Patient recruitment
In this study, we recruited 48 colon cancer patients seeking treatment at our hospital. The tumor tissues of these patients were collected for genotyping of the rs67085638 polymorphism (see below). Based on the results of genotyping of the rs67085638 polymorphism, the patients were grouped into a CC group (N=28) and a CT group (N=20). The demographic and clinicopathological characteristics of each patient were also collected and compared between the two patient groups. The Human Research Ethics Committees of Yangling Demonstration Zone Hospital has approved this research and all methods were performed in accordance with the last vision of the Declaration of Helsinki.
Animal model
Cancerous colon cells were collected from the colon cancer patients genotyped as CC or CT. Then, three cell groups were set up, i.e., an rs67085638-CC group (cancerous colon cells genotyped as CC), an rs67085638-CT+NC shRNA group (cancerous colon cells genotyped as CT and then treated with NC shRNA), and an rs67085638-CT+lncRNA-CCAT1 group (cancerous colon cells genotyped as CT and then treated with lncRNA-CCAT1). The mice were also divided into three groups corresponding to the three groups of cells, i.e., 1. rs67085638-CC mice; 2. rs67085638-CT+NC shRNA mice; and 3. rs67085638-CT+shRNA-CCAT1 mice, to generate a mouse model of colon cancer, which was then treated with PTX at a dose of 12 mg/kg injected via the intravenous injection through the tail every three days for a total of 21 days to study the expression of target genes. In brief, 4-week old BALB/c male nude mice with a body weight of 19 ± 1 g were purchased from the animal facility of our hospital. This research study was approved by the Ethics Committee of our hospital. The mice were given unlimited access to a standard pellet diet as well as unlimited drinking water. The mice were placed in an airy room with a regulated temperature of 25 ± 2 ˚C and a 12 h/12 h dark/light cycle. After 7 days of adaptation, the mice were arbitrarily divided in 2 groups (n= 15 in each group). Then, the cells described above were administered via subcutaneous injection into the rear side of each mouse. After 21 days of treatment, the mice were killed through deep anaesthesia and the tumor in each mouse was removed to measure its weight as well as volume by making use of the formula: V = (W x W x L)/2, where V was the tumor volume, W was the tumor width, while L was the tumor length. All animal experiments were performed in line with the Guide for the Care and Use of Laboratory Animal by International Committees and were approved by the Animal Ethics Committee of Yangling Demonstration Zone Hospital.
Cell culture and transfection
Primary colon cancer cells were isolated from colon cancer patients and maintained in a modified Dulbecco's Eagle medium (DMEM) supplemented with 10% of fetal bovine serum and antibiotics of trypsin and streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA). Then, the cells were divided into 3 groups, i.e., 1. rs67085638-CC group; 2. rs67085638-CT+ NC shRNA; and 3. rs67085638-CT+ shRNA-CCAT1. In the next step, the cells in groups rs67085638-CT+ NC shRNA and rs67085638-CT+ shRNA-CCAT1 were transfected with corresponding shRNAs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the instructions provided by the manufacturer. At 48 h after transfection, the cells were collected to assay the expression of target genes. In addition, some of the cells were treated with different concentrations of PTX, and the survival status of the cells was measured using an MTT assay. In brief, primary colon cancer cells were treated with different concentrations of PTX for two days. Then, an MTT assay kit bought from Aladdin (Shanghai, China) was used according to the instructions provided by the manufacturer. Ultimately, the absorbance in each well was read at a wavelength of 490 nm by utilizing a microplate reader (Bio-Rad, Hercules, CA) to plot the survival curve and calculate the survival of cells at different time points, thus calculating the IC50 of PTX.
RNA isolation and real-time PCR
Collected tumor tissues and cell samples were treated with Trizol (Invitrogen, Carlsbad, CA) according to the instructions provided by the manufacturer to extract total RNA of each sample. Then, a QuantScript reverse transcription assay kit (Tiangen Biotech, Shanghai, China) was used for 60 minutes of reverse transcription at 37 °C to obtain cDNA, which was subjected to real time PCR by using a qPCR assay kit (Tiangen Biotech, Shanghai, China) according to the instructions provided by the manufacturer. The real time PCR reactions were carried out in a Step OnePlus Real-Time PCR machine. Finally, the relative expression of lncRNA-CCAT1, miR-24-3p, and FSCN1 mRNA in each sample was calculated by using the 2−ΔΔCt approach.
Luciferase assay
The Target Scan software was used to predict the binding sites of miR-24-3p on FSCN1 mRNA and lncRNA-CCAT1. Then, wild type lncRNA-CCAT1 sequence and 3’ UTR of FSCN1 mRNA containing the miR-24-3p binding sites were respectively cloned into pcDNA3.1 vectors (Promega, Madison, WI) to create wild type lncRNA-CCAT1 and FSCN1 mRNA vectors. At the same time, a Quick Change mutagenesis kit (Stratagene, San Diego, CA) was used to create site directed mutations in the miR-24 binding sites of lncRNA-CCAT1 and FSCN1 mRNA, and the mutated sequences were also respectively cloned into pcDNA3.1 vectors to create mutant type lncRNA-CCAT1 and FSCN1 mRNA vectors. Finally, CACO-2 cells were co-transfected with lncRNA-CCAT1 and miR-24-3p mimics or FSCN1 mRNA and miR-24 -3p mimics, and a dual-luciferase reporter gene assay kit (Promega, Madison, WI) was used 24 h later according to the instructions provided by the manufacturer to determine the luciferase activity of transfected cells.
Survival rate
The rates of apoptosis and survival were determined with FCM analysis by making use of an Annexin V-APC/PI Apoptosis Assay kit (KeyGen Biotech, Nanjing, China) according to the instructions provided by the manufacturer. The reading of results was done on a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA) utilizing the FlowJo X.10.0.7-1 software.
Western blot analysis
The expression level of FSCN1 in each sample was determined through Western blotting. In brief, the samples were lysed in a radioimmunoprecipitation buffer to extract total protein, which was then resolved on 10% SDS-PAGE. In the next step, the resolved proteins were electrophoretically blotted onto polyvinylidene fluoride (PVDF) membranes, which were then blocked with 5% skim milk at ambient temperature for 1 h. Then, the membranes were incubated along with anti-FSCN1 (1:1000, Abcam, Cambridge, MA) primary antibodies and HRP-labeled secondary antibodies under conditions suggested by the antibody manufacturer. Finally, the membranes were developed in an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Waltham, MA) and the densitometry analysis of protein bands was done using ImageJ software to determine the relative protein expression of FSCN1 in each sample.
IHC assay
The collected tissue samples were fixed by using 10% neutral formalin (Thermo Fisher Scientific, Waltham, MA) according to the instructions provided by the manufacturer. After embedding in paraffin and partitioning into 5 µm slices, the tissue sections were dewaxed, rehydrated and subject to antigen retrieval. Then, the slides were blocked by using bovine albumin and treated with anti-FSCN1 primary antibodies and biotin labeled secondary antibodies under conditions suggested by the antibody manufacturer. Finally, the slides were observed under a fluorescence microscope to determine the positive expression rate of FSCN1 in each sample.
TUNEL assay
Cell apoptosis was analyzed by using a terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling (TUNEL) assay kit (Roche, Basel, Switzerland) according to the instructions provided by the manufacturer.
Statistical analysis
All statistical analyses were done by utilizing GraphPad Prism 7.0 software (GraphPad, La Jolla, CA). All results were presented as the mean ± standard errors. Comparisons of between-group differences were done by making use of Student's t tests. P < 0.05 was taken into consideration to show a statistically significant difference.