br All animal maintenance and operational procedures were
All animal maintenance and operational procedures were carried in accordance with the animal licence protocol approval by Animal Care and Ethics Committee of Dalian Medical University. Male nude mice (Balb/c) aged 4–6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were randomized for xenograft tumor growth and experimental metastasis assays. For xe-nograft tumor formation, the mice were randomly divided into three groups, sh-control, sh-BPTF-2, sh-BPTF-3. 4 × 10 6 Hep3B cells with stable knockdown of BPTF gene or control vector expressing mCherry protein were respectively suspended in 100ul PBS and injected sub-cutaneously into the right flank of each mouse. The tumor volume was measured after two weeks of injection. The tumor volume was calcu-lated as V= (width2 ×length)/2 and the data was recorded every two days within two weeks. Mice were sacrificed and tumors were taken from mice for weighting and photographing. For the metastasis assays, the mice were randomly divided into four groups. PBS（No Hep3B group） or 4 × 106 Hep3B cells with stable knockdown of BPTF gene (Hep3B-sh2 or Hep3B-sh3 group) or control vector expressing mCherry protein (Hep3B-control group) were respectively injected into the tail vein of nude mice. 45 days after injection, the distribution of tumor cells in mice body was detected by in vivo imaging system (CRI maestro). Parameter setting: Excitation: 523 nm, Emission: 560–660 nm, Filter: 560 nm, Exposure time: 1.000 s. Then the mice were sacrificed and lung tissues were taken. Partial tissues were transferred to liquid nitrogen immediately and lysed for western blot analysis, and partial were fixed in formalin for IHC assay.
2.15. Immunohistochemistry staining assay
The examined tissues were fixed in 4% paraformaldehyde, washed with PBS three times, transferred to 70% ethanol, cut into small pieces and then embedded in paraffin in accordance with standard procedures. After being dewaxed and antigen retrieval, the tissue sections were stained using SP kit (SP-9000, ZSGB-BIO, China) according to its
instructions. Briefly, the sections were blocked with blocking reagents, and then were exposed overnight at 4 ℃ to 475473-26-8 against BPTF, hTERT, EpCAM, or CD44. The slides were washed with PBS and in-cubated with anti-mouse/rabbit biotin antibodies for 1 h. After washing, the slides were added with HRP-conjugated streptavidin, de-veloped with HRP substrate(DAB), and counterstained with hematox-ylin. In the end, the paraffin sections were dehydrated by using Gradient alcohol, sealed by means of neutral balsam (Solarbio) and photographed with microscope.
2.17. RNA extraction from frozen tissues and PCR
Total RNA was extracted from xenografts of HCC cells in mice and hepatic carcinoma tumors and adjacent normal tissues from patients using Trizol (Invitrogen Life Technologies), following the manufac-turer's protocol. Briefly, 50–100 mg of tissue sections were added to 1 ml Trizol, centrifuge at 12000 rpm for 10 min to remove insoluble matter. After chloroform extraction and precipitation with isopropanol, RNA was washed twice with 75% ethanol, and finally the RNA pellet was dissolved in 15 μl of RNase-free water.
concentration was measured using the NanoDrop spectro-photometer. cDNA synthesis was performed using Prime-ScriptTM RT-PCR Kit (TaKaRa) according to the protocol described. The sequence of primer as following: BPTF (forward primer, 5′- AATCGGAGAAGTCCA ACGGG-3′, Reverse primer, 5′-TTGCCCTATGTGATGC CCAG-3′); hTERT (forward primer, 5′- AGGCTCACGGAGGTCATCG-3′, Reverse primer, 5′-GGCTGGAGGTCTGTCAAGGTA).
2.18. Statistical analysis
All results were presented as the mean ± SE. A Student t-test was performed to compare the two independent groups of data. The asso-ciations between BPTF or hTERT expression and categorical variables were compared by Pearson Chi-squared test. Survival curves were cal-culated using the Kaplan Meier method. The log-rank test was used to analyze overall survival (OS) time between different clinicopathological factors in hepatocellular carcinoma. Univariate and multivariate ana-lysis were performed using the Cox regression model. Data was ana-lyzed by the SPSS 20 software (Inc, Chicago, IL). P < 0.05 was con-sidered to be significant.
3.1. BPTF was highly expressed in HCC cells and tumor tissues and positively correlated with patients’ advanced stages
We firstly detected the expression of BPTF in human HCC cells and tumor tissues. Western blot analysis indicated that BPTF was highly expressed in five different HCC cell lines and one immortalized liver cell line (L-O2) (Fig. 1A), as well as tumor tissues from 9 cases of patients with HCC, by comparison to the levels of BPTF in the corresponding adjacent non-cancer tissues (Fig. 1B). These results were further con-firmed by the obtained data from Oncomine database. The BPTF mRNA level of clinical tissue samples collected in Wurmbach Liver showed