Real-time PCR was performed around the TP800 qPCR System (Takara, SW, Akron, OH 44314, USA). a transcriptional regulator was further confirmed in a gene expression microarray study in deletion strain because a broad range of genes was found to be up- or downregulated after Med19 functional disruption. Furthermore, Med19 has been demonstrated to be a component of the Mediator complex  and is essential for mediator binding and its activation of RNA Pol II [9,10]. Structural analysis showed that Med19 is usually involved in head-module subunits in mammalian mediator complex and plays an important role in the whole mediator stabilization. The potent function of Med19 as a transcription coactivator for regulating gene expression pattern suggests its role in the development of malignancies. Recently, Med19 was reported to promote the proliferation of bladder malignancy, hepatocellular carcinomas, prostate malignancy, gastric malignancy and breast malignancy cells [11-15]. However, the functional role of Med19 in tongue malignancy cell growth and migration has not been reported. In the present study, we constructed recombinant lentivirus delivering short hairpin RNA (shRNA) against silencing in tongue malignancy cell proliferation, tumor formation and cell migration was investigated and siRNA contamination Med19 siRNA (5-AAGGTGAAGGAGAAGCTAAGT-3) or unfavorable control siRNA (5- TTCTCCGAACGTGTCACGT-3) were inserted into pGCSIL-GFP lentiviral vector, respectively. The siRNA plasmids were transfected together into HEK293T cells with lentiviral helper plasmidto generate the respective lentiviruses using Lipofectamine 2000 (Invitrogen, Grand Island, NY 14072, USA). Bephenium Viral stocks were made and used to infect tongue malignancy cells. Cells were collected for mRNA and protein levels detection after 72 h after contamination. Reverse transcription polymerase chain reaction Total mRNA samples of tongue malignancy cells were prepared with Trizol reagent (Invitrogen, Grand Island, NY 14072, USA) according to the manufacturers instructions. Samples (2.0 g) were used as templates to perform the RT-PCR assay using M-MLV-RTase (Promega, Madison, WI 53711, USA). The producing cDNA was amplified by using the SYBR-Green Grasp PCR Mix (Applied Biosystem, Grand Island, NY 14072, USA) in triplicates. Real-time PCR was performed around the TP800 qPCR System (Takara, SW, Akron, OH 44314, USA). Primers utilized for real-time PCR were as follows: Actin-forward, 5-GGCGGCACCACCATGTACCCT-3, Actin-reverse, 5-AGGGGCCGGACTCGTCATACT-3; Med19-forward, 5-GTAACTTCCTGCCTGACCTG-3, Med19-reverse, TGTGCTTGTGCTTATTCTTCTTC-3. Western blot analysis Cells were lysed in 1 SDS lysis buffer (1 M Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 1 mM aprotinin, 1 mM PMSF and 10 g/mL leupeptin). The protein samples were separated by electrophoresis in SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with Tris buffer saline (TBS) made up of 5% nonfat milk and 0.1% Tween 20 overnight, the membrane was subsequently incubated with primary antibodies at room temperature for 2 h or at 4C overnight and with secondary antibody for another 2 h, respectively. The membrane was then developed using the ECL+plus? Western blotting system (Amersham). Cell proliferation assay Tca8113 cells were infected with Med19 siRNA lentivirus or control lentivirus for 3 days. About 2,000 cells were seeded Bephenium MRK into each well in 96-well plates. An MTT cell proliferation assay was performed for 5 consecutive days and a BrdU incorporation assay was performed at 24 h and 48 h. Results were expressed as the absorbance at 570 nm and 490 nm, respectively. Colony formation assay Med19 siRNA lentivirus or mock control infected Tca8113 cells were collected 3 days after lentivirus contamination. For the plate clone forming experiment, 500 cells were mixed in culture medium, and seeded in 6-well plates and each with three duplicate wells. Afterward, the cells were incubated at 37C in air flow with 5% CO2 and the media were renewed every 3 days. Two weeks later, the colonies were stained Bephenium with Giemsa and the colony number was statistically analyzed. Cell cycle analysis Lentivirus infected tongue malignancy cells were Bephenium fixed with 70% pre-chilled ethanol at 4C for 1 h after 3 days of lentivirus contamination. The fixed cells were washed and stained with propidium iodide (PI) combination made up of 50 g/mL PI and 100 g/mL ribonuclease in PBS for 45 min at 37C. The cells were exceeded through Bephenium a 300-mesh nylon net before the DNA content was determined by quantitative circulation cytometry with standard optics.