MicroRNA-215-3p Suppresses the Growth, Migration, and Invasion of Colorectal Cancer by Targeting FOXM1.

Previous investigations have indicated that microRNA-215-3p is dysregulated in many kinds of cancers and functions as oncogene or tumor suppressor. However, the potential role of microRNA-215-3p in the progression of colorectal cancer remains not well known. Herein, we demonstrated that microRNA-215-3p was downregulated in human colorectal cancer tissues and was reversely correlated to the lymph node metastasis of colorectal cancer. Overexpression of microRNA-215-3p inhibited the clonogenic abilities and metastasis-relevant traits of colorectal cancer cell in vitro. Consistently, upregulation of microRNA-215-3p inhibited the growth and metastasis of colorectal cancer cell in vivo. Forkhead box protein M1 was identified as a direct target of microRNA-215-3p and reexpression of forkhead box protein M1 reversed the suppressive impacts of microRNA-215-3p on the growth, mobility, and invasion abilities of colorectal cancer cell. Altogether, these results revealed the vital role of microRNA-215-3p in the tumorigenesis and metastasis of colorectal cancer.

Introduction

Colorectal cancer (CRC) is still one of the most common and deadly cancers worldwide.[1,2] Owing to the great advancement in the therapeutic strategies and imaging techniques, the 5-year survival rate for all stages of patients with CRC has increased to 66%. Nevertheless, the survival rate of patient with distant-stage CRC remains very poor and the 5-year survival rate is only nearly 10% to 15% owing to the distant metastasis.[3,4] Thus, deep investigations of cancer cell metastasis and the underlying mechanism are very urgent for combating CRC metastasis and improving the prognosis. Metastasis, which is a multistep complex process, composed of migration, invasion, intravasation, survival in the circulation, escaping immunologic surveillance, extravasation, initial growth, and finally metastatic colonization.[5,6]

MicroRNAs (miRNAs), which are small, noncoding RNAs (18-23 nucleotides), modulate the expressions of target proteins via binding to the 3′-untranslated region (3′-UTR) of target messenger RNAs.[7,8] MicroRNAs play vital functions in various biological processes, including cell growth, apoptosis, and their dysregulation are correlated with the progression of several diseases.[9-11] Recent reports have verified that miRNAs contribute to the progression of various cancer types, including tumor growth, cell metastasis, and chemotherapy resistant.[12,13] For example, miR-215 has been implicated in the pathogenesis of several human malignancies and is upregulated in gastric cancer, pancreatic carcinoma, glioma, and acts as a potential oncogene in these tumors.[14-16] By contrast, miR-215 expression has been observed to be significantly reduced in human ovarian cancer, nonsmall cell lung cancer, and colon cancer; and in these cases, it acts as a tumor suppressor.[17-19] In CRC, overexpression of miR-215-5p significantly inhibited the proliferation, clonogenicity, and migration of CRC cell via targeting epidermal growth factor receptor ligand epiregulin and its transcriptional inducer homeobox protein Hox-B9.[20] Previous investigation has demonstrated that overexpression of miR-215-3p sensitizes CRC to 5-fluorouracil-induced apoptosis through regulating C-X-C chemokine receptor type 1.[21] Until now, whether miR-215-3p is involved in the progression of human CRC remains not well explored.

In this research, we demonstrated that miR-215-3p was downexpressed in CRC tissues and cell lines. Overexpression of miR-215-3p significantly inhibited the clonogenic abilities and metastasis-relevant traits of CRC cells. Furthermore, upregulation of miR-215-3p inhibited the tumor growth and metastasis of CRC cell in vivo. Our investigations further deciphered that the target gene of miR-215-3p, forkhead box protein M1 (FOXM1), was regarded to promote the growth, migration, and invasion abilities of CRC cell. Altogether, our results elucidate that miRNA-215-3p suppresses the growth, migration, and invasion of CRC by targeting FOXM1.

Materials and Methods

Colorectal Cancer Tissues and Cells

Forty-eight primary CRC and paired adjacent nontumor tissues were obtained from the Second Affiliated Hospital of Anhui Medical University. No patients received chemotherapy or radiotherapy before surgery. This study was approved by the Institute Research Medical Ethics Committee of the Second Affiliated Hospital of Anhui Medical University. Written consent was obtained from all participants who were involved in the study. The clinicopathological data of all the patients were listed in Supplemental Table 1. Human CRC cell lines (SW480, HT-29, and LOVO) and normal colon epithelial cell line, HCoEpiC, were obtained from Nanjing Cobioer Biotechnology Co, Ltd (Nanjing, Jiangsu, China) and were grown in 1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The HEK-293T cell was obtained from Nanjing Cobioer Biotechnology Co, Ltd) and was cultured in Dulbecco Modified Eagle Medium (Thermo Fisher Scientific) containing 10% FBS and 1% penicillin–streptomycin at 37°C with 5% CO2.

Quantitative Real-time Polymerase Chain Reaction Assay

Total RNAs were extracted using TRIzol Plus RNA Purification Kit (Thermo Fisher Scientific). RNA was reverse transcribed to complementary DNA (cDNA) using the 1-step PrimeScript miRNA cDNA synthesis kit (TaKaRa, Dalian, Liaoning, China), and quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using the SYBR Premix Ex Taq II (TaKaRa)[Please provide the city and state (if USA) or city and country (if other than USA) for “TaKaRa, BD Biosciences, Ambion, and Promega.”]. The level of miRNA was normalized against the endogenous U6 small nuclear RNA. The levels of miR-215-3p and FOXM1 were calculated using the 2−ΔΔCt method.[22] The primers used in qRT-PCR were summarized as following: GAPDH: 5′-AATGGATTTGGACGCATTGGT-3′ and 5′-TTTGCACTGGTACGTGTTGAT-3′; U6: 5′-GCCAGCTCCTACATCTCAGC-3′ and 5′-AGCCTGACTTGCTAGTGGATTAT-3′; miR-215-3p: 5′-ACGCGTTGCTCAAAGACACTCATT-3′ and 5′-CTCGAGTAACATATAAGGGAGATTCC-3′; FOXM1: 5′-CGTCGGCCACTGATTCTCAAA-3′ and 5′-GGCAGGGGATCTCTTAGGTTC-3′.

Cell Transfections

The miR-215-3p and miR-215-3p-negative control (miR-NC) were obtained from GenePharma (Shanghai, China); 2 × 105 SW480 or HT-29 cells were transfected with miRNAs uisng Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). The coding sequences of FOXM1 were amplified using polymerase chain reaction and was inserted into a pcDNA3.1 vector (Thermo Fisher Scientific) to generate the FOXM1 overexpression vector.

Cell Proliferation Assay

SW480 or HT-29 cells (3 × 104) were cultured in 96-well cell culture plates. After incubation for 24, 48, 76, or 96 hours, 25 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide stock solution was added to each well and incubated for 4 hours. The absorbance was detected at 570 nm using Synergy HT Multi-Mode Microplate Reader (Bio-Tek, Winooski, Vermon).

Colony Formation Analysis

Colony formation assay was performed to measure the capacity of cell proliferation; 1000 SW480 or HT-29 cells were cultured into a 6-well plate for 2 weeks. After that, cell colony was fixed using paraformaldehyde and stained using 1% crystal violet. The number of visible cell colonies was counted.[23]

Wound Healing Assay

Either 1 × 103 SW480 or HT-29 cells was cultured into 6-well plate and were cultured using serum-free medium for 24 hours . After that, a wound was made using a 100 μL pipette tip. Then, cells were cultured in serum-free medium. The wound closures were visualized under an inverted microscope (CarlZeiss, Hallbergnoos, Germany).

Invasion Assay

Either 2  ×  103 SW480 or HT-29 cells was cultured into the Transwell chambers with Matrigel-coated (BD Biosciences); 100 μL 20% FBS-containing media was added into the lower chamber as an attractant. After 18 hours, the invaded cells were fixed using methanol and stained using 1% crystal violet.

Luciferase Reporter Gene Assay

The wild-type (wt) 3′-UTR of FOXM1 or mutant type (mut) 3′-UTR of FOXM1 containing the binding site of miR-215-3p was constructed and inserted into the downstream of the firefly luciferase gene in a pGL3-promoter vector (Ambion); HEK-293T cell (1  ×  105) was cultured in 96-well plates for 24 hours and cells were transfected with wt or mut 3′-UTR of FOXM1 and miR-NC or miR-215-3p using Lipofectamine 2000 reagent (Thermo Fisher Scientific). Cell lysates were collected 24 hours after transfection, then luciferase activity was detected using a Luciferase Reporter System (Promega).

Immunoblotting

Total proteins were extracted from cells using radioimmunoprecipitation assay buffer (Beyotime, Naming, Jiangsu, China). A total of 25 μg proteins were loaded on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat milk, the membrane was incubated with primary antibodies against with FOXM1 (#sc-271746, Santa Cruz Biotechnology, California) or β-actin (#sc-58673, Santa Cruz Biotechnology) overnight at 4 °C. Then, membrane was incubated with horseradish peroxidase-conjugated immunoglobulin G (1:10000, Beyotime) for 2 hours. The target bands were detected using the enhanced chemiluminiscence (ECL) system (Millipore, Braunschweig, Germany) and visualized with the ChemiDoc XRS system (Bio-Rad, Hercules, California).

In Vivo Tumorigenesis

SW480 cells (2  ×  107) were suspended in 50 μL phosphate buffered saline and were subcutaneously inoculated into 5-week-old BALB/c nude mice (n = 6 in each group). The length and width of tumor tissue were measured each week. The tumor volume was calculated as 0.5 × length × width.[2] Five weeks after CRC cells inoculation, nude mice were killed and tumor tissue was isolated and weighed.

Experimental Metastasis Assay

SW480 cells (2  ×  107) were injected into 5-week-old BALB/c nude mice via the lateral tail vein (n = 6 in each group). Mice were killed 2 weeks after inoculation and lung metastatic nodules were analyzed under a dissecting microscope. The lungs were fixed in 4% polyoxymethylene for hemotoxylin and eosin staining assay. Animal handling and experimental procedures were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Anhui Medical University.

Statistical Analysis

All data are shown as mean (standard deviation). Statistical tests are 1-sided or 2-sided, and the differences between 2 groups were assessed using Student t tests, while analysis of variance test was conducted in multiple comparable groups. Pearson correlation analysis was utilized to evaluate the correlation between miR-215-5p level and FOXM1 level in CRC tissues. P < .05 was considered significant.

Results

MicroRNA-215-3p Is Downregulated in CRC

Firstly, we detected the levels of miR-215-3p in human CRC cell lines using qRT-PCR assay. As shown in Figure 1A, when compared to colon epithelial cell line, HCoEpiC, miR-215-3p was significantly downexpressed in all human colon cancer cell lines. Next, we investigated the levels of miR-215-3p in 48 cases of CRC tissues and corresponding normal tissues of patients with CRC. Consistently, miR-215-3p was downregulated in CRC tissues compared to that in normal tissues (Figure 1B). To confirm the relationship between miR-215-3p expression and lymph node metastasis (LNM) of CRC, we analyzed the level of miR-215-3p in colon cancer tissues from patients with or without LNM. As shown in Figure 1C, the level of miR-215-3p was significantly reduced in CRC tissues with LNM when compared to that without LNM. Finally, the Kaplan-Meier survival analysis indicated that the overall survival of patients who had low level of miR-215-3p was poor than patients who had high level of miR-215-3p (Figure 1D). All these data indicated that miR-215-3p was downexpressed in human CRC cells and tissues.

Figure 1.

The expression pattern of miR-215-3p in CRC and its association with prognosis. A, The expression of miR-215-3p in normal colonic epithelial cell line, HCoEpiC, and colon cancer cell lines was measured using qRT-PCR. **P < .01 compared to HCoEpiC. B, The expression of miR-215-3p in 48 pairs of primary colon cancer tissues and adjacent colonic epithelium tissues was detected using qRT-PCR. **P < .01 compared to adjacent. C, The expression of miR-215-3p in colon cancer tissues with LNM and colon cancer tissues without LNM. **P < .01 compared to no metastasis. D, Kaplan-Meier survival analysis of the correlation between miR-215-3p expression level and overall survival of 48 colon cancer patients. CRC indicates colorectal cancer; LNM, lymph node metastasis; miR-215-3p, microRNA-215-3p; qRT-PCR, quantitative real-time polymerase chain reaction.

MicroRNA-215-3p Suppresses the Metastasis-Relevant Traits of CRC Cell In Vitro

To decipher the role of miR-215-3p in the development of CRC, miR-215-3p or miR-NC mimics were transfected into CRC cells HT-29 and SW480 (Figure 2A). Then, we observed that overexpression of miR-215-3p significantly inhibited the proliferation of CRC cell (Figure 2B). Consistently, upregulation of miR-215-3p significantly suppressed the colony formation of CRC cell (Figure 2C). Nevertheless, miR-215-3p overexpression had no significantly effect on the growth and apoptosis of normal colonic epithelial cell line, HCoEpiC (Supplemental Figure 1). The 5-bromo-2′-deoxyuridine (BrdU) incorporation assay also verified that miR-215-3p transfection significantly decreased the proliferation of HT-29 and SW480 cell (Supplemental Figure 2). To elucidate the mechanism underlying the miR-215-3p-mediated inhibition of CRC cell growth, the cell cycle was analyzed in cells transfected with miR-215-3p and miR-NC. The fluorescence-activated cell sorting analysis showed that the numbers of cells at G2/M phase in the miR-215-3p-overexpressing SW480 and HT-29 cells were significantly increased compared to the controls (Supplemental Figure 3). However, miR-215-3p overexpression had no effect on the cell cycle of HCoEpiC cells (Supplemental Figure 3). Consistently, the levels of cyclin-dependent kinase 1 and cyclin B2, which are general G2/M cell cycle arrest regulatory molecules, were significantly decreased by miR-215-3p transfection as demonstrated by qRT-PCR assay (Supplemental Figure 4). We next investigated the role of miR-215-3p on the metastasis-relevant traits of CRC cell in vitro. In the wound healing and Transwell invasion assay, overexpression of miR-215-3p reduced the HT-29 and SW480 cell migration and invasion when compared to control (Figure 2D and E). All these findings demonstrated that miR-215-3p transfection inhibited the malignant phenotypes of CRC cell.

Figure 2.

Overexpression of miR-215-3p suppresses colonigenic ability and metastasis-relevant traits of CRC cell in vitro. A, SW480 or HT-29 cells were transfected with miR-NC or miR-215-3p and the level of miR-215-3p was detected using qRT-PCR assay. B, HT-29 and SW480 cells were transfected with miR-215-3p or miR-NC, respectively. The role of miR-215-3p on the proliferation of HT-29 and SW480 cell was assessed using MTT assay. C, Influence of miR-215-3p on colony formation of HT-29 and SW480 cells. Representative images were presented (left panel). The number of cell colonies was quantified. D, Wound-healing assay of HT-29 and SW480 cells transfected with miR-NC or miR-215-3p. E, Invasion assays of HT-29 and SW480 cells transfected with miR-NC or miR-215-3p. **P < .01 compared to control. CRC indicates colorectal cancer; miR-215-3p, microRNA-215-3p; miR-NC, miR-215-3p-negative control; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; qRT-PCR, quantitative real-time polymerase chain reaction.

MicroRNA-215-3p Suppresses the Growth and Metastasis of CRC Cell in Mice

We next explored whether miR-215-3p could decrease the tumor growth of SW480 cells in transplanted tumor model. Transplanted tumor model was constructed using miR-NC or miR-215-3p-transfected SW480 cells. The tumor growth in mice that was injected with miR-215-3p-transfected cell was significantly suppressed when compared to miR-NC-transfected group (Figure 3A and B). Consistently, the tumor weight in mice which was inoculated with miR-215-3p-transfected cell was obviously reduced when compared to miR-NC-transfected group (Figure 3C). To investigate the influence of miR-215-3p on the distant metastasis of CRC cell in nude mice, the experimental lung metastasis assay was conducted. SW480 cells were transfected with miR-NC or miR-215-3p and were injected into mice via tail vein. After 14 days, mice were killed and we observed that miR-NC-transfected SW480 cells resulted in the formation of obviously lung colonies, whereas miR-215-3p-transfected SW480 cells significantly suppressed pulmonary metastasis (Figure 3D and E). These results indicated that miR-215-3p attenuated the tumorigenicity of CRC cell in vivo.

Figure 3.

MicroRNA-215-3p overexpression inhibits tumorigenesis and metastasis in vivo. A, MicroRNA-NC or miR-215-3p-transfected SW480 cell was implanted into nude mice to establish the xenograft CRC model. Representative picture in different group is shown. B, The tumor growth of SW480 was significantly suppressed in mice that were injected with miR-215-3p-transfected SW480 cells. C, Thirty-five days after implantation, the mice were killed and tumor tissue was weighed. D, Representative pictures of lungs from mice were taken after 2 weeks of injection with miR-NC or miR-215-3p-transfected SW480 cells. Arrow indicates the lung metastatic foci. E, The numbers of lung metastasis were quantified and showed. **P < .01 compared to miR-NC. CRC indicates colorectal cancer; miR-215-3p, microRNA-215-3p; miR-NC, miR-215-3p-negative control.

Forkhead Box Protein M1 Is a Direct Target of miR-215-3p

To further investigate the molecular mechanism of miR-215-3p-mediated pathological functions in the progression of CRC, TargetScan (Release 7.2) was selected to find the target gene of miR-215-3p. We observed that miR-215-3p has putative binding sites with the 3′-UTR region of FOXM1 (Figure 4A). Then, the 3′-UTR of FOXM1 containing the binding sites was cloned into the pGL3-promoter vector. The luciferase assay indicated that the luciferase activity in HEK-293T cell transfected with wt 3′-UTR of FOXM1 was reduced by miR-215-3p. However, the luciferase activity in HEK-293T cell transfected with mut 3′-UTR of FOXM1 was not inhibited by miR-215-3p (Figure 4B). Consistently, the future luciferase assay in HT-29 and SW480 cells suggested that miR-215-3p significantly reduced the luciferase activity in cell transfected with wt 3′-UTR of FOXM1 (Supplemental Figure 5). The results of immunohistochemical analysis indicated that the expression of FOXM1 was significantly increased in clinical CRC sample than that in the adjacent tissue (Supplemental Figure 6). Furthermore, miR-215-3p transfection significantly decreased the expressions of FOXM1 in HT-29 and SW480 cells (Figure 4C). Finally, we observed that there is an inverse correlation between FOXM1 and miR-215-3p level in CRC tissues and The Cancer Genome Atlas (TCGA) (Figure 4D and Supplemental Figure 7). These data indicated that miR-215-3p directly regulated the expression of FOXM1 in CRC.

Figure 4.

Forkhead box protein M1 is the direct target of miR-215-3p. A, Schematic diagram of putative miR-215-3p binding site in the 3′-UTR regions of FOXM1. B, HEK-293T cells were cotransfected with vectors containing either the wild-type (wt) or mutant (mut) target sites of FOXM1 and either miR-215-3p mimics or miR-NC. Luciferase reporter assay was performed to verify the binding between miR-215-3p and FOXM1. C, Western blot analysis of FOXM1 expression in CRC cell with or without miR-215-3p overexpression. D, Correlation of miR-215-3p and FOXM1 in paired clinical CRC samples. CRC indicates colorectal cancer; FOXM1, forkhead box protein M1; miR-215-3p, microRNA-215-3p; miR-NC, miR-215-3p-negative control; 3′-UTR, 3′-untranslated region.

Downregulation of FOXM1 Imitates miR-215-3p-Mediated Phenotypes in CRC cell

To investigate the functional contribution of FOXM1 to the phenotypes of miR-215-3p, we then analyzed whether downregulation of FOXM1 inhibited the aggressive phenotype of CRC cell. First, small interfering RNA (siRNA) against FOXM1 (siFOXM1 #1 or siFOXM1 #2) was transfected into SW480 or HT-29 to reduce the level of FOXM1. As shown in Figure 5A and Supplemental Figure 8, transfection of siRNA against FOXM1 in SW480 and HT-29 cells decreased the expression of FOXM1. Meanwhile, knockdown of FOXM1 suppressed the proliferation and colony formation of SW480 and HT-29 cells (Figure 5B and C and Supplemental Figure 8). Furthermore, the BrdU incorporation assay verified that siFOXM1 significantly suppressed the proliferation of HT-29 and SW480 cells (Supplemental Figure 9). In addition, siRNAs against FOXM1 reduced the migration and invasion abilities in SW480 and HT-29 cells (Figure 5D and E and Supplemental Figure 8). Collectively, inhibition of FOXM1 inhibited the metastasis-related phenotype of CRC cell.

Figure 5.

Knockdown of FOXM1 inhibits the growth, migration, and invasion of CRC cell. A, Detection of endogenous FOXM1 expression by Western blotting after transfection of siCon or siFOXM1, respectively, in SW480 cell. B, Effects of transfection of siFOXM1 on the proliferation of SW480 cell. C, Influence of siFOXM1 on the colony formation of SW480 cell. D, Wound healing migration assay of SW480 cell transfected with siCon or siFOXM1, respectively. E, Transwell invasion assay of SW480 cell transfected with siCon or siFOXM1, respectively. **P < .01 compared to control cell (parental SW480 cell). CRC indicates colorectal cancer; FOXM1, forkhead box protein M1.

Overexpression of FOXM1 Reverses the Suppressive Effect of miR-215-3p

Finally, to confirm whether miR-215-3p suppressed the progression of CRC cell via regulating FOXM1, SW480 cells were cotransfected with FOXM1 in combination with or without miR-215-3p. Reexpression of FOXM1 significantly increased the level of FOXM1 in miR-215-3p-transfected SW480 cell (Figure 6A). We further observed that reexpression of FOXM1 partially rescued the growth as well as colony formation of SW480 cell which was reduced by miR-215-3p (Figure 6B and C). Furthermore, the BrdU incorporation assay verified that reexpression of FOXM1 rescued the proliferation of SW480 cell which was reduced by miR-215-3p of HT-29 and SW480 cells (Supplemental Figure 10). To confirm whether overexpression of FOXM1 reversed the inhibition of miR-215-3p on the mobility and invasiveness of SW480 cell, SW480 cell was cotransfected with FOXM1 with or without miR-215-3p mimics and the migration and invasion assays were performed. As shown in Figure 6D and E, upregulation of FOXM1 abrogated the inhibitory effects of miR-215-3p on the migration and invasion of SW480 cell. The similar results were obtained in HT-29 cell (Supplemental Figure 11). Collectively, these findings indicated that miR-215-3p inhibited the progression of CRC cell via regulating the expression of FOXM1.

Figure 6.

Re-expression of FOXM1 reverses miR-215-3p-dependent phenotypes. A, SW480 cells were cotransfected with pcDNA3.1 containing FOXM1 or control vector after transfection of miR-NC or miR-215-3p. The expression of FOXM1 was assessed using Western blotting assay. B, The proliferation of SW480 cell was assessed using the MTT assay. C, Colony formation assays for SW480 cells cotransfected with pcDNA3.1 containing FOXM1 or control vector after transfection of miR-NC or miR-215-3p. D, Wound healing analysis of SW480 cells cotransfected with NC or miR-215-3p mimics together with either pcDNA3.1-FOXM1 or control vector, respectively. E, Invasion assays of SW480 cells cotransfected with NC or miR-215-3p mimics together with either pcDNA3.1-FOXM1 or control vector, respectively. **P < .01 compared to control cell (parental SW480 cell), ## P < .01 compared to SW480 transfected with miR-215-3p. FOXM1 indicates forkhead box protein M1; miR-215-3p, microRNA-215-3p; miR-NC, miR-215-3p-negative control; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide.

Discussion

Accumulating reports have suggested miRNAs play crucial regulatory roles in the progression of various cancers.[24,25] In our work, we proved that miR-215-3p was markedly downexpressed in clinical CRC tissues and cells. Furthermore, the level of miR-215-3p was negatively associated with the LNM and prognosis of patients with CRC. The functional assays indicated the involvement of miR-215-3p in the growth, migration and invasion of CRC in vitro, and upregulation of miR-215-3p suppressed the lung metastasis and growth of CRC cell in vivo.

Our clinical data indicated that miR-215-3p was downregulated in CRC tissues. Recent investigations demonstrate that miR-215-5p is downexpressed in CRC, gastric cancer, and lung cancer.[14,17,18] Nevertheless, no report has revealed the expression of miR-215-3p in CRC. In our study, the qRT-PCR assay was performed to visualize the expression pattern of miR-215-3p and we observed that miR-215-3p was downexpressed in human CRC tissues and cells when compared to normal control. To explore whether miR-215-3p level is related to the overall survival in patient with CRC, the Kaplan-Meier analysis was performed. The result suggested that the overall survival of patient who had low level of miR-215-3p was worse than patient who had high level of miR-215-3p.

MicroRNA serves as tumor-suppressor genes or oncogenes depending on their target genes.[26] Herein, the bioinformatics algorithms, TargetScan, was utilized to find the target gene of miR-215-3p and we finally demonstrated FOXM1 was the direct target gene of miR-215-3p using luciferase reporter gene assay.[27] Meanwhile, the expression of FOXM1 in SW480 and HT-29 cells transfected with miR-215-3p or miR-NC was significantly reduced by miR-215-3p mimics. Functional analysis proved that downregulation of FOXM1 significantly reduced the aggressiveness phenotypes of CRC cell. Most importantly, we demonstrated that reexpression of FOXM1 reversed the suppressive impacts of miR-215-3p on the growth and aggressiveness of CRC cell.

The human FOXM1 gene, which consist of 10 exons, is mapped to chromosome 12p13-3 and plays vital roles in the initiation, promotion, and progression of several cancers.[28-30] Extensive investigations have demonstrated that higher expression of FOXM1 is found in patients with lung cancer, breast carcinoma, gastric carcinoma, and pancreatic cancer.[31-34] Forkhead box protein M1 modulates the expression of various targeted genes, which are important for cancer cell differentiation, proliferation, and apoptosis; cell-cycle progression; and tumor angiogenesis and metastasis, which suggesting that it have a general role in cancer progression.[35] Herein, this work proved that miR-215-3p decreased the growth and aggressive of CRC cell through downregulating its target gene, FOXM1.

In summary, our study demonstrated that miR-215-3p was a tumor suppressor in human CRC. Overexpression of miR-215-3p participated into the progression of CRC through regulating the expression of FOXM1.

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Supplementary_materials for MicroRNA-215-3p Suppresses the Growth, Migration, and Invasion of Colorectal Cancer by Targeting FOXM1 by Xiaowei Tang, Xianfeng Shi, Nianfei Wang, Wanren Peng and Zhendong Cheng in Technology in Cancer Research & Treatment