Abstract
To investigate the regulative effect of microRNA-338-3p on colorectal carcinoma cell invasion and migration.
The microRNA-338-3p expression pattern of colorectal carcinoma tissues and cell lines was detected by real-time reverse transcriptase polymerase chain reaction. The protein level of smoothened was detected by western blot analysis. Furthermore, colorectal carcinoma cells were pretreated with or without anti-smoothened-small interfering ribonucleic acid prior to the addition of pre-microRNA-338-3p or anti-microRNA-338-3p. The status of colorectal carcinoma cell invasion and that of migration were detected by transwell assay and wound healing assay, respectively.
The expression of microRNA-338-3p was significantly down-regulated in colorectal carcinoma tissues in comparison with those in the adjacent non-tumorous tissues, and the value was negatively related to advanced tumor, node, metastasis stage and local invasion. The expression of microRNA-338-3p in colorectal carcinoma cells transfected with pre-microRNA-338-3p p was significantly increased. Furthermore, over-expression of microRNA-338-3p inhibited the expression of smoothened protein in colorectal carcinoma cells, which showed obviously suppressed invasion and migration ability. The expression of microRNA-338-3p in colorectal carcinoma cells transfected with anti-microRNA-338-3p was significantly decreased. Moreover, the down-regulated expression of microRNA-338-3p caused the up-regulated expression of smoothened protein in colorectal carcinoma cells, which showed significantly enhanced invasion and migration ability. However, anti-smoothened-small interfering ribonucleic acid largely, but not completely, reversed the effects induced by blockage of microRNA-338-3p, suggesting that the regulative effect of microRNA-338-3p on colorectal carcinoma cell invasion and migration was indeed mediated by smoothened. Additionally, smoothened was identified as a direct target of microRNA-338-3p by luciferase assay.
MicroRNA-338-3p could inhibit colorectal carcinoma cell invasion and migration by inhibiting smoothened expression.
INTRODUCTION
Colorectal carcinoma (CRC) is one of most common gastrointestinal malignancy in China (1). Widespread metastasis has been a major reason for the dismal outcome of CRC patients. Metastasis is a complex, multi-step process whereby cancer cells migrate from the primary neoplasm to a distant location (2). The process starts when primary tumor cells invade adjacent tissue, followed by cells entering into the blood stream, translocating through the vasculature, exiting blood vessel into the surrounding tissue parenchyma, initiating micrometastases and finally proliferating to form macroscopic secondary tumors (3). Further insight into the molecular mechanisms responsible for CRC invasion and migration could lead to the identification of novel therapeutic targets for CRC (4). One of the critical regulators involved in this process is the microRNA (miRNA) (5).
miRNAs are a class of endogenous and small non-coding regulatory ribonucleic acids (RNAs), which regulate genes at post-transcriptional level (6). Mature miRNAs can be transcribed by RNA polymerase II and are generated from the sequential processing of primary miRNA transcripts by Drosha and Dicer; they then serve as post-transcriptional regulators of gene expression through complementary base pairing to messenger ribonucleic acids (mRNAs) (7). Many reports have shown that miRNAs play key roles in various biological processes, including cell differentiation, proliferation, apoptosis, stress resistance, fat metabolism, tumorigenesis and tumor metastasis (8). Genome-wide studies have revealed that miRNAs may be potential diagnostic or prognostic tools for cancer, and the identification of target mRNAs is a key step in assessing the role of aberrantly expressed miRNAs in human cancer (9,10).
miR-338-3p is a recently discovered miRNA and is involved in cell proliferation and differentiation. Although miR-338-3p is known to be specifically expressed in neuronal tissue, little is known about its abundance and function during carcinogenesis in certain cancers (11,12). We have found that miR-338-3p was down-regulated in several CRC samples compared with adjacent non-tumorous tissues in our previous study, suggesting that miR-338-3p might act as tumor suppressor in CRC; however, the targets it regulated in CRC have not been reported. Smoothened (SMO), a distant relative of G protein-coupled receptors, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis (13). SMO activation triggers a series of intracellular events, culminating in the stabilization of the transcription factor Cubitus interruptus (Ci) and the expression of Ci-dependent genes. These events are recapitulated during mammalian development and tumorigenesis through multiple protein homologs, including three distinct Hh family members (Sonic, Indian and Desert), two Ptc proteins (Ptch1 and Ptch2) and three Ci-like transcription factors (Gli1, Gli2 and Gli3) (14). Although the cellular mechanisms that regulate SMO function remain unclear, the direct inhibition of SMO by cyclopamine, a plant-derived steroidal alkaloid, suggests that endogenous small molecules may be involved (15). Moreover, with the application of bioinformatic predictions, we found that miR-338-3p and SMO mRNA 3′-untranslated region (3′-UTR) had complementary binding sites. Consequently, we inferred that the non-coding RNA, miR-338-3p, acts as a local regulator of SMO by binding to the 3′-UTR of its mRNA, thereby modulating CRC metastasis. In order to verify this hypothesis, we investigated the regulative effect of miR-338-3p on cell invasion and migration in CRC. We aimed to reveal a new regulating mechanism of miR-338-3p in the metastasis of CRC and provide a new miRNA and target gene for clinical application.
PATIENTS AND METHODS
Patients
Forty samples of CRC and matched non-tumorous tissues were harvested from patients who underwent their operation in our hospital, and were confirmed by the post-operative pathological examination. The patient's gender, age, tumor site, tumor, node, metastasis (TNM) stage, local invasion, vessel invasion, differentiation and sera carcinoembryonic antigen (CEA) were obtained from surgical and pathological records. The mean age was 53.8 years (ranging from 33 to 68). Local invasion was classified as tumor invading submucosa (T1), muscularis propria (T2), through muscularis propria into subserosa or into non-peritonealized pericolic or perirectal tissues (T3) and into other organs or structures and/or perforated visceral peritoneum (T4). Differentiation was graded as better (including well- and moderately differentiated tumors) and worse (including poorly differentiated, mucinous and signet-ring cell carcinoma). Non-tumorous tissues were harvested from the intestinal mucosa that were 5 cm away from the cancer site, while normal controls were the intestinal mucosa that were over 10 cm away from the cancer site. All cases did not receive radiotherapy or chemotherapy before operation. The samples were snap frozen in liquid nitrogen after collection and stored at −80°C. All the participants of this study signed an informed consent approved by the Institutional Review Board.
Cell Culture and Transfection
Human CRC-derived cell lines, including HT-29, Lovo, Caco2 and SW-620, and control human umbilical vein endothelial cells (HUVEC), provided by Shanghai Institutes for Biological Science, were resuscitated and resuspended with Dulbecco's modified Eagle medium high glucose (H-DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, USA), 100 kU/ml penicillin G and 100 mg/ml streptomycin. The cells were then plated in 25 cm2 culture bottles and incubated in a 5% CO2 humidified atmosphere at 37°C. The media were changed every 3 days, and the cells were trypsinized using trypsin–edetic acid when they reached 80–90% confluence. Cells aged at passages 4–8 were used for experiments. The day before transfection, cells were seeded in antibiotic-free medium at a density of 1 × 104 cells per well in 96-well plates, incubated for 24 h and then transfected with 80 nmol/l anti-SMO SMARTpool siRNAs or the control siRNA (Dharmacon) using Oligofectamine (Invitrogen, Carlsbad, CA, USA). Secondary transfection was performed 24 h after the first transfection, using 50 nmol/l negative control oligonucleotides, pre-miR-338-3p or anti-miR-338-3p, respectively (Shanghai GenePharma Co. Ltd). miRNA transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). The above experiment was repeated at least three times.
Detection of miR-338-3p Expression by Real-Time RT–PCR
Total RNA from tissues or CRC cells were extracted with routine Trizol reagent (Invitrogen, Carlsbad). The precipitate was dissolved in DEPC-treated water. Nucleic acid protein analyzer (Beckman Coulter, USA) was used to determine RNA concentration. The purity and integrity of RNA were identified by two aspects: A260nm/A280nm was ≥1.8 and the band ratio of 28S RNA to 18S RNA was ≥1.5 in formaldehyde denaturing gel electrophoresis. miR-338-3p were quantified with the methods described previously (16). The comparative 2−ΔΔCT method was used for relative quantification and statistical analysis. The above experiment was repeated at least three times.
miRNA Target Prediction
The analysis of miR-338-3p-predicted targets was performed using the algorithms TargetScan (http://targetscan.org/), PicTar (http://pictar.mdc-berlin.de/) and MiRanda (http://www.microrna.org/microrna/home.do).
Luciferase Activity Assay
The human 3′-untranslated region (3′-UTR) of SMO gene was amplified by polymerase chain reaction (PCR) and cloned into the XbaI site of the pGL3-Control vector (Promega, Madison, WI, USA), downstream of the luciferase gene, to generate the vector pGL3-SMO. For luciferase assay, the CRC cells were cultured in 24-well plates and transfected with 500 ng of either pGL3-SMO or pGL3-control vector and 50 pmol of pre-miR-338-3p or anti-miR-338-3p or negative controls, respectively (Shanghai GenePharma Co. Ltd). Transfection of miRNAs was carried out using Lipofectamine 2000 in accordance with the manufacturer's procedure (Invitrogen). At 24 h after transfection, firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega). The above experiment was repeated at least three times.
Detection of SMO Protein Expression by Western Blot Analysis
Cells were rinsed twice with cold phosphate buffered saline (PBS) buffer and were then lysed in ice-cold lysis buffer containing 150 mmol/l NaCl, 50 mmol/l Tris–HCl (pH 7.6), 0.1% SDS, 1% Nonidet P-40 and protease inhibitor cocktail (Boehringer Mannheim, Lewes, UK). The samples were cleared by centrifugation at 13 000g for 10 min. The cellular protein (50 µg) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidine fluoride (PVDF) membranes (Immobilon, Bedford, MA, USA). After blocking in 20 mmol/l Tris–HCl (pH 7.6) containing 150 mmol/l NaCl, 0.1% Tween-20 and 5% non-fat dry milk, the membranes were incubated overnight at 4°C with primary antibodies against SMO or β-actin, which was used as a sample loading control. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody. The blot was developed using the ECL detection kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The above experiment was repeated at least three times.
Transwell Assay
Briefly, an 8 µM pore size polycarbonate membrane filter was inserted into each Transwell chamber (Corning, NY, USA) and coated with 50 µl of Matrigel (Sigma, St Louis, MO, USA) that had been previously diluted with serum-free medium to obtain a final concentration of 4 mg/ml. The transfected CRC cells (3.0 × 104) were then seeded into the upper chamber with 100 µl of serum-free medium; in the bottom chamber, 1 ml of growth medium containing 10% FBS was added as a chemoattractant. The cells were incubated at 37°C for 48 h and then the Matrigel-coated upper surface of the filter was thoroughly wiped with a cotton swab. The cells that invaded the lower surface of the filter were fixed with 4% paraformaldehyde and stained with crystal purple; cells from three random fields per filter were counted under a microscope at ×100 magnification. The above experiment was repeated at least three times.
Wound Healing Assay
Briefly, CRC cells (5.0 × 105) were seeded into 24-well plates, grown in growth medium overnight without any antibiotic supplement and then transfected with pre-miR-338-3p or anti-miR-338-3p. After 48 h, the culture reached about 90% confluence and an artificial homogeneous wound was created on the monolayer with a 200 µl plastic micropipette tip; cell debris was removed by washing with PBS twice. Closing of the wound (cell migration) was then observed at indicated times and images were captured under an inverted microscope with ×40 objective. The above experiment was repeated at least three times.
Statistical Analysis
All data in the experiment were presented as average ± standard deviation ( ± s). Comparisons between the groups were analyzed with a one-way ANOVA and Student–Newman–Keuls q test using the SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered statistically significant.
RESULTS
miR-338-3p is Aberrantly Down-Regulated in CRC Tissues and CRC-DerivedCell Lines
To study the expression pattern of miR-338-3p in CRC, we performed real-time RT–PCR to detect miR-338-3p expression in 40 CRC samples and adjacent non-tumorous tissues, and in four CRC-derived cell lines. All the 40 cases exhibited miR-338-3p down-regulation in CRCs compared with non-tumorous tissues (0.125 ± 0.091 vs 0.906 ± 0.411) and the difference showed statistical significance (P < 0.01). The expression values of miR-338-3p in HT-29, Lovo, Caco2 and SW-620 cells were 0.896 ± 0.223, 0.102 ± 0.041, 0.904 ± 0.302 and 0.291 ± 0.117, respectively. A significant down-regulation of miR-338-3p was observed in all the four CRC-derived cell lines relative to HUVEC (2.224 ± 0.347, P < 0.01). The whole of these results gave us a first hint that the expression of miR-338-3p might be one of the mechanisms underlying negative regulation of development in CRC cells. The SW-620 cell line was chosen for both pre-miR-338-3p and anti-miR-338-3p transfection in the subsequent experiment because of its intermediate miR-338-3p expression level among the four tested cell lines.
To determine the biological impact of miR-338-3p in the CRC-derived cell line, SW-620 cells were transfected with pre-miR-338-3p or anti-miR-338-3p to increase or reduce miR-338-3p level, respectively. Real-time RT–PCR analysis revealed that introduction of pre-miR-338-3p caused a significant increase in miR-338-3p value (1.985 ± 0.441, P < 0.01); conversely, anti-miR-338-3p caused a significant decrease in miR-338-3p value (0.101 ± 0.038, P < 0.01). These strategies were then used as the basis for the remaining experiments.
Relationship Between miR-338-3p Expression and Clinicopathological Features of CRC
We then investigated the correlation between miR-338-3p expression profile and the clinicopathological features of CRC. We used samples from and associated clinicopathological information of 40 CRC patients who underwent curative surgery. As shown in Table 1, the miR-338-3p expression value was negatively related to TNM stage (P = 0.031) and local invasion (T1, 3 cases, T2, 7 cases, T3, 12 cases, T4, 18 cases, P = 0.014). The miR-338-3p expression was lower in tumors in more advanced TNM stage and having deeper invasion, indicating that miR-338-3p might act as a local regulator in the modulation of CRC invasion and migration.
Relationship between miR-338-3p expression and clinicopathological features in CRC patients
| Variable | No. | miR-338-3p ( | P value |
|---|---|---|---|
| Gender | 0.453 | ||
| Male | 21 | 0.132 ± 0.099 | |
| Female | 19 | 0.110 ± 0.101 | |
| Age | 0.210 | ||
| ≤50 | 12 | 0.134 ± 0.098 | |
| >50 | 28 | 0.111 ± 0.100 | |
| Site | 0.589 | ||
| Colon | 24 | 0.095 ± 0.072 | |
| Rectum | 16 | 0.154 ± 0.129 | |
| TNM stage | 0.031 | ||
| I + II | 15 | 0.185 ± 0.137 | |
| III + IV | 25 | 0.083 ± 0.049 | |
| Local invasion | 0.014 | ||
| T1 + T2 + T3 | 22 | 0.148 ± 0.106 | |
| T4 | 18 | 0.075 ± 0.062 | |
| Differentiation | 0.142 | ||
| Better | 25 | 0.143 ± 0.112 | |
| Worse | 15 | 0.064 ± 0.023 | |
| CEA (µg/l) | 0.785 | ||
| ≤5 | 22 | 0.112 ± 0.092 | |
| >5 | 18 | 0.133 ± 0.111 |
| Variable | No. | miR-338-3p ( | P value |
|---|---|---|---|
| Gender | 0.453 | ||
| Male | 21 | 0.132 ± 0.099 | |
| Female | 19 | 0.110 ± 0.101 | |
| Age | 0.210 | ||
| ≤50 | 12 | 0.134 ± 0.098 | |
| >50 | 28 | 0.111 ± 0.100 | |
| Site | 0.589 | ||
| Colon | 24 | 0.095 ± 0.072 | |
| Rectum | 16 | 0.154 ± 0.129 | |
| TNM stage | 0.031 | ||
| I + II | 15 | 0.185 ± 0.137 | |
| III + IV | 25 | 0.083 ± 0.049 | |
| Local invasion | 0.014 | ||
| T1 + T2 + T3 | 22 | 0.148 ± 0.106 | |
| T4 | 18 | 0.075 ± 0.062 | |
| Differentiation | 0.142 | ||
| Better | 25 | 0.143 ± 0.112 | |
| Worse | 15 | 0.064 ± 0.023 | |
| CEA (µg/l) | 0.785 | ||
| ≤5 | 22 | 0.112 ± 0.092 | |
| >5 | 18 | 0.133 ± 0.111 |
CRC, colorectal carcinoma; CEA, carcinoembryonic antigen.
Relationship between miR-338-3p expression and clinicopathological features in CRC patients
| Variable | No. | miR-338-3p ( | P value |
|---|---|---|---|
| Gender | 0.453 | ||
| Male | 21 | 0.132 ± 0.099 | |
| Female | 19 | 0.110 ± 0.101 | |
| Age | 0.210 | ||
| ≤50 | 12 | 0.134 ± 0.098 | |
| >50 | 28 | 0.111 ± 0.100 | |
| Site | 0.589 | ||
| Colon | 24 | 0.095 ± 0.072 | |
| Rectum | 16 | 0.154 ± 0.129 | |
| TNM stage | 0.031 | ||
| I + II | 15 | 0.185 ± 0.137 | |
| III + IV | 25 | 0.083 ± 0.049 | |
| Local invasion | 0.014 | ||
| T1 + T2 + T3 | 22 | 0.148 ± 0.106 | |
| T4 | 18 | 0.075 ± 0.062 | |
| Differentiation | 0.142 | ||
| Better | 25 | 0.143 ± 0.112 | |
| Worse | 15 | 0.064 ± 0.023 | |
| CEA (µg/l) | 0.785 | ||
| ≤5 | 22 | 0.112 ± 0.092 | |
| >5 | 18 | 0.133 ± 0.111 |
| Variable | No. | miR-338-3p ( | P value |
|---|---|---|---|
| Gender | 0.453 | ||
| Male | 21 | 0.132 ± 0.099 | |
| Female | 19 | 0.110 ± 0.101 | |
| Age | 0.210 | ||
| ≤50 | 12 | 0.134 ± 0.098 | |
| >50 | 28 | 0.111 ± 0.100 | |
| Site | 0.589 | ||
| Colon | 24 | 0.095 ± 0.072 | |
| Rectum | 16 | 0.154 ± 0.129 | |
| TNM stage | 0.031 | ||
| I + II | 15 | 0.185 ± 0.137 | |
| III + IV | 25 | 0.083 ± 0.049 | |
| Local invasion | 0.014 | ||
| T1 + T2 + T3 | 22 | 0.148 ± 0.106 | |
| T4 | 18 | 0.075 ± 0.062 | |
| Differentiation | 0.142 | ||
| Better | 25 | 0.143 ± 0.112 | |
| Worse | 15 | 0.064 ± 0.023 | |
| CEA (µg/l) | 0.785 | ||
| ≤5 | 22 | 0.112 ± 0.092 | |
| >5 | 18 | 0.133 ± 0.111 |
CRC, colorectal carcinoma; CEA, carcinoembryonic antigen.
SMO is a Target of miR-338-3p in CRC
Most miRNAs are thought to control gene expression by base-pairing with the miR-recognizing elements (miR-RE) found in their messenger target. We then used all the three currently available major prediction programs, including TargetScan, Miranda and PicTar, to analyze the potential interaction between miR-338-3p and SMO. SMO gene is predicted by all of the algorithms and reveal potential miR-338-3p target site in its 3′-UTR region (Fig. 1A).
miR-338-3p regulates the expression of smoothened (SMO) in SW-620 cells. (A) SMO 3′-UTR site potentially targeted by miR-338-3p as predicted by TargetScan. (B) Luciferase activity assay showing direct interaction between miR-338-3p and SMO 3′-UTR site. Firefly luciferase reporter activity in the presence of both pGL3-SMO vector and pre-miR-338-3p or anti-miR-338-3p was compared with those of the controls. Luciferase activity in SW-620 cells co-transfected with pGL3-SMO vector and pre-miR-338-3p was decreased markedly compared with negative control. On the contrary, luciferase activity in SW-620 cells transfected with anti-miR-338-3p was increased significantly compared with negative control. cP < 0.01 vs control group. (C) Western blot analysis showing SMO protein expression in SW-620 cells. β-Actin was used as house-keeping gene to normalize SMO protein expression. Lane 1, SW-620 cells transfected with pre-miR-338-3p; lane 2, blank control; lane 3, SW-620 cells transfected with negative control; lane 4, SW-620 cells transfected with anti-miR-338-3p.
To demonstrate that the direct interaction between miR-338-3p and SMO mRNA was responsible for the expression of SMO, we cloned a 278-bp SMO 3′-UTR segment, which includes a potential target site for miR-338-3p, downstream of the pGL3 luciferase reporter gene to generate the pGL3-SMO vector. This vector was co-transfected into SW-620 cells together with pre-miR-338-3p or anti-miR-338-3p. Luciferase activity in tumor cells co-transfected with pGL3-SMO vector and pre-miR-338-3p was decreased markedly compared with negative control. On the contrary, luciferase activity in tumor cells transfected with anti-miR-338-3p was increased significantly compared with negative control (Fig. 1B). These results support the bioinformatic prediction indicating the 3′-UTR of SMO mRNA as a target for miR-338-3p.
To check whether miR-338-3p actually affects SMO expression in CRC cells, we analyzed the consequence of the ectopic expression of miR-338-3p. We transfected the pre-miR-338-3p and anti-miR-338-3p into SW-620 cells as described above, and we searched for changes in SMO protein levels by Western blot analysis. Introduction of pre-miR-338-3p caused a significant increase in miR-338-3p value and decreased SMO protein levels in SW-620 cells. Conversely, anti-miR-338-3p caused a significant decrease in miR-338-3p value and increased SMO protein amounts (Fig. 1C). This result strongly validates a post-transcriptional regulation of SMO protein by miR-338-3p.
miR-338-3p Inhibits Cell Invasion and Migration in CRC Cells
Since SMO has a key role in invasion and migration of cancer cells, we further tested whether the cell invasion and migration potential of transfected CRC cells was modified as a consequence of the demonstrated SMO alteration. To evaluate the effect of miR-338-3p on CRC cell invasion and migration, the well-growing SW-620 cells were transfected with pre-miR-338-3p or anti-miR-338-3p for 48 h and the status of cell invasion and that of migration were determined by transwell assay and wound healing assay, respectively. Our data showed that pre-miR-338-3p transfection significantly reduced the invasion and migration ability of SW-620 cells, whereas knockdown of miR-338-3p expression promoted the invasion and migration of SW-620 cells (Fig 2). These results confirm the potential metastasis-suppressor activity of miR-338-3p in CRC.
Effects of miR-338-3p on cell invasion and migration in CRC cells. (A) The status of cell invasion was determined by Transwell assay. SW-620 cells were transfected with pre-miR-338-3p or anti-miR-338-3p, respectively. After 48 h, the cells that transversed to the reverse side of the Matrigel-coated filter were counted and the data were summarized. (B) The status of cell migration was determined by wound healing assay. SW-620 cells were transfected with pre-miR-338-3p or anti-miR-338-3p and the cells were subjected to wound healing assay. Photographs of these cells were taken and the remaining unhealed gap was measured. (C) Cell invasion was decreased in cells treated with pre-miR-338-3p, whereas cell invasion was increased in cells treated with anti-miR-338-3p. n = 3. Mean ± SD. cP < 0.01 vs control group.
miR-338-3p Suppresses CRC Cell Invasion and Migration by Targeting SMO
If miR-338-3p suppress CRC cell invasion and migration was indeed mediated by SMO, we would expect that the SMO-specific and irreversible antagonist, anti-SMO-siRNA, would abolish this effect. To test this hypothesis, we measured the invasion and migration variations induced by pre-miR-338-3p or anti-miR-338-3p in CRC cells previously transfected with anti-SMO-siRNA. The aim of this experiment was to study whether and how the SMO-depleted cellular environment responds to pre-miR-338-3p or anti-miR-338-3p addition. SW-620 cells were pretreated with or without anti-SMO-siRNA (50 nmol/l) for 24 h prior to the transfection of pre-miR-338-3p or anti-miR-338-3p and the status of cell invasion and that of migration were determined by transwell assay and wound healing assay. The data showed that a reduction in SMO dosage by means different from miR-338-3p over-expression leads to analogous outcomes: when we transfected SW-620 cells with anti-SMO-siRNA that were able to reduce SMO protein of about 80% (Fig. 3A), we observed a sharp decrease in cell invasion and migration compared with negative control (Fig. 3B–D, P < 0.01). Thus, reducing SMO protein levels in CRC cells, either by miR-338-3p over-expression or by anti-SMO-siRNA transfection, is sufficient to induce a comparable cell invasion and migration decrease.
Ectopic expression of miR-338-3p affects the invasion and migration of CRC cells by targeting SMO. SW-620 cells were pretreated with or without anti-SMO-siRNA (50 nmol/l) for 24 h prior to the transfection of pre-miR-338-3p or anti-miR-338-3p. (A) Western blot analysis showing that SMO protein reduced markedly after transfection with anti-SMO-siRNA. Equal loading was confirmed by using β-actin. (B) The status of cell invasion was determined by Transwell assay. After 48 h co-transfection, the cells were subcultured and seeded into Transwell upper chambers for cell invasion assay. Cells that transversed to the reverse side of the Matrigel-coated filter were counted and the data were summarized. (C) The status of cell migration was determined by wound healing assay. After 48 h co-transfection, the cells were subjected to wound healing assay. Photographs of these cells were taken and the remaining unhealed gap was measured. (D) The enhancement of SW-620 cell invasion by anti-miR-338-3p was largely, but not completely, abrogated by anti-SMO-siRNA (number of invasive cells per field from 65.8 ± 4.4 to 39.4 ± 3.1). n = 3. Mean ± SD. cP < 0.01 vs negative control group. dP < 0.01 vs sole anti-miR-338-3p group.
When pre-miR-338-3p was transfected into SW-620 cells previously treated with anti-SMO-siRNA, we observed that anti-SMO-siRNA and pre-miR-338-3p seemed to co-operate to inhibit the invasion and migration (Fig. 3D). However, when anti-miR-338-3p was transfected into SW-620 cells previously treated with anti-SMO-siRNA, we observed that the enhancement of cell invasion and migration by anti-miR-338-3p was largely abrogated by anti-SMO-siRNA (Fig. 3B–D, P < 0.01). These results indicated that the promotive effect of anti-miR-338-3p on CRC cell invasion and migration was largely, but not completely, mediated by SMO, suggesting that anti-miR-338-3p could also activate some SMO-independent signaling pathway to promote CRC cell invasion and migration in addition to the up-regulation of SMO.
DISCUSSION
The survival and prognosis of CRC are still relatively poor, partly due to relapse and metastasis. Therefore, clarification of the molecular pathogenesis of CRC is crucial for developing effective therapy strategies to improve the outcome of patients with this disease. Recently, studies have shown that miRNAs may act as activators or inhibitors of tumor metastasis (17). Previously, we have reported that miR-221 could interact with a target site on the 3′-UTR of CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote CRC occurrence and progress (18). Additionally, miR-155 can promote tumor invasion and metastasis in breast cancer by down-regulating its target, RhoA (19). The miRNA-200 family (miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141 and miRNA-429) can inhibit tumor invasion and metastasis by regulating the epithelial-to-mesenchymal transition (20). miR-126 was found to inhibit cell adhesion, migration and invasion partially through the suppression of proto-oncogene CT10 regulator of kinase in an in vitro model of non-small-cell lung carcinoma (21). It also has been shown that miR-29c is significantly reduced in highly invasive and metastatic nasopharyngeal carcinoma (22). Although many studies have been done to investigate the mechanism of miRNA and tumor metastasis, the mechanism was not clear. Most importantly, few studies have been done on the mechanisms of CRC metastasis regulation by miRNA.
The miR-338 gene is located on chromosome 17 and produces two mature forms (miR-338-3p and miR-338-5p). miR-338-3p is involved in a variety of physiological and pathological processes, and is down-regulated in several malignancies. A highly characterized example is hepatocellular carcinoma, in which miR-338-3p down-regulation was significantly associated with TNM stage, vascular invasion, intrahepatic metastasis, tumor size and tumor grade (23). Interestingly, in this study, we also found miR-338-3p down-regulation in some selective CRC samples. Moreover, the miR-338-3p expression was not only related to TNM stage but also related to tumor invasion. The level of miR-338-3p expression at TNM Stages III and IV was lower than that at Stages I and II, and the tumors which invaded adjacent tissues or organs had less miR-338-3p expression value than those limited to the wall of the colon and rectum. Furthermore, miR-338-3p was also dramatically down-regulated in four human CRC-derived cell lines compared with HUVEC, which was in accordance with the results of clinical samples. However, the relevance of abnormally expressed miR-338-3p to CRC biology and the exact underlying molecular mechanisms had not been well understood.
To extend our previous observation, we focused on the role of miR-338-3p in regulation of invasive and migrative capacity in CRC. In this study, we found that miR-338-3p up-regulation represses the invasion and migration of CRC cells. Conversely, miR-338-3p inhibition promotes CRC cell invasion and migration. These data demonstrated that miR-338-3p is a potential metastasis suppressor for CRC. However, the exact mechanisms of miR-338-3p remain unknown. To understand possible mechanisms underlying miR-338-3p-mediated suppression of invasion and migration in CRC, we used TargetScan, PicTar and MiRanda to identify potential gene targets of miR-338-3p and we found that miR-338-3p and the 3′-UTR of SMO mRNA had complementary binding sites. From this, we deduced that SMO may be a new target of miR-338-3p in CRC; however, this finding has not yet been reported.
SMO, a protein that is related to G-protein-coupled receptors, is the key activator of Hh signaling pathway (24). Up-regulation of SMO in CRC was shown to correlate with a higher biological aggressiveness, advanced stage, poor differentiation, larger size and high proliferative activity (25). Furthermore, it is also well known that SMO regulation, both in physiological and pathological conditions, is exerted mostly at post-transcriptional level (26). Despite this central role of SMO as mediator of all Hh signaling, the mechanisms by which SMO activation is regulated and coupled to downstream components remain enigmatic. In this study, SMO is down-regulated in response to pre-miR-338-3p transfection in CRC cells, and a significant up-regulation of SMO occurs in response to anti-miR-338-3p transfection. Additionally, a direct interaction between miR-338-3p and the target site in the 3′-UTR of the SMO mRNA was also demonstrated by the luciferase assay. Consistent with Huang et al. (27), our results suggest that SMO is a direct target of miR-338-3p in CRC cells.
We hypothesized that miR-338-3p inhibited CRC cell invasion and migration likely through down-regulating SMO. To confirm this, we performed RNA interference to knock-down SMO in CRC cells before the transfection with anti-miR-338-3p. Our data showed that anti-SMO-siRNA could significantly, but not completely, inhibit anti-miR-338-3p-induced invasion and migration of the CRC cells. These results confirmed that miR-338-3p inhibited CRC cell invasion and migration in vitro through down-regulating SMO, which made it a novel potential strategy for CRC treatment. Moreover, these results also demonstrated that the inhibitory effect of miR-338-3p on CRC cell invasion and migration was largely, but not completely, mediated by SMO, suggesting that miR-338-3p could regulate other SMO-independent signaling pathways to promote CRC metastasis.
In conclusion, our study demonstrates that miR-338-3p can suppress the invasion and migration of CRC by directly binding the 3′-UTR of SMO, its target. Though there is still much to learn about the role of miR-338-3p in CRC tumorigenesis, miR-338-3p provides us with a new potential target for CRC treatment.
Funding
This work was supported by National Natural Science Foundation of China (81101896) and National Research Foundation for the Doctoral Program of Higher Education of China (20124433110010).
Conflict of interest statement
None declared.
References
Author notes
Dr Qi Xue and Kai Sun contributed equally to this work.
