Long-chain Non-coding RNA MIR4435-2HG Expression Correlates With Size of Intestinal Polyps in Children and With Metastasis of Colon Cancer

Materials and Methods

Patient specimens and cell culture. Sixty-five colonic polyps specimens were obtained from patients at the Second Affiliated Hospital of Nanjing Medical University with confirmatory diagnoses based on histopathology. Informed written consent prior to operation was obtained from the parents of the children. The human colon-cancer cell line HCT116 was purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, PR China). HCT116 cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Lifesciences, Shanghai, PR China), one hundred U/ml penicillin (Solarbio, Beijing, PR China) and 100 μg/ml streptomycin (Yuanye Bio-Technology, Shanghai, PR China), and uncubated at 37°C with 5% CO₂. The study was approved by the Ethics Committee of The Second Affiliated Hospital of Nanjing Medical University.

LncRNA sequencing. The sequencing of the patient’s lncRNA specimens was done by Shanghai Major Biological Co. The screening criteria for lncRNAs were: 1) differential expression >2, 2) p-value ≤0.05, 3) lncRNAs with known gene sequences, 4) target genes predicted to be cis-regulated by the of Majorbio Cloud Platform.

Data collection & processing. The data containing expression of MIR4435 in 570 colorectal cancer and 42 normal intestinal mucosal tissues were retrieved from TCGA (https://portal.gdc.cancer.gov). Gene-expression profiling and interactive analyses (GEPIA, http://gepia.cancer-pku.cn) were used for analyzing RNA sequencing data of tumors and normal tissues.

RNA extraction and quantitative reverse transcription PCR (RT-qPCR). Total RNA was extracted from patient specimens and cells using the TRIzol reagent (Life Technologies). cDNA was synthesized using reverse transcriptase from VazymE using the following conditions: 15 min at 50°C and 2 min at 85°C, before stopping the reaction at 4°C. The PCR reaction conditions were as following: 5 min at 95°C, followed by 5 s at 95°C, and 31 s at 60°C. The primers for MIR4435 and the endogenous control GAPDH were synthesized by Sangon Biotech Co., Ltd (Shanghai). Sequences of primers were as following: MIR4435 forward, 5′-TGGGAATGGAGGGAAATA-3′ and reverse, 5′-CGGGCAAC AGGTAGAGGT-3′; GAPDH forward, 5′-AAGGTGAAGGTC GGAGTCA-3′ and reverse, 5′-AATGAAGGGGTCATTGATG-3′. The relative expression levels of MIR4435 were normalized to the GAPDH endogenous control and expressed as 2-ΔΔCq.

RNA-interference-vector transfection. For silencing of MIR4435 expression, three RNA interference vectors (shMIR4435-1, shMIR4435-2, and shMIR4435-3) targeting the MIR4435 gene were used to transfect HCT116 cells. An empty vector (shCtrl) was used as negative control. The transfection was performed according to standard procedures. Eighteen h post-transfection, the medium was replaced with RPMI 1640 medium containing 10% FBS. Cells were then observed under fluorescence microscopy 48 h after transfection. The transfected cells with the highest green fluorescent protein (GFP) expression from the RNA interference vectors were selected and treated with medium containing 1 μg/ml of puromycin (Solarbio). Stable GFP expression was observed for more than 10 passages. Images were obtained under fluorescence microscopy. Transfection efficiency of the RNA interference vector in HCT116 cells was confirmed based on down-regulation of MIR4435 expression by qPCR.

Cell-proliferation assay. Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8); (Dojindo Molecular Technologies, Inc., Rockville, MO, USA). HCT116 cells in logarithmic growth phase were seeded in 96-well plates with 5×10³/200 μl. Cells were cultured at 37°C in 5% CO₂. After 24 h, 48 h and 72 h culture, 10 μl of CCK-8 solution was added to each well and the culture was continued for 4 h. The absorbance at 450 nm was measured and the inhibition rate was calculated.

Cell-adhesion assay. A cell adhesion kit (BestBio, Shanghai, PR China) was used for the cell-adhesion assay. One hundred μl of coating solution was added to each well of 96-well plates and incubated overnight at 4°C. The coating solution was then removed, and the plate was placed in a fume hood for a few minutes to complete drying, followed by 3 washes. Next, 5×10⁴ untransfected and transfected cells were plated in the coated wells and incubated at 37°C with 5% CO₂ for 60 min. The medium then was removed, and the wells were washed 3 times before adding 10 μl cell-staining solution and incubation at 37°C for 3-4 h. Colorimetric quantification of the dye was performed at 450 nm using an ELISA plate reader.

Wound-healing assay. A total of 1.5×10⁵ untransfected and transfected cells were plated in 12-well plates for 24 h. The cell layers were then scratched with a sterile pipette tip to make a wound in the monolayer. Cell debris were washed out, and fresh medium was replenished. The cells started migrating from the edge of the wound and repopulated the gap area. Cells were then observed for ‘wound closure’ after 24 h under a light microscope. Image J Software (NIH, Bethesda, MD, USA) was used for analysis and quantification of the healing rate. The wound-area closure was measured in three-independent wound sites per group. The wound-healing rate was calculated as follows: (0 h wound area − 24 h wound area)/0 h wound area ×100%.

Cell invasion assay. The upper-chamber surface of the bottom membrane of the transwell was coated with 50 mg/l Matrigel (Solarbio) (1:4 dilution), air-dried at room temperature and placed under UV light for 30 min. Cells from each experimental group, in the logarithmic-growth phase, were inoculated in the chambers with 10,000 cells per well. Serum-free medium (Lonza, Basel, Switzerland) (200 μl) was added to the upper chamber and complete medium (600 μl) containing 20% FBS was added to the lower layer. After 24 h incubation, cells were washed with PBS 2 times, and fixed with ethanol for 20 min. The cells remaining on the upper membrane were removed with cotton wool, whereas the cells that had invaded through the membrane were stained with crystal violet. After staining, cells on the back of the transwell membrane were imaged and counted using an inverted microscope.

Western blotting. Total proteins from tissues or cells were lysed with RIPA lysis buffer (Beyotime, Haimen, Jiangsu, PR China) containing phenylmethanesulfonyl fluoride (Beyotime) and a protease inhibitor cocktail (Beyotime). Proteins (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Boston, MA, USA). The membranes were incubated with the diluted primary antibodies specific for desmoplakin, β-catenin, E-cadherin, and Smad (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight followed by an a horse-radish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Shanghai) after blocking with 5% bovine serum albumin (Yuanye Bio-Technology) for 1 h. Anti-GAPDH antibody (Santa Cruz Biotechnology) was used as an internal control. Blots were measured with an ECL chromogenic substrate (Beyotime). Protein-expression levels were measured by densitometry analysis of the developed bands.

Statistical analyses. All data are expressed as mean±standard deviation (SD). The t-test was used to analyze the statistical significance of the two groups. The statistical significance of multiple groups was analyzed by ANOVA followed by Bonferroni correction. p≤0.05 was considered significant.