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Cell Biology International (2012) 36, 765–770 (Printed in Great Britain)
Vector-based miR-15a/16-1 plasmid inhibits colon cancer growth in vivo
Lixia Dai*1, Wei Wang*1, Shuang Zhang*1, Qingyuan Jiang†, Ruibo Wang*, Lei Dai*, Lin Cheng*, Yang Yang*, Yu‑Quan Wei* and Hong‑Xin Deng*2
*State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, People's Republic of China, and †Sichuan Provincial Hospital for Women and Children, Chengdu, Sichuan, People's Republic of China


miR-15 (microRNA 15) and miR-16 are frequently deleted or down-regulated in many cancer cell lines and various tumour tissues, suggesting that miR-15a/16-1 plays important roles in tumour progression and might be a method for cancer treatment. We have developed a vector-based plasmid to explore the anti-tumour efficacy of miR-15a/16-1 in colon cancer in vivo. It is proposed that miR-15a and miR-16-1 target cyclin B1 (CCNB1), which associates with several tumorigenic features such as survival and proliferation. The levels of miR-15a and miR-16-1 in colon cancer cells were inversely correlated with CCNB1 expression, and there was consensus between miR-15a/16-1 and CCNB1 mRNA sequences by analysing homology. Vector-based miR-15a/16-1 expression plasmid was constructed and transfected into HCT 116 and SW620 colon cancer cells in vitro. The effects produced on cell viability and angiogenesis were analysed using flow cytometric analysis, colony formation analysis and tube formation analysis. CCNB1 expression down-regulation was checked by Western blotting. Systemic delivery of miR-15a/16-1 plasmids encapsulated in cationic liposome led to a significant inhibition of subcutaneous tumour growth and angiogenesis in tumour tissues, whereas no effects were observed with liposome carrying the non-specific plasmid. In summary, miR-15a/16-1 has been applied in colon cancer treatment in vivo, and resulted in effective colon tumour xenografts growth arrest and angiogenesis decrease. These findings suggest that systemic delivery of vector-based miR-15a/16-1 expression plasmid can be an approach to colon cancer therapy.


Key words: anti-angiogenesis, cancer therapy, CCNB1, miR-15a/16-1

Abbreviations: CLL, chronic lymphocytic leukaemia, DMEM, Dulbecco's modified Eagle's medium, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, HUVEC, human umbilical-vein endothelial cell, miR/miRNA, microRNA, MVD, microvascular density, PCNA, proliferating-cell nuclear antigen, qRT-PCR, quantitative real-time PCR, UTR, untranslated region

1These authors have contributed equally to this work.

2To whom correspondence should be addressed (email denghongx@scu.edu.cn)


1. Introduction

Colon cancer is the most common cancer in the Europe and the second most common cause of cancer-related death in the USA and Europe (Ferlay et al., 2010; Jemal et al., 2010). The current situations reflect an unmet need in the treatment of this cancer.

miRNAs (microRNAs) are short non-coding single-strand RNA found in a variety of species, which can repress or degrade specific protein-coding genes through imperfectly or completely binding to the 3′-UTR (untranslated region) of target mRNAs (Bartel, 2004; Kim et al., 2006). They participate in cell proliferation, differentiation, apoptosis, angiogenesis and play an important role in the development of many serious diseases, including cancers (Cheng et al., 2005; Kloosterman and Plasterk, 2006; Wang et al., 2008). Thus, miRNAs have the potential to be developed for cancer diagnosis and treatment (Esquela-Kersche and Slack, 2006; Chen et al., 2009; Di Leva and Croce, 2010).

miR-15a and miR-16-1 are transcribed as a cluster that resides in human 13q14 chromosomal region and consequently shares identity in the seed region thought to be the primary determinant of target recognition. They function in CLL (chronic lymphocytic leukaemia) and prostate carcinoma by targeting BCL2, CCNE1 and CCND1, which triggers apoptosis and cycle arrest (Calin et al., 2002, 2008; Bonci et al., 2008). We have therefore investigated up-regulation of miR-15a/16-1 in colon cancers with a plasmid-based miRNA expression system to assess its effect on colon cancer cells in vitro and its anti-tumour efficacy in vivo.

2. Materials and methods

2.1. Cell culture and transfection

HCT 116 (ATCC CCL-247) and SW620 (ATCC CCL-227) cells obtained from ATCC maintained in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS. NCM460 (normal human colon mucosa cell line) was obtained from Incell Corporation and maintained in M3:10TM medium (Incell, Cat No. M300A500). HUVECs (human umbilical-vein endothelial cells) were isolated from human umbilical cord veins which were a gift from West China Women and Children Hospital, Sichuan University, and maintained in EGM-2 medium (Lonza Walkersville) (Jaffe et al., 1973). All cells were incubated at 37°C in a humidified air atmosphere containing 5% CO2. For transfection experiments, cells were transfected with Fugene HD Transfection Reagent (Roche Applied Science) according to the manufacturer's protocol.

2.2. Plasmid construction

A 362 bp HindIII/SacI genomic fragment spanning the miR-15a/16-1 resides in human chromosome 13 was cloned into the Gateway entry vector pENTR1A, which was modified to express exogenous sequences under an H1 promoter (pENTR1A H1). The control sequence was named EV and had no homology to any mammalian sequence. The sequence of plasmid was determined by DNA sequencing.

2.3. RNA extraction and qRT-PCR (quantitative real-time PCR)

Total RNA was extracted from colon cancer cells by using TRIzol® reagent (Invitrogen). qRT–PCR analysis for the expression of candidate miRNAs was performed with the TaqMan miRNAs assays kit (Applied Biosystems). For normalization, U6 snRNA (small nuclear RNA) was the reference, and the fold expression of candidate miRNAs was calculated following the method of Schmittgen and Livak (2008).

2.4. Flow cytometry assay

HCT 116 and SW620 cancer cells were plated in 6-well plates. Cells were synchronized by thymine (Sigma) for 24 h and treated with blank, EV or miR-15a/16-1. Two days after transfection, cells were collected and their cycling analysed by flow cytometry after PI (propidium iodide) staining.

2.5. Colony formation assay

HCT 116 and SW620 cells transfected with miR-15a/16-1, EV or untreated were trypsinized and seeded at 500 cells/well in 6-well plates. After 14 days, cells were stained with Crystal Violet and aggregates of >50 cells were scored as colonies.

2.6. Tube formation assay

The anti-angiogenic effects of miR-15a/16-1 were analysed by tube-formation assay using 96-well plate (Pang et al., 2009), HUVECs were seeded at 2×104 per well on polymerized Matrigel (BD Biosciences) and cultured with the supernatant (200 μl) from the cancer cells transfected for 48 h with blank, EV or miR-15a/16-1. After 6 h, tube formation by HUVECs was photographed and evaluated with Image-Pro Plus software (Media Cybernetics) (Qian et al., 2004).

2.7. Western blotting assay

Cells transfected for 48 h were lysed on ice with RIPA buffer (Beyotime Biotechnology). Aliquots of protein from each sample were used for SDS/PAGE, followed by the standard immunoblotting procedure and subsequent probing with the specific primary antibodies: CCNB1 (Abcam) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Sigma), followed by incubation with HRP (horseradish peroxidase)-conjugated secondary antibodies (Abcam). Blots were visualized on Kodak X-Ray films using an enhanced chemiluminescence kit (Pierce).

2.8. Liposome and liposome–DNA complexes preparation

Liposome for treatment of animals was prepared as described by Templeton et al. (1997). Briefly, the final liposome was a small multilamellar cationic liposome (DOTAP/cholesterol) of 100±20 nm. The liposome–DNA complexes used for in vivo administration were prepared by liposome and plasmid (diluted in 5% glucose) gently mixed at a 5:1 weight ratio of liposome/DNA (100 μl total volume per mouse). The resulting mixture was incubated at room temperature for 30 min before intravenous injection.

2.9. Animal studies

The animal procedures were approved by the Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China). Female athymic BALB/c nude mice, 5–6 weeks old, were maintained in pathogen-free conditions. HCT 116 (5×106 cells) and SW620 (2×106 cells) in 0.1 ml serum-free DMEM was injected subcutaneous into the right flank of each mouse. When the tumour was ∼50 mm3, the mice were randomly assigned to three independent treatment groups (n = 5): (a) 5% glucose (5% GS); (b) 10 μg of EV/50 μg of liposome complexes (EV); (c) 10 μg of miR-15a/16-1/50 μg of liposome complexes (miR-15a/16-1). Athymic mice bearing HCT 116 or SW620 xenografts were treated intravenously through the tail vein every 2 days for 3 weeks. The tumour volume was measured with a calliper every 3 days and calculated using the formula: volume = length×width2/2.

2.10. Histologic analysis

The primary antibodies to CCNB1 (Abcam), PCNA (proliferating-cell nuclear antigen; Santa Cruz Biotechnology) and CD31 (BD Biosciences) were used for immunohistochemistry as previously described (Zhang et al., 2009). MVD (microvascular density) was determined by counting the number of microvessels per high-power field in the sections, and the number of CCNB1-positive, PCNA-positive cells were counted in six random fields.

2.11. Statistical analysis

SPSS 11.5 was used for statistical analysis. Data were analysed statistically using one-way ANOVA. Differences were considered significant at P<0.05.

3. Results

3.1. Effect of miR-15a/16-1 on tumour cells in vitro

To detect the effects of miR-15a/16-1 produced on colon cancer cell viability and angiogenesis in vitro, flow cytometry, colony formation and tube formation analyses were performed. qRT-PCR (quantitative real-time PCR) was used to detect mature miR-15a and miR-16 in the colon cancer cell lines 48 h after transfection. Compared with the controls, cells transfected with miR-15a/16-1 had significantly increases in miR-15a and miR-16 levels (Figures 1A and 1B). Based on the results of subsequent experiments, an increase in G2/M phase cells (Figure 1C, HCT 116; Figure 1D, SW620), a marked decrease in the number of cell colonies (Figure 2A, HCT 116; Figure 2B, SW620; P<0.05) and poorly organized tube-like structures (Figure 2C) were observed in the group transfected with miR-15a/16-1. To evaluate the possible toxicity of miR-15a and miR-16-1 overexpression, NCM460, HCT 116 and SW620 cells were transfected in parallel. Transfection in colon tumour cell lines resulted in marked loss of cell viability, whereas treatment of miR-15a and miR-16-1 in NCM460 cells caused no sign of toxicity (data not shown).

3.2. miR-15a/16-1 targeted CCNB1 in vitro

By analysing homology between miR-15a/16-1 and the CCNB1 mRNA sequences, the nucleotides from the 5′-end of miR-15a/16-1 proved complementary to bases 447–458 from the 3′-UTR region of CCNB1 mRNA (Figure 3A). The levels of miR-15a/16-1 were inversely correlated with CCNB1 expression. CCNB1 expression was sharply reduced in SW620 cells transfected with miR-15a/16-1 (Figure 3B); the results indicate that CCNB1 might be targeted by miR-15a/16-1 in colon cancer cells.

3.3. Effect of miR-15a/16-1 on tumour growth in vivo

To investigate the effects of miR-15a/16-1 on growth of human colon cancer in vivo, subcutaneous xenografts in nude mice were established and treated as described in the Materials and methods section. Three days after the last treatment, mice were killed and their tumours excised. Compared with control groups, tumour growth was significantly suppressed (Figures 4A and 4B) and mean tumour weight was reduced (Figures 4C and 4D) in the miR-15a/16-1 group (P<0.05); however, no significant difference was observed between the two control groups (P>0.05). To explore the effects of miR-15a/16-1 on CCNB1 expression, cell proliferation and tumour angiogenesis in the xenografts, CCNB1, PCNA and CD31 were detected by immunohistochemistry. In agreement with the in vitro results, CCNB1 expression was markedly decreased, and the average MVD and proliferation rate (with brown staining) were significantly reduced in mice treated with miR-15a/16-1 (Figure 4E, P<0.05).

4. Discussion

The classical models of tumorigenesis postulate imbalance between oncogenes and tumour suppressor genes. miRNAs may function as tumour suppressors (as is the case for miR-15a and miR-16-1 or let-7 family) or oncogenes (as is the case for miR-155 or members of the miR-17-92 cluster or miR-378) (Zhang et al., 2007; Croce, 2008; Ruan et al., 2009). Thus they have the potential to impact on cancer treatment. miR-15a/16-1s appears to function as tumour suppressors in CLL and prostate carcinoma, and to play important roles in cell proliferation and apoptosis in vitro (Cimmino et al., 2005; Bonci et al., 2008; Calin et al., 2008). Our investigation on the effect of miR-15a/16-1 in colon cancer in vivo shows that systematic delivery of miR-15a/16-1/liposome complex to subcutaneous colon tumour xenografts induced significant suppression on tumour growth (approximately 60% in volume and 50% in weight) and angiogenesis (70%). This is convincing evidence that miR-15a/16-1 can inhibit colon cancer growth in vivo and suggests that miR-15a/16-1 offers a clinically feasible approach for cancer therapy.

miR-15a/16-1 may induce cell apoptosis and cell cycle arrest by targeting BCL2 (Cimmino et al., 2005) and CCND1 in many cancer cells (Bonci et al., 2008; Lerner et al., 2009; Klein et al., 2010). The entry of eukaryotic cells into mitosis is controlled by activation of cdc2 (cell division cycle 2 kinase), and activation of cdc2 is regulated by several steps, including CCNB1 binding. In HCT 116 and SW620 cells, miR-15a/16-1 inhibited cell proliferation and induced cycle arrest by targeting CCNB1, which increased the percentage of cells in G2/M phase. It appears that miR-15a/16-1 down-regulated not only CCND1 and BCL2, but also CCNB1 in colon cancer cells, which is important for colon tumour growth.

miRNAs not only play important roles in cell progression, but are also involved in angiogenesis (Fish and Srivastava, 2008; Kuehbacher et al., 2008). They may directly or indirectly regulate factors involved in angiogenesis. Angiogenesis is a key event of tumour growth and metastasis (Folkman, 1971; Carmeliet and Jain, 2000; Cristofanilli et al., 2002). We found that the vascular density in tumour tissues of miR-15a/16-1 group was much lower than in controls, which suggests that miR-15a/16-1 affects one or more of the angiogenesis factors to block angiogenesis in the colon tumour environment. Altogether, miR-15a/16-1 down-regulated cell cyclin and angiogenesis-regulator proteins, which were critical to tumour progression in vivo.

Over the past few years, RNA interference has become one of the most promising approaches to disease treatment; however, it is still in the exploratory stage. Because of its uncertainty, acute liver toxicity and mortality in mice, it can cause serious side effects in the course of treatment (Pai et al., 2006). In our studies, toxic pathologic changes in liver, lungs, spleen, kidneys and heart were detected by microscopic examination, but we observed no side effects of the delivery system of DNA–cationic liposome complexes, which had now advanced into phase I clinical trial for treatment of NSCLC (Lu et al., 2009). Thus, the safe and efficient delivery of DNA–cationic liposome complexes to tumour seems attractive in cancer therapy.

Mechanisms of the anti-tumour efficacy remain to be fully elucidated; however, two mechanisms might be involved. Significantly decreased CCNB1 expression and angiogenesis may contribute to the efficacy of miR-15a/16-1 in vivo, which is supported by our results. In conclusion, this is novel evidence that the vector-based miR-15a/16-1 plasmid can safely and efficiently inhibit colon-xenografts growth in vivo, making miR-15a/16-1 of considerable potential as a gene suppressor in cancer therapy.

Author contribution

Lixia Dai, Wei Wang and Shuang Zhang designed the procedure of the study, carried out the study design and drafted the manuscript. Qingyuan Jiang and Ruibo Wang assisted in animal study and immunohistological analysis. Lei Dai, Lin Cheng and Yang Yang participated in literature research and data analysis. Hong-xin Deng and Yu-quan Wei supervised the whole experimental work and revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by Hi-tech Research and Development Program (863 Program) of China [grant numbers 2007AA021008 and 2009ZX09102-241], and the National Key Basic Research Program (973 Program) of China [grant number 2010CB529900].

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Received 22 July 2011/5 April 2012; accepted 11 May 2012

Published as Cell Biology International Immediate Publication 11 May 2012, doi:10.1042/CBI20110404


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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