HCC tissue specimens collection.
Thirty-eight paired tissue specimens of HCC and adjacent non-tumor (ANT) tissues were obtained from Tangdu Hospital. Ethical approval was obtained from the Ethics Committee of the Fourth Military Medical University, and informed consent was obtained from each patient. The thirty-eight HCC tissues were divided into 2 groups: metastatic HCC tissues (patients with intrahepatic metastasis or portal vein cancer embolus, n = 22) and non-metastatic HCC tissues (n = 16). All the tissues were obtained at the time of surgery and immediately stored in liquid nitrogen until use.
Cell Lines And Culture Conditions
The HCC cell line MHCC-97H (HCC cells with high metastatic potential) was used in this study. All cell lines were purchased from Shanghai Institute for Biological Sciences (Shanghai, China). All cell lines were routinely cultured in RPMI-1640 medium (Hyclone Laboratories, Logan, UT) supplemented with 10% fetal calf serum (Gibco BRL, Rockville, MD, USA) at 37 °C in a humidified atmosphere of 5% CO2.
Cells were seeded in 4-well 35-mm dishes (Greiner Bio-One North America Inc., Monroe, NC, USA) at a density of 1,000 cells/well and grown for 48 h in culture medium. Then cells were fixed in 4% paraformaldehyde for 20 min and permeabilized in phosphate-buffered saline (PBS) supplemented with 0.5% Triton X-100. After blocking, cells were incubated with the indicated antibodies for 2 h. Cells were washed in PBS, incubated with their corresponding FITC-labeled secondary antibodies (Pierce, Rockford, IL, USA) for 1 h at room temperature and stained with DAPI (Vector Labs, Burlingame, CA, USA). Finally, the cells were mounted using glycerol and observed using a Nikon A1 laser scanning confocal microscope (Tokyo, Japan).
Cell samples were lysed with RIPA buffer (Beyotime, China). Equal amounts (10 µg) of total protein were loaded, and then subsequently immunoblotted with the primary antibodies, including anti-E-cadherin (BD Biosciences, Franklin Lakes, USA), Vimentin (Invitrogen, Carlsbad, CA, USA), Snail, MAPK1, slug and tubulin (Santa Cruz, CA, USA). Proteins were detected using the Amersham enhanced chemiluminescence system (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.
Real-time RT-PCR was performed as described previously . Expression data were uniformly normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, and the relative expression levels were evaluated using the ΔΔCt method [16, 17]. Primers were used as described previously [11, 18, 19].
Vector Construction And Luciferase Reporter Assay
The miR-22 overexpression vector was constructed according to previous .Cultured cells were transfected with miR-22 expression vector, antisense miR-22 (anti-miR-22), scramble miRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The sequence were described as before .
The 3’UTR segment of Snail and MAPK1 were subcloned into the pmirGLO vector (Promega, Madison, WI, USA), respectively [11, 12]. The mutant constructs were generated using a QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA). All constructs were further confirmed by sequencing. Snail and MAPK1 overexpression plasmids were constructed as previous  Cell transfection and dual luciferase reporter assay were performed as described previously.
All statistical analyses were performed using the SPSS statistical software package (SPSS, Chicago, IL, USA). The significance of the data was determined using Student’s t test. All the statistical tests were two-sided, and a P value < 0.05 was considered significant.