Tissue specimens and microarray data
Collected forty-one pairs of histologically confirmed liver cancer and adjacent cancer specimens in the Second Affiliated Hospital of Nanchang University. The collected specimens required no chemotherapy, radiotherapy and immunotherapy. The collected specimens were immediately stored in liquid nitrogen and tissue fixation solution , The study was based on the Helsinki Declaration of 1964 and all subsequent amendments. All patients received written informed consent from the Ethics Committee of the Second Affiliated Hospital of Nanchang University. Three-hundred and seventy-four cases of liver cancer and 50 cases of normal liver tissue were accessed via the Starbase database, and the expression of TUG1 was compared.
Cell lines and cell culture
Normal liver cells (HL-7702), liver cancer cell lines MHCC-97H and HCC-LM3 were selected. All cells were purchased from Shanghai Cell Research Institute (Shanghai, China). HCC-LM3 and MHCC-97H cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), and HL-7702 cells were cultured in RPMI 1640 medium containing 10% FBS. The cells were maintained in an incubator at 37 ℃, 5% CO2, and 95% humidity. Cells in the logarithmic stage of growth were used.
TUG1 siRNA, miR-29a inhibitor, and negative control (NC) were purchased from Guangzhou RiboBio Biotechnolog (Guangzhou). IFITM3 siRNA and NC were purchased from Shanghai Gene Pharmaceutical. MHCC-97H and HCC-LM3 cells were finally selected as the cell lines and divided into the NC group and treatment groups. Each gene interference fragment and inhibitor was transfected into cells using the Lipofectamine 3000 kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The TUG1 siRNA sequence was as follows: sense, 5' GTTGACCTTGCTGTGAGAA 3’ and antisense, 5’ AACCTGGGAACCTTGGATTG 3'. The miR-29a inhibitor sequence was as follows: UAACCGAUUUCAGAUGGUGCUA. The IFITM3 siRNA sequence was as follows: IFITM3‐s1 sense, 5'‐CCA UUC UGC UCA UCG UCA UTT‐3' and antisense, 5'‐AUG ACG AUG AGC AGA AUG GTT‐3'.
Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) and western blotting
All RNA was extracted with TRIzol reagant, then reverse transcribed into cDNA using a reverse transcription kit (Takara, Tokyo, Japan), and then qRT-PCR was run using a PrimeScript RT kit (Takara). All proteins were extracted with RIPA lysate and protease inhibitor in a 100: 1 mixed liquid. The relative expression of each gene was normalized using the expression of housekeeping genes and calculated using the 2-ΔCt method. The RiboBio-designed primers were TUG1, IFITM3, beta-actin. miR-29a and U6, The primary antibodies,IFITM3 (Ab109429), Bax (ab32503), Bcl2 (ab32124), tubulin (ab15246), MMP19 (ab53146), VEGFA (ab52917), N-cadherin (ab76011), and E-cadherin (ab40772) , All antibodies were purchased from Abcam. Western blotting was used to calculate the amount of protein expressed.
Scratch experiments to detect cell migration ability. First seed the cells into a six-well plate,. When the cells grew to 80–90% confluence, a 200-µl sterile tip was used to form a scratch in each well. Then, the separated cells were washed away with PBS, and the width of the scratches at 0 h was observed under a microscope. The cells were cultured for 24 h in new medium, Then measure the width of the scratches taken twice to calculate the ratio of cell healing The healing ratio of the scratches was calculated.
Transwell migration and invasion assays
According to the reagent instructions, the matrix glue was added, and 60–80 µl matrigel was added to the inner chamber; then, the wells were placed in the incubator for half an hour. First, a cell suspension for 12–24 h of starvation was prepared, and the cells were uniformly added to the inner chamber with or without matrigel. Then, 500 µl of serum-containing medium (DMEM) was added to each well in the outer chamber and incubation was continued for 24–48 h in the incubator. Thereafter, the cells in the chamber were washed, the cells were fixed with formaldehyde, and the outer cells were stained with 0.1% crystal violet. Finally, a picture was taken with a microscope after the water had dried.
Measurement of apoptosis via ﬂow cytometry
The cells were cultured in suspension for 48 h and digested with trypsin without EDTA; then, flow cytometry analysis was performed using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's instructions. Data were collected on a BD FACS Canto system and analyzed using Flow Jo software.
Cell cycle assays
The cells were cultured in suspension for 48 h, digested with trypsin without EDTA, and flow cytometry analysis was performed using a cycle kit (BD Biosciences) according to the manufacturer's instructions.
The cells were first seeded into a 96-well plate, then the cells were fixed, then EDU stained according to the manufacturer's instructions (RiboBio), and finally observed and photographed under a fluorescence microscope.Blue represents all cells, red represents proliferating cells, and the EdU positive rate indicates the ratio of red cells to blue cells.
In vivo experiment
Adult male nude mice of 6-8 weeks were selected as experimental objects and purchased from Hunan SJA Experimental Animal Co., Ltd. (Hunan, China). Nude mice were injected with a phosphate solution containing 1 × 107 cells. Tumor volume was measured with calipers every 4 days: tumor volume = (shortest diameter 2 × longest diameter) / 2. After 4 weeks of photographing, the mice were anesthetized, the tumors were collected and weighed, and the expression of IFITM3 in the subcutaneous tumors was detected by immunohistochemistry, qRT-PCR, and western blotting. To assess lung metastasis, a phosphate solution containing 1 × 106HCC-LM3 was injected into the tail vein of nude mice. Three weeks later, the mice were sacrificed after anesthesia, and nude mice lung tissues were taken for hematoxylin and eosin (H & E) staining and immunization.
Hematoxylin & eosin and immunohistochemical staining
The prepared tissue was fixed with a Tissue fixation fluid, placed in a paraffin block, and cut into paraffin sections. Dewaxing was first carried out with xylene, and then the tissue sections were dehydrated with gradient alcohol. Sections were stained with H&E to determine if their morphology changed and then rehydrated and microwaved in sodium citrate buffer (10 mmol/L, pH 6.0) to restore the antigen. Sections were incubated with 0.3% hydrogen peroxide/phosphate buffered saline for 30 min and then blocked with serum. Subsequently, tissues were incubated with a 1:200 dilution of rabbit monoclonal antibody IFITM3 (ab15592, Abcam, Cambridge, MA, USA) at 4 ̊C overnight. Wash 3 times with phosphate buffered saline (PBS) every 5 minutes, and drop the secondary antibody at 37 ° C for 30 minutes. Next, the sections were stained with diaminobenzidine (DAB) and hematoxylin dye, then the excess dye was rinsed with running water, then rehydrated with gradient alcohol, and sealed with neutral resin. Finally, observe and take pictures under the microscope.
Dual-luciferase reporter assay
The dual-luciferase reporter assay (DR) has become an effective means to study the involvement of transcription factors in gene regulation. By analyzing the DNA fragment of the promoter, the transactivation ability of the promoter-binding element is verified, and transcription is investigated. The molecular mechanism of the factor in signal transduction can be observed since the miRNA acts mainly through the 3' UTR on the target gene, and the 3' UTR region of the target gene can be constructed behind the reporter gene luciferase, by comparing or overexpressing the miRNA. Reporter gene expression changes (monitoring changes in luciferase activity) can quantitatively reflect the inhibitory effect of miRNA on the target gene, combined with site-directed gene mutations and other methods to further determine the site of action of the miRNA and the target gene 3' UTR. Dual luciferase reporter assays were used to determine if miR-29a is a direct target gene for TUG1.
Fluorescence in situ hybridization (FISH) is sensitive, accurate, and can detect multiple genes at a time. It can be used to determine the exact position of the target gene, the positional relationship between several genes, and the relationship between genes and telomeres. The relationship between genes and centromeres is an essential element in the construction of genetic maps. The nucleus stained by DAPI is blue under the excitation of ultraviolet light, and the positive expression is the corresponding fluorescein-labeled fluorescence. FAM (488) is green and cy3 is red. In situ hybridization of mRNA showed that the results were positive for the cytoplasm, and a few positive nuclear results were normal. There were different expression levels of microRNA and lncRNA. The fluorescence intensity was different depending on the amount of expression. The TUG1 probe used was as follows: 5'-DIG-AATCTACCTCCAGTGTTCCTGCCGCATCGTG-DIG-3'. The miR-29a-3p probe used was as follows: 5'-DIG-TAACCGATTTCAGATGGTGCTA-DIG-3’.
By combining the antibody with some tracer substances, the antigen-antibody reaction is used to locate the antigenic substance in the tissue or cell. Immunofluorescence steps include, cell fixation and permeation, blocking, incubation of primary antibodies, secondary antibodies, etc. Immunofluorescence can localize the expression of TUG1 and IFITM3 whether it is intranuclear or extranuclear.
Statistical analysis software using GraphPad Prism 7.0 and SPSS 22.0.The expression of TUG1 in HCC tissues and adjacent tissues was compared using the Wilcoxon paired test. The correlation between TUG1 and miR-29a expression levels was found to be statistically significant using Spearman correlation analysis. Differences in overall survival were assessed using the log rank (Mantel Cox) test. The t-test was used to analyze the difference in expression of tumor tissues and paracancerous tissues. The chi-square test was used to compare the data from the two groups. Considered statistically significant when p-value is less than 0.05.