Hepatocellular carcinoma cell lines, including BEL-7404 and SK-HEP-1 (Shanghai Cell Biology Institute of Chinese Academy of Sciences, Shanghai, China) were incubated in DMEM (Thermo Fisher Scientific, MA, USA) and supplemented with 10% FBS (Thermo Fisher Scientific, MA, USA) at an atmosphere of 5% CO2 at 37 °C.
Construction of KNTC1 knockdown cell model
Short hairpin RNAs (shRNAs) targeted against KNTC1 mRNA (NM_014708) were generated, and scrambled control shRNA (shCtrl) was produced as negative control. The sequences were summarized listed as below: 5’-CCGGTGGGGCATTCGTCTTGGTAAATTCAAGAGATTTACCAAGACGAATGCCCCATTTTTG-3’. After shRNA sequences were synthetized cloned into the BR-V108 (Shanghai Yibeirui Biomedical Science and Technology Co., Ltd) plasmid vector. Then, pHelper1.0, pHelper2.0 vectors, and the shRNA plasmids were co-transfected into 293T cells for 48 hours. The recombinant lentivirus was collected, which included LV-shKNTC1 (LV expressing KNTC1 shRNA) and LV-shCtrl (LV expressing scrambled shRNA), respectively. The hole‐by‐dilution titer assay was applied to access infectious titer. BEL-7404 and SK-HEP-1 cells (3 × 105 cells/well) were incubated into 6‐well plates and then infected with LV-shKNTC1 or LV-shCtrl (1× 108 TU/ml) at 70% to 80% confluences. The infection efficiency was estimated after 72 hours post infection.
RNA extraction and qPCR
The cells mRNA was obtained using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Subsequently, superscript first-strand synthesis system (Life Technologies) was applied to reverse transcription. Quantitative reverse transcription polymerase chain reaction (qPCR) (Applied Biosystems, Foster City, CA, USA) was used for relative quantification of KNTC1 gene expression. The relative expression values of KNTC1 were calculated using 2−ΔΔCp method and housekeeping gene GAPDH was internal reference. The primers sequences were listed: KNTC1: 5’‐ TGAAACGCTGCTCCACAAC‐3′ and 5′‐TGCTCGTCAGTAAAGGAACCAT‐3′; GAPDH: 5′‐TGACTTCAACAGCGACACCCA‐3′ and 5′‐CACCCTGTTGCTGTAGCCAAA‐3′.
This study was approved by the Medical Ethics Committee of the Ruijin Hospital of Shanghai Jiao Tong University School of Medicine and was conducted in accordance with the Declaration of Helsinki. The specific anti- KNTC1 monoclonal antibody (biorbyt) was carried out to estimate the KNTC1 expression in 20 para-carcinoma tissues and 152 tumor tissues from different HCC patients by immunohistochemical staining. The main staining procedures were as follows: formalin-fixed paraffin-embedded 3-μm tissue microarray sections were subjected to deparaffinized and hydrated. 3% H2O2 incubation for 10 min was used to eliminate endogenous peroxidase activity. After that incubation with anti- KNTC1 overnight at 4 °C and then with secondary antibody for 60 min at room temperature. DAB staining and hematoxylin counter staining were performed. The stained fields were photographed using microscopy.
Western blot assay
Total proteins extraction from BEL-7404 or SK-HEP-1 cells according to routine methods . The proteins were separated by 8% SDS-PAGE (Sigma-Aldrich, St. Louis, MO, USA) and transferred onto nitrocellulose membranes (GE Healthcare Life sciences, Pittsburgh, PA, USA). After the membranes were sealed with 5% non-fat milk at 37°C for 1 hour, primary antibodies (anti-KNTC1, biorbyt, 1:500; anti-GAPDH, Bioworld, 1:3000; anti-Akt, CST, 1:1000; anti-p-Akt, Bioss, 1:1000; anti-CCND1, CST, 1:2000; anti-CDK6, abcam, 1:1000; anti-PIK3CA, abcam, 1:1000) were incubated at 4°C overnight. The blots were detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rochford, IL, USA) after incubating with the secondary Goat Anti-Rabbit antibody (Beyotime, 1:3000) for 2 hours.
The infected cells were distributed into 96‐well plate at 2000 cells per well and incubated to 4 hours with MTT (20 μL, Genview, Craigieburn, Victoria , Australia) at 5 mg/ml concentrations. 100μL of dimethyl sulfoxide were added to dissolve the methyl nitrogen crystals formed by living cells. After shaking for 2 to 5 minutes, the absorbance at 490nm was detected by microplate reader (Tecan Infinite, Tecan GmbH, Groedig, Austria). The detection time points were 1, 2, 3, 4, and 5 days after cultivation.
Annexin V assay
Fluorescein isothiocya-nate (FITC)‐conjugated Annexin V (eBioscience; Thermo Fisher Scienti c, Inc.) was used for apoptosis detection. The infected cells (2×105 cells/well) were grew to about 70% coverage rate at 6‐well plate, harvested and then stained with (FITC)‐conjugated Annexin V. At least 5×105 cells were used to detect the FITC fluorescence signals via flow cytometry (Billerica, MA, USA).
Colony formation assay
The infected cells were seeded into 6-well plates at 400 to 1000 cells/well after transfection. After 2 weeks cultivation, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 500 μL Giemsa (Dingguo Changsheng, Beijing, China) for 20 min. The total number of colonies were imaged and counted.
3×104 infected cells were cultured to at least 90% confluence at 96‐well plate. The scratch was formed at the central part of the 96 well plate by the scratch instrument. serum-free medium was used to rinse gently for 2-3 times. 0.5% FBS/DMEM was added in to the wells. Wound widths of cell migration were obtained through microscope binding Image J software (NIH, Bethesda, MD, USA). The detection time points of BEL-7404 and SK-HEP-1 were at 0h, 24h, 50h and 0h, 8h, 24h respectively after scratching.
Transwell migration assay
Incubation 1×105 infected cells in 24-well plate contained cell culture inserts (24-well, pore size 8μm; Sigma-Aldrich Co.) and supplement with 100 μL media without FBS. 600ul 30% FBS/DMEM was added into the lower chamber. After incubation for 48 hours (BEL-7404) or 16 hours (SK-HEP-1), noninvasive cells from the upper chamber were removed with a cotton swab. Invasive cells on the surface of the lower membrane were fixed with 5% crystal violet solution for 5minutes and then photographed with an inverted microscope.
Human apoptosis antibody array
The expression levels of apoptosis related proteins involved in Lv-shKNTC1cells were detected by Human Apoptosis Antibody Array kit (abcam) according to the manufacturer’s protocols after collection and lysis.
Tumorigenesis and in vivo imaging in nude mice.
Four-week-old BALB/c nude mice (15–20 g) were used and conducted strictly in accordance with the guidelines of the Animal Care and Use Committee of Shanghai Jiao Tong University. To evaluate SK-HEP-1 (2 × 106) cell proliferation in vivo, Lv-shKNTC1 or Lv-shCtrl were subcutaneously injected into armpit of right forelimb of the mice. Tumor growth curves were measured at 7, 12, 14, 16, 19 days after injection. Volume was accessed through the following formula: V = (length × width 2 ) / 2. At 19 days following injection of HCC cells, 10 μL/g D‑Luciferin (Yeasen, Shanghai, China) were injected intraperitoneally into the mice. After 20 minutes, intraperitoneal injection of 0.7% pentobarbital sodium in anesthetized mice. Fluorescence results were observed using animal imaging system (PerkinElmer, Waltham, USA). All mice were sacrificed, and the tumors were incised, photographed and analyzed by IHC.
GraphPad Prism 6.0 software (GraphPadPrism 6.0 Software Inc., San Diego, CA, United States) were used in all statistical analyses. We used Mann-Whitney U test to analyze the differences between groups and the Spearman rank correlation test was used to confirm the correlations. The differences between the Lv-shKNTC1 group and Lv-shCtr group were analyzed by Student t test. And differences between groups of shKNTC1 and shCtrl were assessed through one-way ANOVA. All data were showed as mean ± SD. P-values < 0.05 were considered statistically significant.