Abstract
Colorectal cancer (CRC) is one of the most common malignancies worldwide. This study aimed to elucidate the clinicopathological significance of miR-338-3p and its association with metastasis-associated in colon cancer-1 (MACC1) in CRC. We evaluated miR-338-3p and MACC1 expression in CRC cell lines and analyzed the clinicopathological features of miR-338-3p in 98 samples of CRC tissues. Subsequent Western blot and cellular biological techniques, and xenograft mouse models were performed to investigate the biological role of miR-338-3p and its association with MACC1 in CRC. Our results show that miR-338-3p expression is lower in CRC cell lines and tissues than that in a human normal colonic epithelial cell line and adjacent normal colorectal tissue, respectively. miR-338-3p expression was significantly associated with histological differentiation, UICC stage, T classification, N classification, and M classification in 98 samples of CRC. The overall survival of CRC patients was significantly less in the low miR-338-3p expression group than in the high miR-338-3p expression group (p<0.01). miR-338-3p mimics suppressed cell proliferation, colony formation, migration, and invasion, but induced apoptosis in CRC cells. miR-338-3p inhibitor reversed these biological phenotypes. miR-338-3p mimics or inhibitor suppressed or increased MACC1 expression in HCT116 and SW620. miR-338-3p mimics reversed the effect of increased MACC1 expression induced by HCT116 with MACC1 over-expression plasmid. Increased cell proliferation, colony formation, and suppressed cell apoptosis caused by MACC1 over-expression were significantly reversed in HCT116 transfected with miR-338-3p mimics, respectively. Suppressed cell proliferation, colony formation, migration, invasion, and increased cell apoptosis caused by MACC1 knockdown were significantly reversed in SW620 transfected with miR-338-3p inhibitor, respectively. In vivo, miR-338-3p agomir significantly inhibited xenograft CRC tumor growth and reversed the effect of increased xenograft tumor growth induced from HCT116 with MACC1 overexpression. In conclusion, our data suggest that miR-338-3p suppresses CRC carcinogenesis and progression by inhibiting MACC1. Targeting miR-338-3p might be a novel treatment strategy for CRC.
Keywords: miR-338-3p, MACC1, colorectal cancer
Introduction
Colorectal cancer (CRC) is one of the most common malignancies worldwide. The prognosis of patients with CRC remains poor despite the improvement of current treatment modalities including surgical resection, radiotherapy, and chemotherapy. The 5-year overall survival rate for CRC patients is just 50-60% mainly because of tumor recurrence and/or metastasis [1]. Thus, it is imperative to find novel biomarkers and develop novel treatment strategies for CRC.
Metastasis-associated in colon cancer-1 (MACC1), a newly identified key regulator of hepatocyte growth factor (HGF)-MET signaling, predicts colon cancer metastasis [2,3]. Overexpression of MACC1 associates with the prognosis of the patients with lung cancer, gastric cancer, and esophageal cancer [4-6]. Our recent study shows that MACC1 promotes carcinogenesis and progression of CRC through β-catenin signaling pathway and mesenchymal-epithelial transition [7].
Increasing evidence shows that miRNAs contribute to development and progression of a variety of human cancers [8-10]. Thirteen miRNAs including miR-338-3p have been significantly differentially expressed in rectal cancer and matched normal rectal mucosa samples by miRNA expression profiles and validated using semi-quantitative real-time PCR [11]. miR-338-3p inhibits the growth and invasion of non-small cell lung cancer cells, ovarian cancer, glioma, and gastric cancer [12-15]. Sun et al. have reported that expression of miR-338-3p is significantly down-regulated in CRCs than in adjacent non-tumor tissues, and the value is negatively related to advanced TNM stage and local invasion [16]. However, there are only 40 samples of CRC in this study. Our present study is to investigate the clinicopathological significance of miR-338-3p in large CRC samples and its association with MACC1 in CRC in vitro and in vivo.
Results
miR-338-3p expression in CRC and its association with clinicopathological features of CRC
To determine miR-338-3p expression level, we first detected miR-338-3p expression in CRC cell lines, our result show that miR-338-3p expression is lower in CRC cell lines including LOVO, SW480, HT29, SW1116, and SW620 except HCT116 compared with human colonic epithelial cell line NCM460 by quantitative real-time PCR analysis. In addition, miR-338-3p expression value was decreased markedly in SW620 which is a metastatic CRC cell line compared with SW480 which is a primary CRC cell line (Figure 1A). To further investigate the association of miR-338-3p expression with clinicopathological features of CRC, we detected miR-338-3p expression in 98 pairs of paraffin-embedded human CRC samples and respective adjacent non-tumor colorectal tissues by real-time PCR analysis. The result showed miR-338-3p expression was significantly lower in CRC than that in adjacent non-tumor colorectal tissues (P<0.01) (Figure 1C). miR-338-3p expression was significantly associated with histological differentiation (P=0.024), UICC staging (P=0.001), T classification (P=0.011), N classification (P=0.044), and M classification (P=0.041) of CRC (Table 1). Kaplan-Meier survival analysis showed that the overall survival of CRC patients was significantly less in low miR-338-3p expression group than that in high miR-338-3p expression group (P<0.01) (Figure 1D).
Figure 1.
A: miR-338-3p expression level in CRC cell lines and normal colon mucosa epithelial cell line NCM460 by quantitative real-time PCR analysis. Data were normalized against the miR-338-3p expression level in NCM460 cells. B: MACC1 mRNA expression level in CRC cell lines and NCM460 cells by quantitative real-time PCR analysis. C: miR-338-3p expression was significantly lower in paraffin-embedded CRC tissues (T) than that in adjacent non-tumor colorectal mucosa tissues (N). D: Overall survival of CRC patients with different levels of miR-338-3p expression by Kaplan-Meier analysis. E: MACC1 expression in CRC by immunohistochemistry staining (a: normal colon mucosa; b: well differentiated CRC; c: moderately differentiated CRC; d: poorly differentiated CRC ). F: miR-338-3p expression in high and low MACC1 expression group of CRC, respectively.
Table 1.
MiR-338-3p expression level and its relationship with clinicopathological features of colorectal cancer
| Variable | No. (%) | Expression of miR-338-3p | P value | ||
|---|---|---|---|---|---|
|
|
|||||
| Low (%) | High (%) | ||||
| Sex | Male | 55 (56.1) | 29 (29.6) | 26 (26.5) | 0.702 |
| Female | 43 (43.9) | 21 (21.4) | 22 (22.5) | ||
| Age(years) | ≤62 | 47 (48.0) | 20 (20.4) | 27 (27.6) | 0.107 |
| >62 | 51 (52.0) | 30 (30.6) | 21 (21.4) | ||
| Histological differentiation | Well | 23 (23.5) | 8 (8.2) | 15 (15.3) | 0.024 |
| Moderately | 47 (48.0) | 22 (22.5) | 25 (25.5) | ||
| Poorly | 28 (28.5) | 20 (20.3) | 8 (8.2) | ||
| UICC stage | I-II | 57 (58.2) | 21 (21.4) | 36 (36.8) | 0.001 |
| III-IV | 41 (41.8) | 29 (29.6) | 12 (12.2) | ||
| T classification | T1-T2 | 31 (31.6) | 10 (10.2) | 21 (21.4) | 0.011 |
| T3-T4 | 67 (68.4) | 40 (40.8) | 27 (27.6) | ||
| N classification | N0 | 45 (45.9) | 18 (18.4) | 27 (27.5) | 0.044 |
| N1-N2 | 53 (54.1) | 32 (32.7) | 21 (21.4) | ||
| M classification | M0 | 53 (94.79) | 22 (22.5) | 31 (31.6) | 0.041 |
| M1 | 45 (45.9) | 28 (28.6) | 17 (17.3) | ||
miR-338-3p inhibits cellular growth, migration, and invasion and induces apoptosis in CRC
As shown in Figure 2A, 2B, miR-338-3p expression was significantly higher in HCT116 and SW620 cells transfected with miR-338-3p mimics compared with the control groups, respectively (P<0.01 and P<0.01). Meanwhile, miR-338-3p expression was significantly lower in HCT116 and SW620 cells transfected with miR-338-3p inhibitor compared with the control groups, respectively (P<0.01 and P<0.01). Cell proliferation was significantly suppressed in HCT116 and SW620 cells transfected with miR-338-3p mimics compared with the control group, respectively (P<0.01 and P<0.01). However, cell proliferation was significantly increased in HCT116 and SW620 cells transfected with miR-338-3p inhibitor compared with the control group, respectively (P<0.05 and P<0.01) (Figure 2C). miR-338-3p mimics significantly suppressed colony formation in HCT116 and SW620 cells compared with the control group, respectively (P<0.05 and P<0.05). miR-338-3p inhibitor significantly increased colony formation in HCT116 and SW620 cells compared with the control group, respectively (P<0.05 and P<0.05) (Figure 2D). Flow cytometry analysis showed that elevated apoptosis was found in HCT116 and SW620 cells transfected with miR-338-3p mimics compared with the control group, respectively (P<0.01 and P<0.05). Decreased apoptosis was found in HCT116 and SW620 transfected with miR-338-3p inhibitor compared with the control group, respectively (P<0.05 and P<0.05) (Figure 2E). miR-338-3p mimics significantly suppressed SW620 cell migration and invasion by cell migration assay and invasion assay, respectively (P<0.01 and P<0.01). Similarly, miR-338-3p inhibitor significantly increased SW620 cell migration and invasion, respectively (P<0.01 and P<0.01) (Figure 2F, 2G).
Figure 2.
(A, B) miR-338-3p mimics (A) or inhibitor (B) significantly increased or suppressed miR-338-3p expression in HCT116 and SW620 cells compared with the control group, respectively. (C, D) miR-338-3p mimics or inhibitor significantly suppressed or increased cell proliferation (C) and colony formation (D) in HCT116 and SW620 cells compared with the control group, respectively. (E) miR-338-3p mimics or inhibitor significantly increased or suppressed cell apoptosis in HCT116 and SW620 compared with the control group, respectively. (F, G) miR-338-3p mimics or inhibitor significantly suppressed or increased SW620 cell migration (F) and invasion (G) compared with the control group, respectively.
miR-338-3p inhibits xenograft CRC tumor growth in vivo
To further investigate the role of miR-338-3p on CRC growth in vivo, we performed assays using SW620 cells and HCT116 with MACC1 overexpression implanted subcutaneously in BALB/c-nu mice, respectively. As shown in Figure 3A-C, miR-338-3p agomir significantly suppressed tumor growth and tumor weight of SW620 cells implanted subcutaneously in BALB/c-nu mice compared with the control group, respectively. Interestingly, miR-338-3p agomir significantly reversed the effect of increased xenograft tumor growth and tumor weight induced from HCT116 with MACC1 overexpression implanted subcutaneously in BALB/c-nu mice compared with the control group, respectively (Figure 3D-F).
Figure 3.
(A-C) miR-338-3p agomir significantly suppressed tumor growth (B) and tumor weight (C) of SW620 cells implanted subcutaneously in BALB/c-nu mice compared with the control group, respectively. (D-F) miR-338-3p agomir significantly reversed the effect of increased xenograft tumor growth (E) and tumor weight (F) induced from HCT116 with MACC1 overexpression implanted subcutaneously in BALB/c-nu mice compared with the control group, respectively.
miR-338-3p suppresses CRC carcinogenesis and progression by inhibiting MACC1
To clarify the molecular mechanism of miR-338-3p in CRC carcinogenesis and progression, we predicted that miR-338-3p UACGACC could completely bind AUGCUGG (position 1932-1938) of MACC1 mRNA 3’UTR using TargetScan, PicTar, and miRanda software. First, MACC1 mRNA expression was higher in CRC cell lines including HCT116, LOVO, SW1116, SW620, SW480 and HT29 than that in NCM460 cell line by real-time PCR analysis (Figure 1B). Immunohistochemistry staining showed that high MACC1 expression was found in 61.2% (60/98) of CRC, low MACC1 expression was found in 38.8% (38/98) of CRC (Figure 1E). miR-338-3p low expression was found in 61.7% (37/60) of the high MACC1 expression group. miR-338-3p high expression was found in 65.7% (25/38) of the MACC1 low expression group (Figure 1F). There was a significant negative correlation between miR-338-3p and MACC1 expression in CRC tissues (r=-0.268, P=0.008, Table 2). Further study showed that MACC1 mRNA and protein expression was suppressed in HCT116 and SW620 cells transfected with miR-338-3p mimics compared with the control group, respectively. MACC1 mRNA and protein expression was increased in HCT116 and SW620 cells transfected with miR-338-3p inhibitor compared with the control group by real-time PCR and Western blot analysis, respectively (Figure 4A, 4B). miR-338-3p mimics dramatically reversed the effect of increased MACC1 mRNA and protein expression induced by HCT116 transfected with MACC1 over-expression plasmid compared with the control group, respectively. miR-338-3p inhibitor reversed the effect of decreased MACC1 mRNA and protein expression induced by SW620 with MACC1 knockdown compared with the control group, respectively (Figure 4C-F).
Table 2.
The correlation between miR-338-3p expression and MACC1 expression in colorectal cancer
| Variable | miR-338-3p expression level | P value | r | ||
|---|---|---|---|---|---|
|
| |||||
| Low (%) | High (%) | ||||
| MACC1 expression level | Low | 13 (13.3%) | 25 (25.5%) | 0.008 | -0.268 |
| High | 37 (37.7%) | 23 (23.5%) | |||
Figure 4.
(A, B) miR-338-3p mimics or inhibitor dramatically suppressed or increased MACC1 mRNA (A) and protein (B) expression in HCT116 and SW620 cells compared with the control group by quantitative real-time PCR and western blot analysis, respectively. (C-F) miR-338-3p mimics dramatically reversed the effect of increased MACC1 mRNA (C) and protein (E) expression induced by HCT116 transfected with MACC1 over-expression plasmid compared with the control group, respectively. miR-338-3p inhibitor reversed the effect of decreased MACC1 mRNA (D) and protein (F) expression induced by SW620 with MACC1 knockdown compared with the control group, respectively.
We next tested whether MACC1 is indispensable to miR-338-3p-mediated malignant phenotype suppression in CRC. Our data show that increased cell proliferation, colony formation, and suppressed cell apoptosis caused by MACC1 over-expression is significantly reversed in HCT116 transfected with miR-338-3p mimics compared with the control group, respectively (Figure 5A-C). Suppressed cell proliferation, colony formation, migration, invasion, and increased cell apoptosis caused by MACC1 knockdown were significantly reversed in SW620 transfected with miR-338-3p inhibitor compared with the control group, respectively (Figure 5D-H).
Figure 5.
(A-C) Increased cell proliferation (A), colony formation (B), and suppressed cell apoptosis (C) caused by MACC1 over-expression were reversed in HCT116 transfected with miR-338-3p mimics compared with the control group, respectively. (D-F) Suppressed cell proliferation (D), colony formation (E), and increased cell apoptosis (F) caused by MACC1 knockdown were significantly reversed in SW620 transfected with miR-338-3p inhibitor compared with the control group, respectively. (G, H) Suppressed cell migration and invasion caused by MACC1 knockdown were significantly reversed in SW620 transfected with miR-338-3p inhibitor compared with the control group, respectively.
Materials and methods
Clinical samples and patient information
Ninety-eight pairs of paraffin-embedded archived CRC and adjacent non-tumor colorectal mucosal tissues (ANT) were collected from our Department between January 2002 and December 2006. No patients had received chemotherapy and/or radiotherapy before operation. The histopathology of the disease was determined by two pathologists according to the criteria of the World Health Organization. Clinical staging was done according to UICC staging. For the research purposes of these clinical materials, prior patient consent and approval from the Institutional Research Ethics Committee was obtained. Detailed clinical information about these patients, including age, gender, clinical stage, histological differentiation, T classification, N classification, and distant metastasis status, is summarized in Table 1. Follow-up information was available for all patients.
Cell lines and small interfering RNA (siRNA) sequences
The human CRC cell lines SW480 and SW620 were maintained in Leibovitz’s L-15 Medium (Invitrogen, Carlsbad, CA). HCT116 was grown in McCoy’s 5A Medium (Invitrogen). LOVO and SW1116 were cultured in RPMI-1640 medium (Invitrogen). HT29 was maintained in Dulbecco’s modified Eagle’s medium (Invitrogen).The human colonic epithelial cell line NCM460 was cultured in RPMI-1640 medium. All medium were supplemented with 10% (v/v) fetal bovine serum (Invitrogen), 1 × antibiotic/antimycotic (100 units/mL streptomycin, 100 units/mL penicillin, and 0.25 mg/mL amphotericin B). All cell lines were cultured in humidified incubator at 37°C with 5% CO2.
The small interfering RNA (siRNA) specifically for MACC1 was chemically synthesized and purified from Ribobio Inc. (Guangzhou, China). The targeted MACC1 sequences were: sense 5’-CAC CAU AGC UUG CAA AGU A dTdT-3’, antisense 5’-UAC UUU GCA AGC UAU GGU G dTdT-3’. The siRNA was transfected using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s instructions. Scrambled siRNA were used as negative control group.
Establishment of stably transfected cell lines
For MACC1 over-expression, ectopic MACC1 coding sequence was amplified by polymerase chain reaction (PCR). The primer sequences were: forward: 5’-CCG CTC GAG ATG CTA ATC ACT GAA AGA AAA C-3’; reverse: 5’-CCG CTC GAG CTA TAC TTC CTC AGA AGT GGA GAA T-3’. The amplified product was cloned into the pBaBb-puromycin plasmid and confirmed by sequencing. For MACC1 silencing, sequences of short hairpin RNA targeting MACC1 (shMACC1) and scrambled siRNA were cloned into the pSUPER-retro-puromycin plasmid, respectively. The shMACC1 sequences were: 5’-TTC ACC CTT CGT GGT AAT AAT-3’, and the scrambled siRNA sequences were: 5’-CAA CAA GAT GAA GAG CAC CAA-3’. CRC cell lines were transfected with aforementioned constructed plasmids or empty vector. Stably transfected cell lines were selected with 0.5 μg/ml puromycin at 48 hours after infection.
Cell proliferation assay
HCT116 and SW620 cells (1 × 103) were plated onto 96-well plates with medium containing 10% FBS and incubated overnight. After transfection, cell proliferation was determined at 0, 24, 48, 72 and 96 h using the Cell Counting Kit-8 (CCK8) (Keygene, China). The absorbance (OD) was measured at a wavelength of 450 nm using a Microplate Autoreader (BioTek Instruments, USA). This experiment was performed in triplicate.
Cell apoptosis analysis
Cells (5 × 105) were seeded in 6-well plates and incubated overnight until 50-60% confluence. The cells were transfected with 100 nM miR-338-3p mimics or inhibitor and harvested at 48 h, washed in cold PBS, fixed with 80% ethanol for 8 h at 4°C, then stained with propidium iodide buffer (50 mg/ml propidium iodide, 0.1% sodium citrate and 0.1% Triton X-100) for 3 h at 4°C. Non-specific control miRNA mimics or inhibitor was used as the control group, respectively. Cells (2 × 104) were analyzed for cell apoptosis using a Becton Dickinson FACS can (Becton Dickinson Immunocytometry Systems, San Jose, CA). The percentage of apoptotic cells was quantified using Cell Quest software. This experiment was performed in triplicate.
Colony formation assay
After 48 h transfection, 400 cells per well were plated in 6-well plates and grown for 2 weeks, respectively. The cells were then washed twice with PBS, fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 15 min. The number of colonies in 10 random view fields was counted under a microscope and the average number of colonies was achieved. The experiment was replicated three times independently.
Transwell migration and invasion assays
Migration and invasion assays were carried out in Transwell chambers containing polycarbonate filters (8 μm pore size; Corning Incorporated, Life Sciences, NY, USA). After transfection with 100 nM miR-338-3p mimics or inhibitor for 48 h, HCT116 and SW620 cells (migration/2 × 104 cells; invasion/2 × 105 cells) in a 500 μl volume of serum-free medium were placed in the upper chambers and incubated at 37°C with 5% CO2 for 24 hours, while a 200 μl volume of medium containing 15% FBS was added to the lower chamber as chemoattractant. Cells were allowed to invade through the Matrigel (BD Biosciences) or migrate for 24 hours at 37°C with 5% CO2. Following invasion or migration, cells were fixed with 4% formaldehyde and stained with 1% crystal violet. Cells on the upper surface of the filters were removed by wiping with a cotton swab. Cells counts were the mean number of cells per fields of view. Three independent experiments were performed and the data are presented as mean ± standard deviation (SD).
Quantitative real time PCR
Reverse transcription was performed using One step PrimeScript miRNA and mRNA cDNA Synthesis Kit (Takara Biotechnology Co.Ltd, Dalian, China), and quantitative real-time PCR was performed using SYBR Premix Ex Taq II (Takara Biotechnology). RNAU6B snRNA and GAPDH was used for sample loading normalization. The primer sequences used for miR-338-3p were followed: forward: 5’-GGG GTA CCG AAT CTT CCC AGT AGG CG-3’; Reverse, 5’-TTG CGG CCG CAA AGG AGA AGG GCC AAA C-3’. The specific forward primer of RNAU6B was 5’-ACG CAA ATT CGT GAA GCG TT-3’. Reverse primer for U6B snRNA was Uni-miR qPCR primer (TakaRa Code D350A). The primer sequences used for MACC1 were followed: forward: 5’-GCA AGC TGG TGT GTC ATC AGC AAA-3’; reverse: 5’-ACA GAG GGC AGC TCA TGT TCT CAT-3’. The quantity of miR-338-3p in each CRC tissues relative to its paired ANT was calculated using the equation [RQ=2-ΔΔCT, ΔΔCT=(CTmiRNA-CTU6)T-(CTmiRNA-CTU6)N]. The expression level of miR-338-3p was classified into low expression and high expression group compared with RQ ratio =RQ (CRC)/RQ (ANM). The geometric mean of housekeeping gene GAPDH was used to normalize the variability at mRNA expression levels. All experiments were performed in triplicate.
Western blot analysis
As we previously described [17], primary MACC1 antibody (Sigma, St. Louis, MO) was used. GAPDH (Cell Signaling Technology) was used as the loading control. After washing, the membranes were incubated with secondary antibody HRP-conjugated goat anti-rabbit (Cell Signaling Technology) for 1 h at room temperature and visualized by enhanced chemiluminescence detection kit (Millipore).
Immunohistochemistry staining
As we previously described [7], the primary MACC1 antibody (Sigma, St. Louis, MO, 1:200) was used. The degree of MACC1 staining was based on both the proportion of positively stained tumor cells and intensity of staining. We evaluated MACC1 expression in CRC specimen by determining the staining index, which scores as 0, 1, 2, 3, 4, 6, 8, 9 and 12. The staining index score of 6 (the cutoff point) was used to distinguish between low and high expression of MACC1.
Xenograft tumor model
Female BALB/c-nude mice (4-5 weeks old and weighing 15-18 g) were housed under pathogen-free conditions. HCT116 cells (1 × 106) with MACC1 overexpression and SW620 cells were trypsinized, washed twice with serum-free medium, and reconstituted in serum-free medium DMEM, mixed 1:1 with Matrigel (Becton-Dickinson) and then inoculated subcutaneously into the right flank of each nude mouse, respectively. A local miR-338-3p agomir treatment was initiated when the tumor was palpable at a volume of approximately 20 mm3. The mice were randomly assigned into treatment and negative control groups (n=5 mice/group) and given intra-tumor injection with 2 nM miR-338-3p agomir or non-specific control miRNA dissolved in 20 μl PBS every 3 days. The treatment time was 21 days. Tumor size was measured every 3 days, using a digital caliper, and the tumor volume was calculated according to the formula: tumor volume (mm3)= length × width2 × 0.5. At the end of the experiment, all mice were sacrificed and the total weights, tumor weights, and the tumor volumes were recorded. All the experiments were performed following the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication).
Statistical analyses
Groups from cell culture and in vivo experiments were compared using an unpaired, two-tailed Student’s tests and results are presented as mean ± SD. For CCK8 assay, comparison was done by univariate variance analysis (two-way ANOVA). Statistical analyses were performed using SPSS 16.0 statistical software. P<0.05 was considered to be statistically significant.
Discussion
Several studies have demonstrated that miR-338-3p suppresses progression of a number of malignant tumors including non-small cell lung cancer, ovarian cancer, glioma, gastric cancer, and hepatocellular carcinoma [12-15,18]. To our knowledge, only Sun and Xue et al. have reported miR-338-3p is expressed differentially in 40 samples of CRC and associated with progression and prognosis of CRC [16,19,20]. Our results showed that miR-338-3p expression was lower in almost all CRC cell lines and tissues than that in NCM460 and adjacent normal colorectal tissue samples, respectively. miR-338-3p expression was significantly associated with histological differentiation, UICC stage, T classification, N classification, and M classification in large samples of CRC. The overall survival of CRC patients was significantly less in low miR-338-3p expression group than that in high miR-338-3p expression group. Further study showed that miR-338-3p mimics suppressed cell proliferation, colony formation, migration, and invasion, but induced apoptosis in HCT116 and SW620 cells. Our data suggest that miR-338-3p plays an important role as tumor suppressor gene in CRC carcinogenesis and progression.
Our recent study shows that MACC1 promotes carcinogenesis and progression of CRC through β-catenin signaling pathway and mesenchymal-epithelial transition [7]. miR-1 downregulation cooperates with MACC1 in promoting MET overexpression in human CRC [21]. MicroRNA-143 targets MACC1 to inhibit cell invasion and migration in CRC [22]. Interestingly, miR-338-3p inhibits malignant biological behaviors of glioma cells by targeting MACC1 gene [14]. miR-338-3p inhibits epithelial-mesenchymal transition in gastric cancer cells by targeting ZEB2 and MACC1/Met/Akt signaling [15]. To study whether miR-338-3p suppresses CRC carcinogenesis by inhibiting MACC1, our data showed that there was a negative correlation between miR-338-3p and MACC1 expression in CRC tissues. miR-338-3p mimics or inhibitor suppressed or increased MACC1 mRNA and protein expression in CRC cells, respectively. miR-338-3p mimics reversed the effect of increased MACC1 expression induced by HCT116 transfected with MACC1 over-expression plasmid. Increased cell proliferation, colony formation, and suppressed cell apoptosis caused by MACC1 over-expression were reversed in HCT116 transfected with miR-338-3p mimics. miR-338-3p agomir significantly inhibited xenograft CRC tumor growth and reversed the effect of increased xenograft tumor growth induced from HCT116 with MACC1 overexpression. Our results suggest that miR-338-3p suppresses CRC carcinogenesis and progression by inhibiting MACC1. The direct evidence of miR-338-3p targeting MACC1 in CRC needs further study by luciferase reporter assay. miR-338-3p inhibits the growth and invasion of non-small cell lung cancer cells by targeting IRS2 or sphingosine kinase 2 [13,23]. miR-338-3p targets pyruvate kinase M2 and affects cell proliferation and metabolism of ovarian cancer [12]. lncRNA Snhg1, a non-degradable sponge for miR-338, promotes expression of proto-oncogene CST3 in primary esophageal cancer cells [24]. Other factors might be involved in miR-338-3p-mediated signaling pathway in carcinogenesis and progression of CRC, this issue needs further study.
Acknowledgements
This study was supported by National Natural Science Foundation of China (81472251, 81272636, 81502021).
Disclosure of conflict of interest
None.
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