Cell strains and animals
The human esophageal squamous carcinoma cell line EC9706 was purchased from the State Key Laboratory of Molecular Oncology, Chinese Academy of Medical Sciences. The experimental cells were divided into seven groups, normal EC9706 cells, EC9706 cells transfected with shPrdx1 lentivirus (shPrdx1), EC9706 cells transfected with negative control lentivirus (shControl), EC9706 cells transfected with OE-Prdx1 lentivirus (OE-Prdx1), EC9706 cells transfected with corresponding negative control lentivirus (OE-Control), EC9706 cells treated with Tripolin A (EC9706+Tripolin A), and EC9706 cells transfected with OE-Prdx1 lentivirus and treated with Tripolin A (OE-Prdx1+Tripolin A). Healthy male BALB/c nude mice (4 weeks, n=20, 10 in each group) were provided by the Animal Center of the Chinese Academy of Science (Shanghai, China). All animals were raised and treated according to guidelines developed by the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The mice were housed under strictly controlled SPF environmental conditions and mice were sacrificed by CO2 inhalation.
Acquisition of lentivirus
The Prdx1 interfering and negative control lentivirus plasmid were constructed by inserting the target sequence (shPrdx1: TGTCTGACTACAAAGGAAA, shControl: TTCTCCGAACGTGTCACGT) into the Age I/EcoR I restriction enzyme cutting sites of the GV112-Lentivirus vector according to previous results (3). The over-express Prdx1 (Accession Number: NM_001202431) and control plasmid were constructed by inserting the target sequence into the Age I/BamHI restriction enzyme cutting sites of the GV287-Lentivirus vector. Plasmid vectors were obtained from the Genechem Co. Ltd (Shanghai, China). The synthesis of the target gene sequence, establishment of the plasmid carrier, and lentivirus packaging were performed as described previously.3
Cell culture and lentivirus transfection
The EC9706 cells were cultured in RPMI-1640 media (Gibco) containing 10% fetal bovine serum (FBS, Gibco) in a humidified atmosphere of 5% CO2 at 37 ℃. The EC9706 cells (MOI=100) were infected with shPrdx1 lentivirus (1×109 TU/ml) or the over-expressed Prdx1 lentivirus (1×108 TU/ml) to decrease or increase the levels of Prdx1. The corresponding negative control lentivirus was similarly transfected into cells. The procedure was as follows. EC9706 cells were inoculated into a 6-well microplate at a density of 1×105/ well. Next, 1 ml of culture medium without antibiotics was added to each well when the cells grew to 30% confluence. Then, 5 mg/ml polybrene and the lentivirus were added and cultured for 12 h at 37 ℃, followed by the addition of 2 ml of fresh culture medium. The expression of GFP fluorescent protein was measured as an indicator of the efficiency of lentivirus transfection using an inverted fluorescence microscope. The cells were collected at 72 h for further experiments.
Gene chip detection was used to detect the changes in gene and signal pathways in ESCC cells following Prdx1 interference. The total RNA of control EC9706 cells and cells in which Prdx1 was inhibited were isolated using the Trizol reagent. The extracted total RNA samples were subjected to quality testing with a NanoDrop 2000 and an Agilent Bioanalyzer 2100. The qualified samples were then applied to a human gene expression profile chip (chip type: GeneChip primeview human, number: 901838) developed by Affymetrix. The hybridization, washing of the gene chip, scanning, and data analysis were completed by the Genechem Co. Ltd (Shanghai, China).
Tumor formation in nude mice
Negative control EC9706 cells in logarithmic growth phase and EC9706 cells with inhibited levels of Prdx1 were prepared as cell suspensions and injected subcutaneously into the right axillary of nude mice (2×107 /ml, 200 μl for each). The body weight and tumor volume of each nude mouse were measured twice each week. Each nude mouse was sacrificed under anesthesia at 23 d and the mice were put into a live imaging device (IVIS® Lumina III) for luciferase luminescence detection, and the tumor was removed, weighed, photographed, and preserved after the mouse was sacrificed.
Immunofluorescence detection of cell cilia
In order to induce cell cilia formation, the cells were inoculated into a 24-well plate. When the cell density was 80%, the culture medium was replaced by fresh serum-free medium for 4-6 d. To measure immunofluorescence, slides with cells were fixed with 4% polyformaldehyde for 30 min, incubated with glycine (2 mg/ml) for 5 min, and then blocked with 5% BSA for 20 min. The rabbit anti-acetyl-α-Tubulin (1:500, Abcam) antibody was then added and incubated overnight at 4 ℃. After washing with PBST three times, the cells were then incubated with sheep anti-rabbit antibody (1:200, Santa Cruz), labeled with IgG/TR for 1 h at 37 ℃, and then washed with PBST three times. Nucleus staining was performed by DAPI reagent for 7 min and then the cover slide was sealed. Positive fluorescence microscopy (ECLIPSE NI, Nikon, Japan) was used to observe and photograph cells.
Quantitative real-time PCR
cDNA was synthesized from total RNA (1 μg) purifified using TRIzol reagent, and then quantitative real-time reverse transcription PCR (qRT-PCR) was performed on a Rotor-Gene 6000 thermocycler (Corbett Research, Sydney, Australia) using a KAPA SYBR FAST Universal 2X qRT-PCR Master Mix (Kapa Biosystems, Woburn, MA, USA) . The primers for FGFR1, IGF1R, ABI2 and NEDD9, Aurora A, HDAC6 were designed by Sangon Biotech (Shanghai, China). PCR reaction parameters were as follows: 1 cycle of 95 ℃ for 3 min, followed by 40 cycles each comprising three steps of 95 ℃ for 3 s, 55 ℃ for 15 s, and 72 ℃ for 30 s. All PCR samples were prepared in triplicate and the relative mRNA expression levels were normalized to GAPDH and determined by the 2−ΔΔCt method.
Transwell Invasion Experiment
Transwell invasion chambers (article number: 354480, Corning) were used to detect cell invasion capacity. Serum-free medium was preheated at 37 ℃, added to the upper chamber, and incubated in a 37 ℃ incubator for 2 h. After discarding the medium, 500 μl fresh medium containing 10% FBS was added to the lower chamber and 300 μl cell suspension (2x105 cells/ml) was added to the upper chamber. After incubation for 24 h, the chamber was removed, washed twice with PBS, and then fixed with 5% glutaraldehyde for 30 min at 4 ℃. The cells were stained with 1% crystal violet staining solution for 20 min and then washed three times with PBS. The cells on the upper surface were cleaned with cotton balls, and we photographed the cells in the under surface of the chamber using an inverted microscope (Nikon, Japan). The mean value was determined by averaging the number of cells counted in nine visual fields.
Western-blotting was used to detect and analyze the levels of proteins in cells and tumor tissues. Total proteins were extracted from cells or tissues, quantified with the BCA reagent method, and then separated by 8% or 10% (w/v) SDS-PAGE. The protein was transferred to a PVDF membrane (Bio-Rad, Hercules), blocked for 1.5 h with 5% (w/v) skim milk in TBST at room temperature, and incubated overnight at 4 ℃ with the Prdx1 antibody (1:10000, Abcam), NEDD9 antibody (1:1000, Cell signaling technology), p-Aurora A antibody (1:1000, Gentex), Aurora A antibody (1:10000, Abcam), HDAC6 antibody (1:10000, Abcam), or the GAPDH antibody (1:10000, Abcam). The membranes were washed three times with TBST and then incubated with horseradish peroxidase-conjugated (HRP) secondary antibody for 1 h at room temperature. The protein signal was detected using a protein gel imaging system (ChemiDocXRS + Imaging System; Bio-Rad).
All data were expressed as the mean ± SEM. A one-way analysis of variance and t test were performed to compare the differences among various samples. All data were analyzed using the software GraphPad Prism 5.0. The difference was statistically significant if P<0.05.