br As the catalytic subunit of the telomerase holoenzyme
As the catalytic subunit of the telomerase holoenzyme complex, human telomerase reverse transcriptase (hTERT), uses its own RNA as template to synthesize telomeres, and then add telomeres to the ends of chromosomes to extend the shortened telomeres and to protect chro-mosomes [18,19]. Therefore, hTERT plays an important role in cell senescence and tumorigenesis. It has been reported to be involved in cell signaling transductions governing cell proliferation, apoptosis, migration and stem cell function [20,21]. In HCC, the serum and tissue expression of hTERT was significantly increased than that in patients with liver cirrhosis , and TERT mRNA was reported to serve as a tumor marker for early detection of hepatocellular carcinoma . Sustained activation of telomerase is even thought to be essential for the growth and progression of HCC, and targeting therapy against hTERT holds great promises in HCC treatment . The expression and function of hTERT gene are known to be regulated at various molecular levels, and among all the regulatory events, transcription modulation is one of the most important. Therefore, the study on how hTERT is transcriptionally regulated, focusing primarily on the discovery and verification of the unknown trans-acting regulators, including tran-scription factors and epigenetic modifiers, will provide new and po-tential therapeutic strategies for HCC treatment. r> In the study, we investigated the functional roles of BPTF in HCC and its effect on the stemness of HCC cancer stem CAY 10566 in vitro and in vivo. We also assessed the response of chemotherapeutic drugs to BPTF knockdown in HCC cells, and evaluated the transcriptional regulation of hTERT by BPTF. Our data show that BPTF promotes stemness maintenance, tumor growth and metastasis by transcriptionally acti-vating hTERT expression in HCC cells. The simultaneous high expres-sion of BPTF and hTERT was positively correlated with the advanced malignancy and predicted poor prognosis for patients with HCC.
2. Materials and methods
2.1. Cell lines and cell culture
Human hepatoma cells Hep3B, HepG2, SNU-449, and human normal liver cell L-O2 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). L-O2 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Hep3B and HepG2 were cultured in Eagle's Minimum Essential Medium (MAC GENE) supplemented with 10% fetal bovine serum. SNU-449 were maintained in RPMI-1640 Medium containing 10% fetal bovine serum. Bel7402 cell line was obtained from Peking University Hepatology Institute (China), and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All re-ferred cell lines were cultured in a humidified atmosphere containing 5% CO2 at 37 ℃.
Cells plated in 96-well plates (2000 cells/well) were transfected with control or specific shRNAs. 48 h later, 10ul MTT reagent (5 mg/ ml) was added in single well and treated for 3.5 h. Then MTT was discarded and 150ul Dimethyl Sulphoxide (DMSO) was added in single well. Finally, the OD value at 490 nm was measured.
2.4. Colony formation assay
Cells transfected by control or specific shRNAs were trypsinized into single cell and seeded in 6-well plates (1000 cells/well) with continuous culture for 8 days. The cells were washed with PBS twice and fixed with the mixture (methanol: glacial:acetic 1:1:8) for 10 min, and then were stained with 0.1% crystal violet for 10 min. The plates were washed with PBS and the colonies were photographed and counted.
2.5. Wound scratch assay
Cells were plated in a 6-well plate and grown to nearly 70–80% confluence. Then the cells were treated with plasmids, or siRNA, or inhibitor for 24 h and scraped in a straight line to create a “scratch”. The images of the cells at the beginning and at regular intervals during cell migration to close the scratch were captured and compared through quantifying the migration rate of the cells.
2.6. Transwell invasion assay
Cell invasion ability was detected using 24-well chemotaxis cham-bers (Corning, CA, USA, Cat: 3422). The cells were washed twice with PBS, resuspended in 100 μl serum-free medium and added into the upper chambers. The lower chambers were filled with 500 μl medium containing 20% fetal bovine serum (FBS). The cells were incubated for 48 h in the upper chamber coated with a mixture of serum-free medium and Matrigel (4:1; BD Biosciences, Cat: 356234). The membrane were fixed in methyl alcohol for 10 min at room temperature, stained with crystal violet for 10 min, washed 3 times with PBS and dried off. The crystal violet was dissolved with 500 μl 33% acetic acid and the OD570 value was measured.