Clinical tissue samples
A total of 30 CRC tissues and 30 adjacent normal tissue samples were obtained from 30 patients aged 30–70. No patients had received chemotherapy or radiotherapy before surgery at the Third Affiliated Hospital of Xinxiang Medical University Hospital. This study was approved by Ethic Committee of Third Affiliated Hospital of Xinxiang Medical University, and the tissues were obtained after the consent of patients. All the tissue samples were immediately frozen in liquid nitrogen and stored at − 80 °C until RNA extraction.
Cell Culture And Transfection
CRC cell lines SW480, HCT116, LS174T, SW620, RKO and HT29 were obtained from the American Type Culture Collection (ATCC). Normal human fetal colonic mucosa cell line (FHC) was established at our laboratory. All cell lines were cultured in RMPI-1640 medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Invitrogen, USA) in 5% CO2 at 37 °C.
Rna Isolation And Real-time Quantitative Pcr (qrt-pcr)
Total RNA from tissues or cultured cells was solated using Trizol reagent (Takara, Japan) according to the manufacturer’s protocols. RNA (1 µg) was reverse transcribed to cDNA with reverse transcription kit (Takara, Japan). qRT-PCR was performed on ABI 7500 RT-PCR system (Applied Biosystems, USA) with SYBR® Premix Ex Taq™ Kit (TaKaRa, Japan) and Mir-X™ miRNA qRT-PCR SYBR® Kit (Clontech Laboratories, USA) in accordance with the manufacturer’s instructions. All experiments were repeated three times. Data were calculated using comparative cycle threshold (CT) (2−ΔΔCT) method. The results were normalized to the expression of GAPDH. The primers for LincHOXA10 (F: CCC AGT AAG CCA AAG TCA AGCC, R: CTG AGG TCA ATG GTG CAA AGG ).
Western Blotting
Proteins were lysed in RIPA buffer (KeyGen Biotech, China) containing 100 mmol/L phenyl methane sulfonyl fluoride (PMSF), and quantified by bicinchoninic acid (BCA) protein quantitative assay (KeyGen Biotech, China). Protein lysates were separated using 10% SDS-PAGE and transferred onto PVDF membranes (Roche, Switzerland). Then, the membranes were incubated with specific antibodies against HOXA10 (Abcam, England), E-cadherin, N-cadherin, Vimentin, p-smad2 and p-smad3 (Cell Signaling Technology, USA) followed by incubation with the appropriate second antibodies, and α-Tubulin was used as an internal control. Finally, the membranes were detected using an enhanced chemiluminescence (ECL) detection system (FDbio, China), according to the manufacturer’s instructions. The results were normalized to the expression of a-tubulin (Proteintech, USA).
Lentiviral Vector Preparation And Plasmid Transfection
The siRNAs targeting LincHOXA10 (si-LincHOXA10) and siRNA negative control (si-NC) were all purchased from GenePharma (China). The full length of HOXA10 was subcloned into pcDNA3.1 to overexpress HOXA10 levels with empty pcDNA3.1 serving as control. The pcDNA3.1 vector was bought from GenePharma (Shanghai). Lipofectamine™ 2000 (Invitrogen) was used for cell transfection according to the manufacturer’s instructions.
Immunohistochemistry (ihc)
According to the specifications of the S-P kit, paraffin-embedded tissues were cut into 5 µm-thick sections, dehydrated with organic solvent, retrieved with citrate buffer, incubated with primary antibody ( Anti-HOXA10 antibody: Abcam, England.) and then detected with an avidin-biotin complex with 3, 3′-diaminobenzidine. The degree of staining was observed and scored independently by two pathologists. Immune staining intensity was rated as follows: 0(no staining), 1(yellow or light brown, weak staining), 2 (brown, moderate staining) and 3(darkbrown, strong staining). Immune staining quality was rated as follows: 0(no staining), 1(< 30%), 2(30%-70%) and 3(> 70%). Tumor tissue intensity was scored via summation as follows: 0–1 (-), 2–3 (+), 4 (++), and 5–6 (+++). Tissues scored 0–1 (-)/2–3 (+) were classified into the low-expression group, and tissues scored 4 (++)/5–6 (+++) were classified into the high-expression group.
Immunofluorescence Staining
For immunofluorescence staining of cultured cells, cells seeded on confocal dish were transfected with adenoviral vectors. 48 h later, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. The cells were then incubated with primary antibodies at 4 °C overnight followed by washes with PBS and incubation with fluorescent secondary antibody in dark at room temperature for 1 h. After final washes with PBS, the confocal dish was mounted using an anti-fade mounting solution containing 4, 6-diamidino-2-phenylindole (DAPI). The staining was examined, and images were captured using an Olympus Confocal laser scanning microscopy FV1200.
Cell Proliferation Assay
Cell proliferation was determined with the 3-(4,5- dimethylthiazol-2 -yl)- 2,5- diphenyltetrazolium bromide (MTT) assay. In brief, 1 × 103 cells were seeded in each well of 96-well plates in 200-µL volume. At 24, 48, and 72 hours after culture, MTT (20µL, 5 mg/mL) was added into each well and incubated for 4 hour at 37 °C. After incubation, MTT was removed and 150 µl dimethyl sulphoxide (DMSO; Sigma, USA) was added to the wells. The absorbance was measured at 570 nm wavelength with a microplate reader (Bio-Rad, Hercules, CA, USA). The experiment was conducted repeatedly for three times.
Transwell Migration And Invasion Assay
Transfected cells (1 × 105) were seeded in serum-free medium in the top chamber of each transwell well (BD Biosciences, USA), which featured a pore size of 8 µm. Matrigel (BD Biosciences) was used to cover the top side of the membrane for invasion assay and Matrigelfree condition was used for migration assay. The matched lower chamber was filled with complete medium supplemented with 10% FBS. The cells were incubated at 37 °C with 5% CO2 for 48 hours, the non-traversed cells were removed from the upper filter with a cotton swab, and the traversed cells were fixed with formaldehyde and stained with hematoxylin for 30 minutes. Then, the cells that migrated or invaded to the basal portion of the membrane in the lower compartment of the chamber were counted in 5 random visual fields using a microscope (× 200). All experiments were performed in triplicates.
In vivo tumor growth assay
CRC cells were harvested by trypsinization, washed twice, and then re-suspended with serum-free medium. To evaluate CRC tumor growth in vivo, 5 × 106 of RKO and HCT116 cells stably expressing control or LincHOXA10 shRNA via lentiviral vector were separately injected subcutaneously into the left and right back flank of nude mice (n = 3 per group). Twenty five days later, tumors were removed and measured.
Statistical analysis
All statistical analyses were performed using SPSS 19.0 (Abbott Laboratories, USA). The quantitative results of all experiments were presented as the mean ± SD. Differences among/between sample groups were analysed by one-way ANOVA or the independent-samples t-test. Relationships between LincHOXA10 expression and clinicopathological characteristics were tested using Pearson’s χ2-test. Differences were considered significant if P < 0.05*; P < 0.01**; P < 0.001***.