2.1 Tissue sample
The cancerous tissue and adjacent non-cancerous tissues of 50 patients with primary HCC surgery were obtained from the clinical sample bank of the First Affiliated Hospital of Zhengzhou University. The collection of human specimens was approved by the Biomedical Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Patients had signed informed consent. The standard was to perform a curative hepatectomy between 201602-201806. HCC was diagnosed pathologically by two senior pathologists and no adjuvant treatment was performed before the operation. All specimens were collected, and were stored at -80 °C.
2.2 Cell culture and transfection
Human liver cancer cell lines (HepG2, MHCC97H, SK-Hep1, HUH7, HepG2, LO2) were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). HCCLM3, MHCC97H and cells LO2 were obtained from Fenghuishengwu(Wuhan,China).The cells were cultured in RPMI 1640 medium supplemented with 10% FBS. All cells were incubated at 37 ° C in a cell incubator containing 5% CO 2.
Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) was used to transfect si-CASC15, sh-CASC15,miR-2355-5p mimics, miR-2355-5p agomir and corresponding controls (GenePharma, Shanghai, China) into cells. The human cDNA sequence of CASC15 was cloned into the pcD-ciR vector to construct CASC15 over-expression plasmid. The vector was purchased from Geneseed Biotech Co., Ltd. (Guangzhou, China), and oligonucleotides were transfected into cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA).
2.3 Cell proliferation detection
Cells were seeded in a 96-well plate at a density of 5,000 cells per well. 100 μLCCK8 solution (Liji, Shanghai, China) was added. After 4 h, the absorbance value was analyzed by a microplate reader (Bio Tek Instruments, USA) at 450nm.
2.4 EdU staining
HCC cells concentration were adjusted to 1X105 / ml, and inoculated in a 96-well for 24 hours. After culture treatment, EdU with a final concentration of 50 μmol / L was added and the cells were incubated for 2 h. Then 4% polyoxymethylene fixing solution was added for 30min, 0.5% Triton X-100 was added for 20min, 100 μ l of Apollo reaction solution was add to each well to incubate for 30min, and Hoechst33342 staining solution was added for 30min. The cells were observed under fluorescence microscope.
2.5 Colony formation
The cells were made into a 1000 cell / mL cell suspension, and placed in a 6-well culture plate. The original culture solution was discarded after 48 hours of culture. Then the methanol fixing solution was taken out and fixed for 10 min. After repeated experiments, the colony formation rate was observed under a microscope. A cell cluster composed of more than 50 cells was counted as 1 colony.
2.6 Transwell analysis
Transwell chambers with 8 μm pores were purchased from Corning (Corning, NY). The transfected cells with a concentration of 1 × 106 cells in 100 μl were resuspended in RPMI 1640 medium without FBS and, afterward, seeded into the upper chambers of the 24‐well plate. For another, the lower chambers were full of 600 μl RPMI 1640 medium added with 10% FBS. Cells were incubated for a whole night. Finally, cells which migrated into the opposite side of the transwell membrane were fixed by methanol, stained using crystal violet, and counted under a light microscope. For the invasion assay, 30 μl Matrigel (BD Biosciences, Heidelberg, Germany) was used to cover the transwell membrane and proceeded similarly as described above.
2.7 Luciferase reporter assay
Total RNA was extracted from the cells, and CASC15 was amplified using WT and MUT primers. The fragment of CASC15 was inserted into the pGL3-Bashc luciferase reporter vector named CASC15-WT-Luc. The mutant plasmid CASC15-Mut-Luc was formed by mutagenesis in the binding region of CASC15 and miR-2355-5p. CASC15-WT-Luc and CASC15-Mut-Luc were co-transfected into cells with miR-30a-5p mimic and pRLTK. After 6 h, Luciferase activity was analyzed by the DualLuciferase Reporter Assay Kit (Promega, Madison, WI).
2.8 Quantitative reverse transcription-PCR (qRT-PCR)
Total RNA in tissues and cells was extracted by TRIzol reagent (Biosntech, Beijing, China). SYBR-Green (Takara Biotechnology, Co., Lt. (Dalian, China) was used in qRT-PCR reaction. Amplification was performed by ABI 7,500 real-time PCR system. A qScript microRNA cDNA synthesis kit (Quantabio, Beverly, California, USA) was used for cDNA synthesis. The expression levels of RSU1P2 and miR-202-5p were analysed by the 2-△△CT method. The expression levels of miRNA were standardized by U6. The expression levels of lncRNA were standardized by GAPDH. The primer sequences were as follows:
lncRNA CASC15: forward 5′- CTTATTCACTTGCCGGGAG-3′,
lncRNA CASC15: reverse 5′- CGCTTAGATCCCAAGGG-3′.
miR-2355-5p: forward: 5′- CTGATTGTGAAGAGAATGT-3′,
miR-2355-5p: reverse: 5′-GTTCTTCGACATCCGGGCCG-3′.
Six1: forward: 5′-CCACAGAATCCGCGAACCT-3′,
Six1: reverse: 5′-GAGTTCATTACAGCGTTGGC-3′.
GAPDH: forward: 5′-CGAGAGAATCCGCGGACAT-3′,
GAPDH: reverse: 5′-TTGTGCAATACAGCGTGGAC-3′.
U6: forward: 5′-GACAGATTCGGTCTGTGGCAC-3′,
U6: reverse: 5′-GATTACCCGTCGGCCATCGATC-3′.
2.9 Western blot
The transfected cells were collected, the total proteins were extracted, and the protein concentration was quantified by the BCA Protein Assay Kit. Next, the loading buffer was added to the protein, and then 50 μg per well was loaded. Proteins were separated using 10% polyacrylamide gel electrophoresis, transferred to PVDF membrane, and blocked with 5% BSA for 1 h. Then the membrane was incubated with anti-Six1 (1: 1000, Amyjet, Wuhan, China), TGF-b (1: 1000 , Amyjet, Wuhan, China) and anti-GAPDH The antibody (1: 1000 , Amyjet, Wuhan, China) overnight. Then it was incubated with anti-rabbit secondary antibody (1: 1000, Amyjet, Wuhan, China) for 1 h .
2.10 In vivo tumor growth assay
To examine the effects of CASC15 and miR-2355-5p on the growth of HCC cells in vivo, sh-CASC15 or miR-2355-5p inhibitor were stably transfected into luciferase-labeled HCC cells. Tumors were inoculated into nude mice. A total of 3 × 106 transfected HCC cells were subcutaneously injected into six-week-old male nude mice (n = 5 per group). The tumor volume was measured with a caliper. 4 weeks after tumor formation, euthanize nude mice, remove tumor samples, collected tumors in mice and measure tumor weight. After 4 weeks of tumor formation, the nude mice were euthanized, and tumor samples were taken out to collect tumor and measure tumor weight. The experiment was approved by the First Affiliated Hospital of Zhengzhou University.
2.11 Statistical methods
The monitoring data were analyzed by SPSS19.0 statistical software. The results of data analysis were represented as mean ± standard deviation (mean ±SD). Multigroup data analysis was founded on one-way ANOVA. LSD test was used for subsequent analysis. P < 0.05 indicated significant difference.