Lentiviral vector‑mediated IL‑9 overexpression stimulates cell proliferation by targeting c‑myc and cyclin D1 in colitis‑associated cancer

Introduction

Colorectal cancer is considered thethird highest malignancy in the United States, causing >55,000 mortalities/year (1). Environmental and genetic factors influence the progression of colorectal cancer, which is characterized by inflammation arising from various causes (2). The most common type of chronic intestinal inflammation associated with colorectal cancer is inflammatory bowel disease, which includes ulcerative colitis (UC) and Crohn's disease (CD). Colorectal cancer caused by inflammatory bowel disease is referred to as colitis-associated cancer (CAC).

CAC in patients with UC is caused by unifocal or multifocal dysplastic mucosa in chronic inflammatory regions (3). UC has been reported to increase the risk of CAC by approximately eight-fold in comparison to the general population (4). The degree and duration of the disease, and the number of flares have been recognized as risk factors, suggesting that uncontrolled inflammation may drive tumorigenesis (5). Chronic inflammation has been proposed to increase epithelial cell turnover in the colonic mucosa, resulting in accelerated aging of colonic mucosa, and enhanced vulnerability for acquiring genetic and epigenetic alternations (2). These results suggest that age-associated inflammation may be used for the identification of patients with UC with an increased risk of manifesting CAC later in life (6).

The molecular mechanisms underlying inflammation-mediated cancer development are not fully understood, and may differ between CAC and other forms of colorectal cancer. The involvement of cytokines and immune mediators is well understood in virtually all stages of colonic tumorigeneses, including tumor initiation, promotion, progression and metastasis (7). The pathogenesis of CAC is affected by several cytokines. Interleukin (IL)-9 forms part of the c-chain family of cytokines. Several types of cells receptive to IL-9 exist, including mast, T, antigen-presenting and epithelial cells, all of which are found in the human lung and gut (8). IL-9 also exhibits downstream regulatory activities on epithelial cells in the airway and intestinal mucosa barrier, where overexpression of IL-9 induces airway hyper-responsiveness and increases intestinal permeability (9). Previous research has recognized that IL-9 serves an important role in the pathogenesis of several chronic inflammatory and autoimmune diseases, including inflammatory bowel disease, multiple sclerosis, lupus erythematous, food allergies and asthma (10). For instance, Gerlach et al confirmed that IL-9 expression is associated with UC, whereby IL-9 expression is markedly increased in patients diagnosed with UC compared with healthy control participants and generally highest in patients with the more severe form of the disease (11). Based on these findings, we hypothesized that IL-9 inhibits the mucosal healing process, the subsequent exacerbation of inflammation and the formation of chronic lesions. In addition, IL-9 may mediate cell proliferation and serve catalytic role in CAC pathogenesis.

Inflammation is considered a cellular stressor that initiates DNA damage or genetic instability. Chronic inflammation may induce genetic abnormalities and epigenetic mechanisms, which can subsequently mediate malignant cell alteration. The nuclear transcription factor MYC proto-oncogene bHLH transcription factor (c-Myc) is part of the Myc gene family and has been reported to have multiple functionsas an important oncogene (12). Given that it is an essential transcription regulator, the activation of c-Myc may accelerate cell cycle progression and induce cell apoptosis (13). c-Myc is upregulated in various types of malignant tumor, including colorectal cancer, and is considered compulsory for the uncontrolled proliferation of cancer cells (14). According to whole-exome sequencing analysis, the c-Myc genomic locus has been reported to be frequently expressed in CAC compared with sporadic colorectal cancer (15). Cyclins were named following their periodic cell cycle-dependent pattern of expression. Notably, cyclin D1 is considered the most popular given its widespread role in human cancer with a larger depth of functional characterization. Cyclin D1 is recognized as a proto-oncogene, whose overexpression is able to cause cell proliferation to become malignant and uncontrolled (16). Studies have reported the overexpression of cyclin D1 gene in a variety of cancer types, including breast cancer, bladder cancer, parathyroid neoplasms, lymphoma, melanoma andlung cancer (17–22).

The present study aimed to examine the expression of IL-9 in CAC colorectal tissue specimens and the determine effect of IL-9 on cell proliferation. The results demonstrated that IL-9 was overexpressed in CAC tissues compared with adjacent tissues, and IL-9 overexpression promoted the growth of colonic epithelial cells by upregulating the expression of c-Myc and cyclinD1.

Materials and methods

Patients and tissue specimens

The present study was approved by the Research Ethics Committee of the First Hospital of Shanxi Medical University (Taiyuan, China). All patients agreed to participate and signed informed consent forms. The study used 12 (8 male and 4 female) colorectal tissue specimens from patients with CAC and tumor-adjacent tissues as a control. The median age of patients was 72.5 years (72.5±17.3 years) at the time of surgery. The specimens were collected from Department of Pathology at the First Hospital of Shanxi Medical University between June 2010 and December 2017. The diagnosis of CAC was founded on medical history, endoscopic findings, histological examination, laboratory tests and clinical disease presentation.

Immunohistochemistry

A total of 12 paired formalin-fixed, paraffin-embedded samples were subjected to immunohistochemistry staining for IL-9. Briefly, the cancer tissues were fixed in 4% paraformaldehyde at 4°C for 48 h. Following dehydration, the tissues were sliced at 4 µm thickness and blocked in 10% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 30 min, followed by incubating with rabbit anti-human IL-9 monoclonal antibody (cat. no. A1894; 1:50-1:200; ABclonal Biotech Co., Ltd., Woburn, MA, USA) at 4°C overnight. The slices were incubated with goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. SPN-9001; 1:100; OriGene Technologies, Inc., Rockville, MD, USA) at room temperature for 2 h. The SCANSCOP Digital Pathology Scanning system (Aperio Technologies, Inc., Vista, CA, USA) was used forscanning of pathological slices. Under alight microscope, five representative high magnification fields of vision were selected for each slice, and the number of IL-9-positive immunization cells was counted. The mean value represents the IL-9 positive degree of the case.

Construction of lentivirus overexpressing IL-9

The gene sequence of IL-9 was based on National Centre for Biotechnology Information (NM_000590.1, http://www.ncbi.nlm.nih.gov/nuccore/NM_000590.1), and the Basic Local Alignment Search Tool (BLAST) sequence was produced using DNAMAN software (version 6.0; Lynnon LLC, San Ramon, CA, USA). Overexpression lentiviral vector pGV367 and AgeI/NheI Restriction endonuclease were purchased from Shanghai GenechemCo., Ltd. (Shangahi, China). Human IL-9 was cloned from the human genome with the following primers: IL-9 forward, 5′-GAGGATCCCCGGGTACCGGTCGCCACCATGCTTCTGGCCATGGTCCTTAC-3′ and reverse, 5′-CACACATTCCACAGGCTAGTCATATCTTGCCTCTCATCCCTCTCATC-3′. The lentivirus vector plasmid pGV367 and the IL-9 gene sequence were digested by AgeI and NheI restriction enzymes. Recombinant lentiviral vector was detected by DNA sequencing. Following exact matching of the sequencing, pGV367-IL-9 was cotransfected with two assistant plasmids pHelp 1.0 and pHelp 2.0 into 293 cells (purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) to establish the recombinant lentivirus expressing IL-9 (LV-IL-9). LV-negative control (NC) contains empty gene sequence was used as lentivirus control group. All the vectors were purchased from Shanghai GeneChem Co., Ltd.

Cell culture and LV-IL-9 transfection

Colorectal cancer cell RKO and Caco2 cells are adherent cells, and purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Caco2 cells were cultured in Dulbecco's modified Eagle medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37°C with 5% CO2. RKO cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin/streptomycin (Life Technologies; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2. RKO and Caco2 cells were infected with LV-IL-9 with amultiplicity of infection (MOI) of 10 and 20 respectively at 80% confluence. Subsequently, the expression of enhanced green fluorescent protein (GFP) was observed by fluorescence microscope at a ×200 magnification (IX71; Olympus Corporation, Tokyo, Japan).

Cell proliferation assay

Cell proliferation was investigated and determined using Cell Counting Kit 8 (CCK8; cat. no. AR1160-500; Wuhan Boster Biological Technology, Ltd., Wuhan, China). RKO and Caco2 cells were seeded into 96-well plates at a density of 2×103 cells/well in 100 µl of culture medium, and infected with LV-IL-9 at a MOI of 20 or with LV-NC (2 µl) (n=6) for 0, 12, 24, 48 and 72 h in a 5% CO2 humidified incubator at 37°C. Subsequently, the cells were then incubated with 10 µl of CCK8/well for 30 min at 37°C. The optical density of each well was assessed at 450 nm using a microplate reader.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from RKO and Caco2 cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA was reverse transcribed the using Advantage® RT-for-PCR kit (cat. no. 639505; Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. Subsequently, the cDNA was subjected to RT-qPCR analysis. Amplification was performed as follows: Initial incubation at 95°C for 10 sec; followed by 40 cycles at 95°C for 5 sec and at 62°C for 45 sec; and extension at 72°C for 3 min. The 25 µl PCR reaction system included 1 µl cDNA temple, 2 µl primers, 2 µl dNTP, 0.5 µl RT/Platinum™ Taqmix (Invitrogen; Thermo Fisher Scientific, Inc.) and distilled water. RT-qPCR was performed using the T100-Thermal Cycler Real-Time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Gene expression was normalized to the levels of the internal reference GAPDH using the 2−ΔΔCq method (23). Each sample was assessed in triplicate and the mean expression level was determined. The following primers were used: c-Myc forward, 5′-TTCGGGTAGTGGAAAACCAG-3′ and reverse, 5′-AGCAGATCGAATTTCTTCCA-3′; cyclinD1 forward, 5′-GAGGAAGAGGAGGAGGAGGA-3′ and reverse, 5′-GAGATGGAAGGGGGAAAGAG-3′; B cell lymphoma-2 (Bcl-2) forward, GGATGCCTTTGTGGAACTGT-3′ and reverse, 5′-AGCCTGCAGCTTTGTTTCAT-3′; Bcl-2 associated X protein (Bcl-xL) forward, 5′-TCTGGTCCCTTGCAGCTAGT-3′ and reverse, 5′-ATTCTGAGGCCAAGGGAACT-3′; surviving forward, 5′-ACCTGAAAGCTTCCTCGAGA-3′ and reverse, 5′-AACCCTTCCCAGACTCCACT-3′; induced myeloid leukemia cell differentiation protein Mcl-1 homolog (Mcl-1) forward, 5′-TGCTGGAGTAGGAGCTGGTT-3′ and reverse, 5′-CCTCTTGCCACTTGCTTTTC-3′; GAPDH forward, 5′-CCACCTTCGATGCCGGGGCT-3′ and reverse, 5′-GGGGCCGAGTTGGGATAGGG-3′.

Western blot analysis

The colorectal tissue specimens from patients with CAC and tumor-adjacent tissues were pulverized and lysed with RIPA buffer (Beyotime Institute of Biotechnology, Beijing, China) containing proteinase inhibitor cocktail (cat. no. P8340; Sigma-Aldrich; Merck KGaA). Protein concentration was measured using a bicinchoninic acid protein assay kit. A total of 60 µg protein were subjected to 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Subsequently, the membranes were blocked in 5% fat-free milk for 2 h at room temperature and incubated with the following primary antibodies overnight at 4°C: Rabbit polyclonal anti-IL9 (1:500; cat. no. ab111915; Abcam, Cambridge, UK) and rabbit monoclonal anti-GAPDH (1:500; cat. no. AF1186; Beyotime Institute of Biotechnology). Following incubation with the HRP-conjugated secondary antibodies from Santa Cruz Biotechnology, Inc. (1:2,000; cat. no. sc-2004, goat anti-rabbit IgG-HRP; cat. no. sc-2005) for 2 h at room temperature, the membranes were visualized using the ECLPlus kit (GE Healthcare, Chicago, IL, USA).

Enzyme-linked immunosorbent assay (ELISA)

Concentrations of IL-9 were detected in the culture medium of RKO cells and Caco-2 cells using solid phase sandwich ELISA according to the manufacturer's protocols of ELISA kit (cat. no. BMS208196T, eBioscience; Thermo Fisher Scientific, Inc.). The hormone assay sensitivity was set at 0.1 pg/ml and the assay range was 3.1–200 pg/ml. For statistical analysis, the culture medium was independently collected three times. The ELISA kit for IL-9 was purchased from eBioscience (Thermo Fisher Scientific, Inc.).

Statistical analysis

All group data are presented as the mean ± standard deviation. Comparisons among the three groups were performed using one-way analysis of variance, using the Fisher's least significant difference test for multiple comparisons. The Student's t-test was applied to analyze the differences between two groups. P<0.05 was considered to indicate a statistically significant difference. All statisticalanalys is was performed using GraphPad Prism 5.0 software (GraphPadSoftware, Inc., La Jolla, CA, USA).

Results

IL-9 expression is upregulated in CAC tissues

A total of 12 patients with CAC were enrolled for the present study, including eight males (66.7%) and four females (33.3%), with a mean age of 72.5±17.3 years, and a history of ulcerative colitis (lasting 25.8±3.8 years). The basic clinical information of the patients was obtained by consulting their disease history.

While IL-9 involvement in atopic diseases has been previously established (24), its expression in patients with CAC remains to be elucidated. To investigate the potential role of IL-9 in CAC, IL-9 expression levels were determined. Among the 12 cases of CAC, all had IL-9 expression in the intestinal mucosal epithelial cells and inflammatory cell cytoplasm (Fig. 1A). It was demonstrated that the number of IL-9-positive cells in the intestinal mucosa was significantly increased compared with that in paracancerous tissues (Fig. 1B; P<0.001). Furthermore, the western blot analysis results demonstrated that IL-9 protein expression was significantly increased in CAC tissues (Fig. 1C; P<0.001).

Figure 1. IL-9 is overexpressed in colonic biopsy specimens from patients with CAC. (A and B) Expression of IL-9 was examined by immunohistochemical staining of IL-9 in (A) CAC and (B) tumor-adjacent tissues. The number of IL-9-positive cells in the intestinal mucosa was significantly increased compared with that in paracancerous tissue. ***P<0.001. (C) Representative western blot and quantitative data demonstrating IL-9 expression in CAC and adjacent tissues. ***P<0.001. IL-9, interleukin-9; CAC, colitis-associated cancer.

Identification of IL-9 gene overexpression usinglentivirus vector

The BLAST results for the IL-9 cDNA sequence determined that the expected IL-9 gene sequence was completely consistent. It was confirmed that the IL-9 was correctly inserted into the vector and that the IL-9 overexpression lentivirus vector was successfully constructed. LV-IL-9-infected 293 cells were detected by fluorescence microscopy through examining the level of GFP-positive cells (Fig. 2A and B).

Figure 2. Lentiviral vector-interleukin-9 efficiently infects 293 cells. Infection efficiency as determined by (A) light and (B) fluorescence microscopy at 72 h postinfection in 293 cells. Scale bar, 50 µm.

LV-IL-9 effectively infected colonic epithelial cells

GFP was observed in RKO and Caco2 cells under a fluorescence microscope indicating that the lentivirus has been successfully transfected (Fig. 3A). The RT-qPCR results demonstrated that IL-9 gene expression in the Caco-2 overexpression (OE) group was 12,508.689 times that of the NCgroup (Fig. 3B; P<0.001). Furthermore, IL-9 gene expression in the RKO OE group was 5,037.700 times that of the NC group (Fig. 3B; P<0.001). IL-9 protein expression was 141.61±12.28 pg/ml in the transfected Caco-2 cell medium as determined by ELISA, whereas IL-9 expression was 25.79±5.22 pg/ml in the untransfected group (Fig. 3C). IL-9 protein level was 106.65±17.45 pg/ml in the transfected RKO medium and 35.23±4.45 pg/ml in untransfected RKO cells (Fig. 3C). The IL-9 protein expression levels were significant increased in the two OE groups compared with their untransfected counterparts (P<0.05).

Figure 3. IL9 overexpression in RKO and Caco2 cells. (A) Infection efficiency as determined by light and fluorescence microscopy at 72 h postinfection in RKO and Caco2 cells. Scale bar, 50 µm. (B) IL-9 mRNA level in RKO and Caco2 cells following overexpression of IL-9. (C) The level of IL-9 protein in the cell culture supernatant following overexpression of IL-9 as determined by ELISA. The level of IL-9 protein was significantly increased following overexpression of IL-9. Data are presented as the mean values ± standard deviation. *P<0.05 and ***P<0.001. IL-9, interleukin-9.

IL-9 overexpression promotes cell proliferation

The CCK8 assay demonstrated that IL-9 overexpression induced a significant increase in the proliferation rates of Caco-2 and RKO cells compared with that of the corresponding NC groups (Fig. 4A and B).

Figure 4. IL-9 promotes RKO and Caco2 cell growth. (A and B) Compared with control group and LV-NC group, overexpression of IL-9 significantly promoted the proliferation of (A) RKO and (B) Caco2 cells. *P<0.05 compared with the control and LV-NC groups. IL-9, interleukin-9; CCK-8, Cell Counting Kit-8; LV, lentiviral vector; NC, negative control.

IL-9 overexpression upregulates c-Myc and cyclinD1 mRNA expression

As presented in Fig. 5A and B, compared with the control and LV-NC groups, the relative levels of c-Myc and cyclinD1 mRNA were demonstrated to be significantly increased in Caco-2 and RKO cells infected with LV-IL-9. However, no significant differences in the expression levels of Bcl-2, Bcl-xL protein, surviving or Mcl-1 were observed following IL-9 OE (Fig. 5C-F).

Figure 5. IL-9 upregulates the expression of c-Myc and cyclinD1. (A and B) A significantincrease in the relative expression of (A) c-Myc and (B) cyclin D1 mRNA was observed in the LV-IL-9 group compared with control and LV-NC groups. (C-F) No significant difference was observed in the expression of (C) Bcl-2, (D) Bcl-xL, (E) surviving or (F) Mcl-1 between the LV-IL-9 and LV-NC groups. **P<0.01 and ***P<0.001. c-Myc, MYC proto-oncogene bHLH transcription factor; Bcl-2, B cell lymphoma-2; Bcl-xL, Bcl-2-associated X protein; Mcl-1, Induced myeloid leukemia cell differentiation protein Mcl-1 homolog; IL-9, interleukin-9; LV, lentiviral vector; NC, negative control.

Discussion

IL-9 is considered as a pleiotropic cytokine that mediates the growth of several types of cells. The role of IL-9 in tumor immunity is controversial, and its involvement in disease remains unclear. Research has confirmed that the OE of IL-9 is associated with the progression of various forms of lymphoma and leukemia (25,26). However, the expression of IL-9 in CAC and its clinical importance remain unclear.

In the current study, the expression of IL-9 in cancer and adjacent colonic mucosa specimens were collected from patients with CAC between 2010 and 2017, and was analyzed using immunohistochemistry. The results confirmed that IL-9 is mainly expressed in intestinal mucosal epithelial cells and inflammatory cell cytoplasm. The expression of IL-9 in cancer tissues was markedly higher than that in adjacent tissues, which suggests that IL-9 may play an important role in the pathogenesis of CAC. Based on these results, we presume that the expression level of IL-9 fluctuates between cancer types. The objective of our study was to analyze the colonic biopsy specimens from patients with CAC and examine how the expression of IL-9 may relate to the proinflammatory effect of IL-9 in the pathogenesis of UC (27).

To investigate the mechanism of IL-9 function in the pathogenesis of CAC, we constructed lentivirus expressing IL-9 and infected RKO and Caco2 cells to study its role on cell proliferation. The lentiviral vector overexpressing IL-9 gene was successfully constructed and the lentivirus successfully infected RKO and Caco2 cells. IL-9 overexpression could promote the proliferation of colonic epithelial cells by upregulating of the expression of c-Myc and cyclinD1. In alung cancer study, Ye et al (28) demonstrated that the proliferation rate of tumor cells increases with the IL-9 intervention, suggesting that IL-9 promotes the growth of lung cancer cells. The present study also showed that the proliferation of tumor cells increased with the intervention of IL-9, suggesting that IL-9 promotes the growth of colon cancer cells, similar to the findings in lung cancer.

The c-Myc gene is typically downregulated in inflammation, and overexpressed in sporadic and colitis-associated colon adenocarcinomas. It regulates cell proliferation or differentiation, and its overexpression is able to induce cell transformation and tumor formation (29). CyclinD1, as one of the cell cycle regulatory proteins, is overexpressed in several human tumor types, including non-small cell lung cancer (30),esophageal cancer (31) and head and neck cancer (32). The observations in the present study provided evidence that IL-9 overexpression may upregulate the expression of c-Myc and cyclinD1 in RKO and Caco2 cells as described above. This evidence is in accordance with the results of a previous study (13).

In conclusion, the results of the present study demonstrated that IL-9 promoted the proliferation of RKO and Caco2 colonic epithelial cells, partially via the upregulation of c-Myc and cyclinD1 expression. The results of this study provide an important experimental basis for further study of the role of IL-9 in CAC.

Acknowledgements

Not applicable.

Funding

The present study was supported by research grants from the National Clinical Research Center for Digestive Diseases (Xi an, China; grant no. 2015BAI13B07).

Availability of data and materials

The datasets generated/analyzed in the present study are available on reasonable request from the corresponding author.

Authors' contributions

LH conceived the study, performed the experimental design and data interpretation, and prepared and revised the manuscript. LT performed the majority of the experiments. YL and RC performed the immunohistochemical assay. PZ and JZ performed the quantitative polymerase chain reaction assay.

Ethics approval and consent to participate

The present study was approved by the Research Ethics Committee of the First Hospital of Shanxi Medical University (Taiyuan, China). All patients agreed to participate and signed informed consent forms.

Patient consent for publication

The study participants provided consent for the publication of any associated data/images.

Competing interests

All authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References