-2and U2OS cells.2.4Med19knockdown inhibited SaOS-2and U2OS cell proliferation by arresting thecell cycle at G0/G1phaseTo better understand the effect of Med19knockdown on decreased cell viability, weused flow cytometry to examine if the cell cycle was affected. As shown in Fig.4A, theknockdown of Med19induced an increase in the percentage of cells in the G0/G1phase ofthe cell cycle (from54.32±0.76%in control SaOS-2cells to72.48±1.46%in RNAi SaOS-2cells), parallel to a decrease in the percentage of cells in the S-phase (from29.65±0.77%in control SaOS-2cells to16.60±0.17%in RNAi SaOS-2cells). Similar results were obtainedwith U2OS cells infected with Med19-shRNA-Lv (Fig.4B). The difference in the percentageof G0/G1or S-phase cells between control cells and non-infected cells was not significant inSaOS-2and U2OS cells. These results indicate that Med19knockdown can inhibit SaOS-2and U2OS cell proliferation by the induction of G0/G1arrest.2.5Med19knockdown reduced the count of S-phase cellsTo further confirm the effect of Med19knockdown in SaOS-2and U2OS cellsproliferation, we measured the rate of DNA synthesis by BrdU incorporation assay. BecauseBrdU is incorporated instead of thymidine into the DNA during DNA synthesis inproliferating cells, the number of cells that incorporate BrdU reflects the percentage of totalS-phase cells. As shown in Fig.5A, after2days of infection, lentivirus-mediated shRNAagainst Med19did not impact the percentage of S-phase cells in SaOS-2cells. However, thepercentage of S-phase cells in SaOS-2cells infected with Med19-shRNA-Lv for four daysdecreased by about53%(**P<0.01) compared with cells infected with control lentivirus.The difference in the percentage of S-phase cells between cells infected with lMed19-shRNA-Lv and non-infected cells (Con) was not significant. Similar results wereobtained in U2OS cells infected with Med19-shRNA-Lv (Fig.5B). Knockdown of Med19substantially decreased the fraction of cells in the S-phase of the cell cycle, indicating thatMed19knockdown can arrest the cell growth.Taken together, the lentiviral expressing Med19-specific shRNA could effectivelydecrease Med19expression in SaOS-2and U2OS cells. Med19knockdown inhibitedSaOS-2and U2OS cell proliferation by the induction of G0/G1arrest. Thus, it is expected tobe a potential therapeutic target for the treatment of osteosarcoma. As to the mechanism ofhow Med19influences cell cycle progression of human osteosarcoma cells, though we caremore about it, investigation on it have not still been accomplished. Thus future studies arewarranted to fully understand its function and complex signaling pathways.
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