Hsa-miR-574-5p negatively regulates MACC-1 expression to suppress colorectal cancer liver metastasis

Four-week-old male athymic BALB/c nu/nu nude mice (weighing from 15 to 18 g) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (China). The bedding materials, drinking water, feed pellets and other items in contact with the animals were all autoclaved. The experiment and feedings were in SPF (specific-pathogen-free) condition according to the SYXK 2007–0001 standard. The animals were cultured in the animal lab in Shanghai Medical College of Fudan University in accordance with Guide for the Care and Use of Laboratory Animals. This study was approved by Ethics Committee of the Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.

Human colorectal cancer cell lines SW1116 and HCT116 were provided by Shanghai Institute of Digestive Disease. Cells were grown in complete medium supplemented with 10% fetal bovine serum (Gibco, CA, USA) and cultured in a 37°C humidified atmosphere of 5% CO₂. One milliliter volume of pCDH cDNA lentivectors/green fluorescent protein (GFP) (5 × 10⁸ TU/mL, Shanghai Ming Hong Biotech Co. Ltd., China) was added to 1 ml single cell suspension (1 × 10⁷/mL cells) of SW1116 and HCT116 to infect cells (multiplicity of infection was 50). The lentivirus infection efficiency was observed using fluorescence microscope 3 days after infection. Cells were collected 5 days after infection for subsequent animal model development.

Prior to trial, 40 nude mice were fasted for 12 h and then weighted. The mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (45 mg/kg) and placed in a supine position on the operating table. The skin was disinfected with 75% alcohol and oblique incision was made on left back (below conjunction of left posterior axillary line and costal margin) for 0.5 to 1.0 cm to expose spleen. Then the spleen was pulled out of the abdominal cavity gently and 5 × 10⁶/mL colorectal cancer cells in single suspension were injected slowly into spleen using five-gauge needle. Each nude mouse was injected for 0.2 ml over 3 min with visible splenic capsule swelling and whitening. After injection and needle withdrawal, 75% ethanol cotton swab was used for hemostasis oppression for 2 min, by which extravasated cancer cells were also killed to prevent intra-abdominal metastasis. The spleen was removed at 10 min after injection and then 3–0 absorbable sutures were used for abdomen closure. The mice were fed normal diet after waking up from anesthesia. The whole operation process followed the principles of aseptic surgery. Thirty-five days later after injection, the mice were sacrificed. The distribution of colorectal cancer cells in various organs were observed with fluorescence dissecting microscope (Qwin, Leica). The percentage of fluorescent area to the liver surface area was used to reflect liver metastasis and calculate the incidence.

We extracted the whole genome sequence of MACC-1 from the National Center for Biotechnology Information (NCBI, Gene ID: 346389), especially the 3′-UTR (untranslated regions) sequence. The miRNAs profile that might regulate MACC-1 expression was predicted using miRbase (http://​www.​mirbase.​org/​index.​shtml) and microRNA.org (http://​www.​microrna.​org/​microrna/​home.​do). Expect (E) value was used to choose the probable regulation miRNA. The smaller the E value is, the more probable the miRNA regulation of MACC-1 is.

Total RNA was extracted from colorectal cancer cell lines (SW1116 and HCT116) with TRIzol reagent (Invitrogen, CA, USA). Housekeeping gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. MACC-1 mRNA level was measured with Access RT-PCR kit (Promega, USA). Hairpin-it miRNAs qPCR kit (GenePharma, China) was used to detect the hsa-miR-574-5p expression in SW1116 and HCT116 cell lines with stem-loop reverse transcription. The primers used in the study were shown as follows: MACC-1 (3′UTR sequence) primers, forward 5′-TTGCGGAGGTCACCATAGC-3′; reverse 5′-TTTCCAACAACGGGCTCA-3′; GAPDH primers, forward 5′-GGGCTGCTTTTAACTCTG-3′; reverse 5′-TGGCAGGTTTTTCTAGACGG-3′.

The amplified PCR products of 3′UTR sequence of wild type MACC-1 or mutant MACC-1 containing the putative has-miR-574-5p binding site were transformed into 293 T cells by pGL3 vector. The procedures were performed according to manufacture’s instruction of lipofectamine 2000 (Invitrogen, USA). At 24 h after transfection, luciferase activity was detected by Dual-Glo™ Luciferase Assay System (E2920, promega) and normalized by Renilla activity.

Lipofectamine 2000 was used for oligonucleotide transfection. Three transfection groups were used for each cell line: control group which was transfected with 5 mg/L liposome only; mimic oligonucleotide group (mimic group) which was transfected with mixture of 100 nM mimic oligonucleotide and liposome; antisense oligonucleotide group (inhibitor group) which was transfected with mixture of 100 nM antisense oligonucleotide and liposome. The transfection was conducted in triplicate for each group. Cells were observed by inverted microscopy 24, 48, 72, and 96 h after transfection. Oligonucleotides expression was confirmed by RT-PCR.

The cells were lysed with 2× lysis buffer on ice for 15 min and broken by ultrasonic disrupter at 4°C. After centrifugation at 12,000 g for 15 min, protein concentration in supernatant was measured. Protein (40 μg) was loaded onto a 10% SDS-PAGE gel (30 mA for 2 h) and transferred onto PVDF membrane (400 mA for 2 h, at 4°C). After probed with 1:1000 diluted rabbit polyclonal MACC-1 antibody (Abcam, MA, USA) at 4°C overnight, the blots were subsequently incubated with HRP (horseradish peroxidase) - conjugated secondary antibody (1:5000). The same results were repeated for seven times.

Log phase cells were digested by trypsin and re-suspended in complete medium to prepare cell suspension. Hemacytometer was used to determine cell count. Cells were seeded at a density of 500 cells per well into 96-well plates in triplicate for each group, and incubated for another 3 days. Medium was changed every 3 days. Cellomics Array Scan was used to scan and image each well to analyze number and size of clones as well as the number of cells in each clone.

For invasion assay, 1.0 × 10⁵ cells in 1% FBS (fetal bovine serum) were added to upper chamber pre-coated with 80 μL diluted matrigel matrix (BD, NJ, USA). Then 600 μl complete medium was added to the matched lower chamber. After 24–72 h incubation, noninvaded cells were removed from the upper surface of the transwell membrane with a cotton swab. Invaded cells on the lower membrane surface were fixed in paraformaldehyde for 15 min, and stained with 500 μL Giemsa solution. Five randomly selected fields were photographed with Image-Pro Plus software, and then counted.

Cell suspension (5 × 10³ cells, 200 μl) was added into each well of a 96-well plate. Relatively loose spheroids were visible after 3 days. The medium was then refreshed and cells were cultured for another 3 days to form bigger spheroids. Inverted phase contrast microscope was used to observe the size and morphology of cell spheroids. The diameter of spheroid was measured at day 7 and the size was compared among experimental groups[23].

All data were expressed as mean ± standard deviation (SD) and processed using SPSS12.0 statistical software. A P-value less than 0.05 was considered to be statistically significant.

The miRNAs that might regulate the MACC-1 gene expression was identified by bioinformatics analysis. The top 15 were shown in Table 1. Among those miRNAs, hsa-miR-574-5p expression level may be correlated to MACC-1 expression for the E value (0.10) was relatively lower.

Two human colorectal cancer cell lines SW1116 and HCT116 labeled with GFP were injected through spleen into male athymic BALB/c nu/nu nude mice to develop liver-metastatic models, respectively and liver-metastatic mice model was successfully constructed. Five weeks after spleen injection, GFP expression was visible in livers from 7 out of 20 nude mice in SW1116 group (7/20). While it was visible in livers only from 6 out of 20 nude mice in HCT116 group (6/20). The mean ratio of tumor volume induced by SW1116 cells (25.0 ± 4.4%) was significantly higher than that induced by HCT116 cells (16.0 ± 2.5%, P < 0.01, Figure 1A, B, C).

We performed qRT-PCR analysis to detect the mRNA level of MACC-1 and hsa-miR-574-5p in SW1116 and HCT116 cell lines. MACC-1 mRNA level in HCT116 cells with lower metastasis potential was significantly lower than that in SW1116 cells, which has higher metastasis potential (P < 0.01, Figure 2A, B). The mRNA level of hsa-miR-574-5p in HCT116 cells was higher than that in SW1116 cells (P < 0.01, Figure 2C). Our results indicated that an inverse correlation between hsa-miR-574-5p and MACC-1 at mRNA level was observed in the SW1116 and HCT116 cell lines.

We co-transfected MACC-1 (3′UTR sequence) vector and hsa-miR-574-5p or negative control into cells. The luciferase activity of wt-MACC1 group was significantly reduced comparing with wt-MACC1-NC group (P < 0.05, Figure 2D). There were no significant differences between mu-MACC1-NC group and mu-MACC1 group (P > 0.05, Figure 2D), demonstrating that miRNA could not combine to the mutant 3′UTR. The results showed that has-miR-574-5p might inhibit MACC1 expression.

Compared to corresponding control group, the protein expression of MACC-1 was down-regulated in mimic groups in both SW1116 and HCT116 cell lines (P < 0.05), as shown in Figure 3A, B. Compared to corresponding control group, the protein expression of MACC-1 was up-regulated in inhibitor group in both SW1116 and HCT116 cell lines (P < 0.05), also shown in Figure 3A, B. The protein expression of has-miR-574-5p was reduced in antisense transfected HCT116 cells, while it was increased in miRNA mimics transfected SW1116 cells (Figure 3C). Thus, it was indicated that hsa-miR-574-5p negatively regulated MACC-1 expression at protein level.

To study the effect of has-miR-574-5p on colony formation, cell invasion and cell spheroid formation, the antisense transfected HCT116 cells and miRNA mimics transfected SW1116 cells were investigated. It showed that the decreased expression of has-miR-574-5p in HCT116 cells stimulated cell proliferation and invasive activity, inducing increased colony formation, cell invasion and cell spheroid formation of HCT116 cells, compared to control group (P < 0.05), as shown in Figure 4. Conversely, the increased expression of has-miR-574-5p in SW1116 cells decreased colony formation, cell invasion and cell spheroid formation, compared to control group (P < 0.05), as shown in Figure 4.