- Email: [email protected]
Overexpression of miRNA-143 Inhibits Colon Cancer Cell Proliferation by Inhibiting Glucose Uptake
Archives of Medical Research
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(2019)
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BRIEF REPORT
Overexpression of miRNA-143 Inhibits Colon Cancer Cell Proliferation by Inhibiting Glucose Uptake Jian Zhao, Yuze Chen, Fang Liu, and Mingdi Yin Department of Colorectal Surgery, Cancer Hospital of China Medical University; Liaoning Cancer Hospital and Institute, Xiaoheyan Road, Dadong District, Shenyang City, Liaoning Province, P R China Received for publication June 30, 2018; accepted December 13, 2018 (ARCMED_2018_186).
MiRNA-143 overexpression is related to upregulated blood glucose level in diabetic mice, and downregulated miRNA-143 has been observed in several types of cancers, indicating its role as a tumor suppression miRNA. Glucose metabolism plays pivotal roles in tumor growth. Therefore, miRNA-143 may target glucose metabolism to inhibit tumor growth. Up to now, the functionality of miRNA-143 in colon cancer is still largely unknown. Our study was carried out to investigate the role of miRNA-143 in colon cancer and to explore the interactions between miRNA-143 and glucose uptake pathway. In this study we observed that miRNA-143 was downregulated in both colon biopsies (tumor tissues for colon cancer patients) and whole blood of colon patients than in healthy controls. Downregulation of miRNA-143 effectively distinguished colon cancer patients from healthy controls. Expression levels of miRNA-143 were found to be significantly correlated with tumor size but not distant tumor metastasis. MiRNA-143 overexpression inhibited glucose uptake and glucose transporter 1 (GLUT1) expression in colon cancer cells. MiRNA-143 overexpression also inhibited colon cancer cell proliferation. We therefore concluded that overexpression of miRNA-143 inhibited colon cancer cell proliferation by inhibiting glucose uptake. Ó 2018 IMSS. Published by Elsevier Inc. Key Words: Colon cancer, miRNA-143, Glucose uptake, Glucose transporter 1.
Introduction Colon cancer, or colorectal cancer, is a type of cancer that develops from large bowel. Incidence of colon cancer ranks the 2nd place among all cancers in females and the 3rd place among cancers in males (1). Colon cancer affects more than 130,000 new cases and causes about 60,000 cancer-related deaths worldwide every year (2,3). With proper treatment, the overall 5-year survival of patients with colon cancer is generally high, while the further improvement of treatment outcomes of colon cancer is limited by the complex unknown pathogenesis (4,5). Tumor growth is the basis of cancer development and progression. Tumor growth inhibition now is considered
as an important therapeutic target for cancer treatment (6). Glucose metabolism provides energy for proliferation of both normal cells and cancer cells and abnormally accelerated glucose metabolism distinguish cancer cells from normal tissue cells (7). How to inhibit glucose metabolism is a major task for the clinical treatment of different types of cancers (8). Downregulation of miRNA-143 has been observed in many types of cancers including colon cancer (9). Recent studies revealed that miRNA-143 is an indirect regulator of blood glucose metabolism (10) and cancer glycolysis (11). In this study we observed that miRNA143 may inhibit colon cancer cell proliferation by inhibiting glucose uptake.
Materials and Methods Address reprint requests to: Mingdi Yin, Department of Colorectal Surgery, Cancer Hospital of China Medical University; Liaoning Cancer Hospital and Institute, No. 44 Xiaoheyan Road, Dadong District, Shenyang City, Liaoning Province, 110042, P R China; Phone: (0086) 02431916243; FAX: (0086) 024-24315679; E-mail: [email protected]
Patients and Specimens A total of 78 patients with colon cancer were selected in Cancer Hospital of China Medical University from January
0188-4409/$ - see front matter. Copyright Ó 2018 IMSS. Published by Elsevier Inc. https://doi.org/10.1016/j.arcmed.2018.12.009
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2015eJanuary 2018. Inclusion criteria: a) patients diagnosed as colon cancer; b) patients received treatment for the first time; c) patients willing to participate. Exclusion criteria: a) patients complicated with other malignancies; b) patients received treatment before admission; c) patients older than 70 years. The 78 patients included 42 males and 36 females, and age ranged from 31e69 years, with a mean age of 49.6 7.1 years. Tumor biopsies and blood (elbow vein) were extracted from all patients. At the same time, colon biopsies and blood were also extracted from 52 healthy volunteers to serve as control group. There were 27 males and 25 females in control group, and age ranged from 29e68 years, with a mean age of 49.1 6.6 years. Age and gender distributions were not significantly different between 2 groups. Ethics committee of Cancer Hospital of China Medical University approved this study, and all participants signed informed consent. Real-time Quantitative PCR To detect the expression of miRNA-143, all miRNA extractions were performed using miRNeasy Kit (Qiagen) with all operations being performed in strict accordance with manufacturer’s instructions. miScript II RT Kit (Qiagen) was used to synthesize cDNA. MiRNA expression was assayed using miScript SYBR Green PCR Kit (Qiagen) with RNU6 (miRNA) as an endogenous control. To detect the expression of GLUT1 mRNA, Trizol reagent (Invitrogen, USA) was used to extract total RNA, Applied BiosystemsÔ High-Capacity cDNA Reverse Transcription Kit was used to perform reverse transcriptio, and SYBRÒ Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA) was used to prepare PCR reaction systems with GAPDH was a endogenous control. Cell Lines, Cell Culture and Transfection Colon carcinoma cell line RKO and normal colon cell line CCD-1Co were purchased from ATCC. Cells were cultured in ATCC-formulated Eagle’s Minimum Essential Medium (Catalog No. 30-2003) containing 10% fetal bovine serum at 37 C in a 5% CO2 incubator. Hsa-miR-143 mimic and negative control siRNA were purchased from SigmaAldrich. LipofectamineÔ 2000 was used to transfect 50 nM siRNA into 5 105 cells. Cells without transfected were used as control cells. Cells transfected with negative control siRNA were used as negative control cells.
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with 2 ml of Krebs-Ringer-HEPES (KRH) buffer (1.2 mM MgSO4, 5 mM KCl, 120 mM NaCl, 25 mM Hepes, pH 7.4, 1.3 mM CaCl2 1.3 mM KH2PO4). Then 1 mCi of (3H)-2deoxyglucose (Perkin Elmer Life Sciences) was added and cells were incubated at 37 C for 20 min (glucose uptake reduced significantly after 20 min). Cells were then washed with pre-cold KRH buffer to stop glucose uptake. Radioactivity was measured by liquid scintillation spectrometry after cell lysis using lysis buffer (0.2% SDS, 10 mM TriseHCl, pH 8.0). Intracellular level of (3H)-2-deoxyglucose was represented by disintegrations per minute (DPM). Cell Proliferation Assay Upregulation of miRNA-143 was confirmed by qRT-PCR after transfection and an overexpression rate of miRNA143 above 200% of that of control cells was reached before each subsequent experiment. Cells were collected and cell suspensions with a cell density of 5 104 cell/ml were prepared. Each well of a 96 well plate was filled with 0.1 ml cell suspension. Cells were cultured at 37 C, and CCK-8 solution (10 uL) was then added into each well 24, 48, 72 and 96 h later. A FisherbrandÔ accuSkanÔ GO UV/Vis Microplate Spectrophotometer (Fisher Scientific) was used to measure OD values (450 nm) to represent cell proliferation rate. Western-blot RIPA solution (Thermo Fisher Scientific, USA) was used for all total protein extractions with all operations performed in strict accordance with manufacturer’s instructions. Protein concentration was measured by bicinchoninic acid (BCA) assay, followed by 10 % SDS-PAGE gel electrophoresis with 40 mg protein in each well. Gel transfer to PVDF membrane was performed. Blocking was performed by incubating membranes with 5% skimmed milk for 1.5 h at room temperature. Then rabbit anti-human primary antibodies of GLUT1 (1: 2000, ab15309, Abcam) and GAPDH (1: 1000, ab8245, Abcam) were then used to incubate with membranes overnight at 4 C. After that, goat anti-rabbit IgG-HRP secondary antibody (1:1000, MBS435036, MyBioSource) was used to incubate with membranes at room temperature for 1h. ECLÔ Select Western Blotting Detection Reagent (Sigma-Aldrich, USA) was added to develop signals. Signals were normalized to GAPDH endogenous control using Image J software.
Glucose Uptake Assay Upregulation of miRNA-143 was confirmed by qRT-PCR after transfection and an expression of miRNA-143 above 200% of that of control cells was reached before each subsequent experiment. Cells were collected and cell suspensions with a cell density of 5 104 cell/mL were prepared. Cell suspension was incubated at 37 C in a 5% CO2 incubator for 24 h. After washing with PBS, 5 105 cells were mixed
Statistical Analysis Data processes were performed using Graphpad Prism 6 software. Measurement data were expressed as mean standard deviation and were compared by unpaired t test (between two groups) and one-way analysis of variance followed by LSD test (among multiple groups). Count data were compared by Chi-square test. Diagnostic values of miRNA-134 for colon
MiRNA-143 Inhibits Colon Cancer
cancer were evaluated by ROC curve analysis. p !0.05 was considered to be statistically significant.
Results MiRNA-143 was Downregulated in Colon Cancer Patient than in Healthy Controls Differential expression of miRNA-143 in cancer tissues and healthy tissues has been observed in several types of malignancies. In this study, expression of miRNA-143 in colon biopsies and blood of both colon cancer patients and healthy controls was detected by qRT-PCR. As shown in Figure 1A, expression of miRNA-143 in colon tissues was significantly downregulated in colon cancer patients than in healthy controls ( p !0.05). In addition, blood levels of miRNA-143 were also significantly lower in colon cancer patients than in healthy controls (Figure 1B, p ! 0.05). In contrast, expression of GLUT-1 was significantly upregulated in colon cancer patients than in healthy controls in both in colon tissues (Figure 1C, p !0.05) and blood (Figure 1D, p !0.05). Downregulation of miRNA-143 Effectively Distinguished Colon Cancer Patients from Healthy Controls ROC curve analysis was performed to evaluate the diagnostic values of miRNA-143 expression in colon biopsies and blood for colon cancer. AUC bigger that 0.65 is
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considered with diagnostic value. For miRNA-143 expression in colon biopsies, the area under the curve (AUC) was 0.9026, with error standard of 0.02708 and 95% confidence interval of 0.8495-0.9557 (Figure 2A). For miRNA143 expression in blood, AUC was 0.8189, with error standard of 0.03846 and 95% confidence interval of 0.7425-0.8933 (Figure 2B). MiRNA-143 Expression was Significantly Correlated with Tumor Size and Distant Tumor Metastasis Correlations between miRNA-143 expression and clinicopathological data of patients with colon cancer were analyzed by Chi-square test. As shown Table 1, miRNA-143 expression in colon biopsies was significantly correlated with tumor size but not age, gender and existing of distant tumor metastasis. Similarly, a significant correlation was also found between blood level of miRNA-143 and tumor size (Table 2). Overexpression of miRNA-143 Inhibited Glucose Uptake and Downregulated GLUT-1 Expression in Colon Cancer Cells Before transfection, miRNA-143 level was significantly higher in cells of colon carcinoma cell line RKO than in cells of the normal colon cell line CCD-1Co (Figure 3A, p !0.05). After transfection, miRNA-143 overexpression was reached in cells of both cell lines (Figure 3A, p ! 0.05). Glucose uptake assay was performed to measure glucose uptake in colon cancer cells after miRNA-143
Figure 1. MiRNA-143 was downregulated in colon cancer patients than in healthy controls. This figure shows the comparison of miRNA-143 expression in colon biopsies (A) and blood (B) between colon cancer patients and healthy control, and the comparison of GLUT-1 mRNA expression in colon biopsies (A) and blood (B) between colon cancer patients and healthy control. Expression of GLUT-1 was significantly upregulated in colon cancer patients than in healthy controls in both in colon tissues (C; p ! 0.05) and blood (D; p ! 0.05).
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Figure 2. Downregulation of miRNA-143 effectively distinguished colon cancer patients from healthy controls. This figure shows the ROC curve analysis of the miRNA-143 expression in colon biopsies (A) and blood (B) for colon cancer.
MiRNA-143 showed downregulated expression and plays a tumor suppression role in different types of cancers
including colon cancer (12,13). It has been reported that miRNA-143 is also key regulator of blood glucose transportation (10) and cancer glycolysis (11). However, crosstalk between miRNA-143 and glucose metabolism in colon cancer is unknown. The key finding of our study is that miRNA-143 may inhibit the proliferation of colon cancer cells by reducing glucose uptake through the downregulation of GLUT-1. The development and progression of colon cancer is a complex process with multiple internal and external factors involved. It is generally believed that genetic factors play pivotal roles in colon cancer (14). In recent year, several tumor suppression genes, such as TET-1 (15), and oncogenes, such as IGFBP7 (16), have been identified in the pathogenesis of colon cancer. Besides messenger RNAs that encode protein products, human genome also transcribes a large set of non-coding RNAs that have critical functions in nearly all aspects of tumor biology (17). In our study we observed than miRNA-143 expression was significantly downregulated in colon cancer patients than in healthy controls in both colon biopsies and blood. Our data were consistent with the findings reported in previous studies (9,13). In addition, we also proved that downregulated expression of miRNA-143 effectively distinguished patients with colon cancer from healthy controls. Previous studies and the findings in our study
Table 1. Correlations between miRNA-143 expression in colon biopsies and clinicopathological data of patients with colon cancer
Table 2. Correlations between miRNA-143 expression in blood and clinicopathological data of patients with colon cancer
overexpression. As shown in Figure 3B, compared with control cells (C) and negative control cells (NC), glucose uptake was significantly decreased after miRNA-143 overexpression in cells of colon carcinoma cell line RKO but not in cells of the normal colon cell line CCD-1Co ( p ! 0.05). GLUT-1 plays pivotal roles in glucose translocation. Compared with control cells (C) and negative control cells (NC), GLUT-1 expression was significantly decreased after miRNA-143 overexpression in cells of colon carcinoma cell line RKO but not in cells of the normal colon cell line CCD-1Co ( p !0.05, Figure 3C). Overexpression of miRNA-143 Inhibited Proliferation Colon Cancer Cells CCK-8 assay was performed to measure cell proliferation rate after miRNA-143 overexpression. As shown in Figure 4, compared with control cells (C) and negative control cells (NC), miRNA-143 overexpression significantly inhibited proliferation of cells of colon carcinoma cell line RKO but not cells of the normal colon cell line CCD-1Co ( p !0.05).
Discussion
Items
Groups
O50 (years) !50 (years) Gender Male Female Primary tumor O2 cm diameter !2 cm Tumor distant Yes metastasis No Age
HighLowCases expression expression 34 44 42 36 49 29 33 45
18 21 18 21 19 20 15 24
16 23 24 15 30 9 18 21
c2
p
0.21 0.65 1.86 0.17 6.64 0.01 0.47 0.49
Items
Groups
O50 (years) !50 (years) Gender Male Female Primary tumor O2 cm diameter !2 cm Tumor distant Yes metastasis No Age
HighLowCases expression expression 34 44 42 36 49 29 33 45
16 23 19 20 20 19 14 25
18 21 23 16 29 10 19 20
c2
p
0.21 0.65 0.83 0.36 4.46 0.03 1.31 0.25
MiRNA-143 Inhibits Colon Cancer
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Figure 3. Overexpression of miRNA-143 inhibited glucose uptake and downregulated GLUT-1 expression in colon cancer cells. Before transfection, miRNA143 level was significantly higher in cells of colon carcinoma cell line RKO than in cells of the normal colon cell line CCD-1Co After transfection, miRNA143 overexpression was reached in cells of both cell lines (A). This figure also shows the effects of miRNA-143 overexpression on glucose uptake (B) and GLUT-1 expression (C) in cells of colon carcinoma cell line RKO and normal colon cell line CCD-1Co. *p !0.05.
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Figure 4. Overexpression of miRNA-143 inhibited proliferation colon cancer cells. miRNA-143 overexpression significantly inhibited proliferation of cells of colon carcinoma cell line RKO (A) but not cells of the normal colon cell line CCD-1Co (B) (p ! 0.05).
both revealed that downregulation of miRNA-143 is likely involved in the pathogenesis of colon cancer and low miRNA-143 expression level may has potential diagnostic value for this disease. In our study we observed that miRNA-143 is significantly correlated with tumor size but not distant tumor metastasis, indicating the potential involvement of miRNA-143 in the regulation of tumor growth but not tumor metastasis in colon cancer. Our in vitro cell proliferation experiments also proved that overexpression of miRNA-143 caused accelerated proliferation of colon cancer cells. Accelerated glucose metabolism is common in different types of cancers and is required for the abnormal growth of tumors. Overexpression of glucose-regulated proteins has been observed in patients with colon cancer (18). In addition, the significant correlation between highglucose diet and increased risk of colon cancer has also been reported (19). MiRNAs are key regulator of glucose metabolism in different cancers including colon cancer (20). In our study, we proved than miRNA-143 is a negative regulator of glucose uptake in colon cancer cells, and the inhibitory role of miRNA-143 on glucose uptake is likely achieved through the downregulation of GLUT-1, which is critical for glucose translocation (21). Therefore, miRNA-143 may inhibit tumor growth in colon cancer by inhibiting tumor growth through the downregulation of miRNA-143. Our study also revealed a miRNA-143- GLUT-1 signaling in glucose uptake in colon cancer. Whether the interaction between miRNA-143 and GLUT-1 is direct or indirect is unknown. In addition, the regulatory role of miRNA-153 on glucose uptake and GLUT-1 expression was not observed in normal colon cancer cells. Therefore, the regulatory role of miRNA-153 on glucose uptake and GLUT-1 expression is likely mediated by disease or colon cancer related factors. Our future studies will focus on the identification of those factors. In conclusion, our study further confirmed the downregulation of miRNA-143 in colon cancer. Our study
identified the involvement of miRNA-143 in colon cancer, which is achieved through the inhibited glucose uptake and GLUT-1 expression.
Acknowledgments We thank the financial support from Shenyang Technology and Science Project in 2018 (18-014-4-69).
References 1. Marisa L, de Reynies A, Duval A, et al. Gene expression classification of colon cancer into molecular subtypes: characterization, validation, and prognostic value. PLoS Med 2013;10:e1001453. 2. Siegel R, DeSantis C, Jemal A. Colorectal cancer statistics. CA Cancer J Clin 2014;64:104e117. 3. Siegel RL, Miller KD, Fedewa SA, et al. Colorectal cancer statistics. CA Cancer J Clin 2017;67:177e193. 4. Liang B, Shahbaz M, Wang Y, et al. Integrinb6-targeted immunoliposomes mediate tumor-specific drug delivery and enhance therapeutic efficacy in colon carcinoma. Clin Cancer Res 2015;21:1183e1195. 5. Mannucci S, Ghin L, Conti G, et al. Magnetic nanoparticles from Magnetospirillum gryphiswaldense increase the efficacy of thermotherapy in a model of colon carcinoma. PLoS One 2014;9:e108959. 6. Tsuru H, Shibaguchi H, Kuroki M, et al. Tumor growth inhibition by sonodynamic therapy using a novel sonosensitizer. Free Radic Biol Med 2012;53:464e472. 7. DeBerardinis RJ, Sayed N, Ditsworth D, et al. Brick by brick: metabolism and tumor cell growth. Curr Opin Genet Dev 2008;18:54e61. 8. Hamanaka RB, Chandel NS. Targeting glucose metabolism for cancer therapy. J Exp Med 2012;209:211e215. 9. Pichler M, Winter E, Stotz M, et al. Down-regulation of KRASinteracting miRNA-143 predicts poor prognosis but not response to EGFR-targeted agents in colorectal cancer. Br J Cancer 2012;106: 1826e1832. 10. Jordan SD, Kr€uger M, Willmes DM, et al. Obesity-induced overexpression of miRNA-143 inhibits insulin-stimulated AKT activation and impairs glucose metabolism. Nat Cell Biol 2011;13:434. 11. Fang R, Xiao T, Fang Z, et al. MicroRNA-143 (miR-143) regulates cancer glycolysis via targeting hexokinase 2 gene. J Biol Chem 2012;287:23227e23235. 12. Ng EKO, Li R, Shin VY, et al. MicroRNA-143 is downregulated in breast cancer and regulates DNA methyltransferases 3A in breast cancer cells. Tumor Biol 2014;35:2591e2598.
MiRNA-143 Inhibits Colon Cancer 13. Chen J, Wang MB. The roles of miRNA-143 in colon cancer and therapeutic implications. Transl Gastrointest Cancer 2012;1:169e174. 14. Ryan BM, Wolff RK, Valeri N, et al. An analysis of genetic factors related to risk of inflammatory bowel disease and colon cancer. Cancer Epidemiol 2014;38:583e590. 15. Neri F, Dettori D, Incarnato D, et al. TET1 is a tumour suppressor that inhibits colon cancer growth by derepressing inhibitors of the WNT pathway. Oncogene 2015;34:4168e4176. 16. Rupp C, Scherzer M, Rudisch A, et al. IGFBP7, a novel tumor stroma marker, with growth-promoting effects in colon cancer through a paracrine tumorestroma interaction. Oncogene 2015;34:815e825. 17. Esteller M. Non-coding RNAs in human disease. Nat Rev Genet 2011; 12:861e874.
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18. Xing X, Lai M, Wang Y, et al. Overexpression of glucoseregulated protein 78 in colon cancer. Clin Chim Acta 2006;364: 308e315. 19. Bruce WR, Giacca A, Medline A. Possible mechanisms relating diet and risk of colon cancer. Cancer Epidemiol Biomarkers Prev 2000; 9:1271e1279. 20. Chen B, Liu Y, Jin X, et al. MicroRNA-26a regulates glucose metabolism by direct targeting PDHX in colorectal cancer cells. BMC Cancer 2014;14:443. 21. Freemerman AJ, Johnson AR, Sacks GN, et al. Metabolic reprogramming of macrophages glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype. J Biol Chem 2014;289:7884e7896.
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BRIEF REPORT
Overexpression of miRNA-143 Inhibits Colon Cancer Cell Proliferation by Inhibiting Glucose Uptake Jian Zhao, Yuze Chen, Fang Liu, and Mingdi Yin Department of Colorectal Surgery, Cancer Hospital of China Medical University; Liaoning Cancer Hospital and Institute, Xiaoheyan Road, Dadong District, Shenyang City, Liaoning Province, P R China Received for publication June 30, 2018; accepted December 13, 2018 (ARCMED_2018_186).
MiRNA-143 overexpression is related to upregulated blood glucose level in diabetic mice, and downregulated miRNA-143 has been observed in several types of cancers, indicating its role as a tumor suppression miRNA. Glucose metabolism plays pivotal roles in tumor growth. Therefore, miRNA-143 may target glucose metabolism to inhibit tumor growth. Up to now, the functionality of miRNA-143 in colon cancer is still largely unknown. Our study was carried out to investigate the role of miRNA-143 in colon cancer and to explore the interactions between miRNA-143 and glucose uptake pathway. In this study we observed that miRNA-143 was downregulated in both colon biopsies (tumor tissues for colon cancer patients) and whole blood of colon patients than in healthy controls. Downregulation of miRNA-143 effectively distinguished colon cancer patients from healthy controls. Expression levels of miRNA-143 were found to be significantly correlated with tumor size but not distant tumor metastasis. MiRNA-143 overexpression inhibited glucose uptake and glucose transporter 1 (GLUT1) expression in colon cancer cells. MiRNA-143 overexpression also inhibited colon cancer cell proliferation. We therefore concluded that overexpression of miRNA-143 inhibited colon cancer cell proliferation by inhibiting glucose uptake. Ó 2018 IMSS. Published by Elsevier Inc. Key Words: Colon cancer, miRNA-143, Glucose uptake, Glucose transporter 1.
Introduction Colon cancer, or colorectal cancer, is a type of cancer that develops from large bowel. Incidence of colon cancer ranks the 2nd place among all cancers in females and the 3rd place among cancers in males (1). Colon cancer affects more than 130,000 new cases and causes about 60,000 cancer-related deaths worldwide every year (2,3). With proper treatment, the overall 5-year survival of patients with colon cancer is generally high, while the further improvement of treatment outcomes of colon cancer is limited by the complex unknown pathogenesis (4,5). Tumor growth is the basis of cancer development and progression. Tumor growth inhibition now is considered
as an important therapeutic target for cancer treatment (6). Glucose metabolism provides energy for proliferation of both normal cells and cancer cells and abnormally accelerated glucose metabolism distinguish cancer cells from normal tissue cells (7). How to inhibit glucose metabolism is a major task for the clinical treatment of different types of cancers (8). Downregulation of miRNA-143 has been observed in many types of cancers including colon cancer (9). Recent studies revealed that miRNA-143 is an indirect regulator of blood glucose metabolism (10) and cancer glycolysis (11). In this study we observed that miRNA143 may inhibit colon cancer cell proliferation by inhibiting glucose uptake.
Materials and Methods Address reprint requests to: Mingdi Yin, Department of Colorectal Surgery, Cancer Hospital of China Medical University; Liaoning Cancer Hospital and Institute, No. 44 Xiaoheyan Road, Dadong District, Shenyang City, Liaoning Province, 110042, P R China; Phone: (0086) 02431916243; FAX: (0086) 024-24315679; E-mail: [email protected]
Patients and Specimens A total of 78 patients with colon cancer were selected in Cancer Hospital of China Medical University from January
0188-4409/$ - see front matter. Copyright Ó 2018 IMSS. Published by Elsevier Inc. https://doi.org/10.1016/j.arcmed.2018.12.009
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2015eJanuary 2018. Inclusion criteria: a) patients diagnosed as colon cancer; b) patients received treatment for the first time; c) patients willing to participate. Exclusion criteria: a) patients complicated with other malignancies; b) patients received treatment before admission; c) patients older than 70 years. The 78 patients included 42 males and 36 females, and age ranged from 31e69 years, with a mean age of 49.6 7.1 years. Tumor biopsies and blood (elbow vein) were extracted from all patients. At the same time, colon biopsies and blood were also extracted from 52 healthy volunteers to serve as control group. There were 27 males and 25 females in control group, and age ranged from 29e68 years, with a mean age of 49.1 6.6 years. Age and gender distributions were not significantly different between 2 groups. Ethics committee of Cancer Hospital of China Medical University approved this study, and all participants signed informed consent. Real-time Quantitative PCR To detect the expression of miRNA-143, all miRNA extractions were performed using miRNeasy Kit (Qiagen) with all operations being performed in strict accordance with manufacturer’s instructions. miScript II RT Kit (Qiagen) was used to synthesize cDNA. MiRNA expression was assayed using miScript SYBR Green PCR Kit (Qiagen) with RNU6 (miRNA) as an endogenous control. To detect the expression of GLUT1 mRNA, Trizol reagent (Invitrogen, USA) was used to extract total RNA, Applied BiosystemsÔ High-Capacity cDNA Reverse Transcription Kit was used to perform reverse transcriptio, and SYBRÒ Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA) was used to prepare PCR reaction systems with GAPDH was a endogenous control. Cell Lines, Cell Culture and Transfection Colon carcinoma cell line RKO and normal colon cell line CCD-1Co were purchased from ATCC. Cells were cultured in ATCC-formulated Eagle’s Minimum Essential Medium (Catalog No. 30-2003) containing 10% fetal bovine serum at 37 C in a 5% CO2 incubator. Hsa-miR-143 mimic and negative control siRNA were purchased from SigmaAldrich. LipofectamineÔ 2000 was used to transfect 50 nM siRNA into 5 105 cells. Cells without transfected were used as control cells. Cells transfected with negative control siRNA were used as negative control cells.
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with 2 ml of Krebs-Ringer-HEPES (KRH) buffer (1.2 mM MgSO4, 5 mM KCl, 120 mM NaCl, 25 mM Hepes, pH 7.4, 1.3 mM CaCl2 1.3 mM KH2PO4). Then 1 mCi of (3H)-2deoxyglucose (Perkin Elmer Life Sciences) was added and cells were incubated at 37 C for 20 min (glucose uptake reduced significantly after 20 min). Cells were then washed with pre-cold KRH buffer to stop glucose uptake. Radioactivity was measured by liquid scintillation spectrometry after cell lysis using lysis buffer (0.2% SDS, 10 mM TriseHCl, pH 8.0). Intracellular level of (3H)-2-deoxyglucose was represented by disintegrations per minute (DPM). Cell Proliferation Assay Upregulation of miRNA-143 was confirmed by qRT-PCR after transfection and an overexpression rate of miRNA143 above 200% of that of control cells was reached before each subsequent experiment. Cells were collected and cell suspensions with a cell density of 5 104 cell/ml were prepared. Each well of a 96 well plate was filled with 0.1 ml cell suspension. Cells were cultured at 37 C, and CCK-8 solution (10 uL) was then added into each well 24, 48, 72 and 96 h later. A FisherbrandÔ accuSkanÔ GO UV/Vis Microplate Spectrophotometer (Fisher Scientific) was used to measure OD values (450 nm) to represent cell proliferation rate. Western-blot RIPA solution (Thermo Fisher Scientific, USA) was used for all total protein extractions with all operations performed in strict accordance with manufacturer’s instructions. Protein concentration was measured by bicinchoninic acid (BCA) assay, followed by 10 % SDS-PAGE gel electrophoresis with 40 mg protein in each well. Gel transfer to PVDF membrane was performed. Blocking was performed by incubating membranes with 5% skimmed milk for 1.5 h at room temperature. Then rabbit anti-human primary antibodies of GLUT1 (1: 2000, ab15309, Abcam) and GAPDH (1: 1000, ab8245, Abcam) were then used to incubate with membranes overnight at 4 C. After that, goat anti-rabbit IgG-HRP secondary antibody (1:1000, MBS435036, MyBioSource) was used to incubate with membranes at room temperature for 1h. ECLÔ Select Western Blotting Detection Reagent (Sigma-Aldrich, USA) was added to develop signals. Signals were normalized to GAPDH endogenous control using Image J software.
Glucose Uptake Assay Upregulation of miRNA-143 was confirmed by qRT-PCR after transfection and an expression of miRNA-143 above 200% of that of control cells was reached before each subsequent experiment. Cells were collected and cell suspensions with a cell density of 5 104 cell/mL were prepared. Cell suspension was incubated at 37 C in a 5% CO2 incubator for 24 h. After washing with PBS, 5 105 cells were mixed
Statistical Analysis Data processes were performed using Graphpad Prism 6 software. Measurement data were expressed as mean standard deviation and were compared by unpaired t test (between two groups) and one-way analysis of variance followed by LSD test (among multiple groups). Count data were compared by Chi-square test. Diagnostic values of miRNA-134 for colon
MiRNA-143 Inhibits Colon Cancer
cancer were evaluated by ROC curve analysis. p !0.05 was considered to be statistically significant.
Results MiRNA-143 was Downregulated in Colon Cancer Patient than in Healthy Controls Differential expression of miRNA-143 in cancer tissues and healthy tissues has been observed in several types of malignancies. In this study, expression of miRNA-143 in colon biopsies and blood of both colon cancer patients and healthy controls was detected by qRT-PCR. As shown in Figure 1A, expression of miRNA-143 in colon tissues was significantly downregulated in colon cancer patients than in healthy controls ( p !0.05). In addition, blood levels of miRNA-143 were also significantly lower in colon cancer patients than in healthy controls (Figure 1B, p ! 0.05). In contrast, expression of GLUT-1 was significantly upregulated in colon cancer patients than in healthy controls in both in colon tissues (Figure 1C, p !0.05) and blood (Figure 1D, p !0.05). Downregulation of miRNA-143 Effectively Distinguished Colon Cancer Patients from Healthy Controls ROC curve analysis was performed to evaluate the diagnostic values of miRNA-143 expression in colon biopsies and blood for colon cancer. AUC bigger that 0.65 is
3
considered with diagnostic value. For miRNA-143 expression in colon biopsies, the area under the curve (AUC) was 0.9026, with error standard of 0.02708 and 95% confidence interval of 0.8495-0.9557 (Figure 2A). For miRNA143 expression in blood, AUC was 0.8189, with error standard of 0.03846 and 95% confidence interval of 0.7425-0.8933 (Figure 2B). MiRNA-143 Expression was Significantly Correlated with Tumor Size and Distant Tumor Metastasis Correlations between miRNA-143 expression and clinicopathological data of patients with colon cancer were analyzed by Chi-square test. As shown Table 1, miRNA-143 expression in colon biopsies was significantly correlated with tumor size but not age, gender and existing of distant tumor metastasis. Similarly, a significant correlation was also found between blood level of miRNA-143 and tumor size (Table 2). Overexpression of miRNA-143 Inhibited Glucose Uptake and Downregulated GLUT-1 Expression in Colon Cancer Cells Before transfection, miRNA-143 level was significantly higher in cells of colon carcinoma cell line RKO than in cells of the normal colon cell line CCD-1Co (Figure 3A, p !0.05). After transfection, miRNA-143 overexpression was reached in cells of both cell lines (Figure 3A, p ! 0.05). Glucose uptake assay was performed to measure glucose uptake in colon cancer cells after miRNA-143
Figure 1. MiRNA-143 was downregulated in colon cancer patients than in healthy controls. This figure shows the comparison of miRNA-143 expression in colon biopsies (A) and blood (B) between colon cancer patients and healthy control, and the comparison of GLUT-1 mRNA expression in colon biopsies (A) and blood (B) between colon cancer patients and healthy control. Expression of GLUT-1 was significantly upregulated in colon cancer patients than in healthy controls in both in colon tissues (C; p ! 0.05) and blood (D; p ! 0.05).
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Figure 2. Downregulation of miRNA-143 effectively distinguished colon cancer patients from healthy controls. This figure shows the ROC curve analysis of the miRNA-143 expression in colon biopsies (A) and blood (B) for colon cancer.
MiRNA-143 showed downregulated expression and plays a tumor suppression role in different types of cancers
including colon cancer (12,13). It has been reported that miRNA-143 is also key regulator of blood glucose transportation (10) and cancer glycolysis (11). However, crosstalk between miRNA-143 and glucose metabolism in colon cancer is unknown. The key finding of our study is that miRNA-143 may inhibit the proliferation of colon cancer cells by reducing glucose uptake through the downregulation of GLUT-1. The development and progression of colon cancer is a complex process with multiple internal and external factors involved. It is generally believed that genetic factors play pivotal roles in colon cancer (14). In recent year, several tumor suppression genes, such as TET-1 (15), and oncogenes, such as IGFBP7 (16), have been identified in the pathogenesis of colon cancer. Besides messenger RNAs that encode protein products, human genome also transcribes a large set of non-coding RNAs that have critical functions in nearly all aspects of tumor biology (17). In our study we observed than miRNA-143 expression was significantly downregulated in colon cancer patients than in healthy controls in both colon biopsies and blood. Our data were consistent with the findings reported in previous studies (9,13). In addition, we also proved that downregulated expression of miRNA-143 effectively distinguished patients with colon cancer from healthy controls. Previous studies and the findings in our study
Table 1. Correlations between miRNA-143 expression in colon biopsies and clinicopathological data of patients with colon cancer
Table 2. Correlations between miRNA-143 expression in blood and clinicopathological data of patients with colon cancer
overexpression. As shown in Figure 3B, compared with control cells (C) and negative control cells (NC), glucose uptake was significantly decreased after miRNA-143 overexpression in cells of colon carcinoma cell line RKO but not in cells of the normal colon cell line CCD-1Co ( p ! 0.05). GLUT-1 plays pivotal roles in glucose translocation. Compared with control cells (C) and negative control cells (NC), GLUT-1 expression was significantly decreased after miRNA-143 overexpression in cells of colon carcinoma cell line RKO but not in cells of the normal colon cell line CCD-1Co ( p !0.05, Figure 3C). Overexpression of miRNA-143 Inhibited Proliferation Colon Cancer Cells CCK-8 assay was performed to measure cell proliferation rate after miRNA-143 overexpression. As shown in Figure 4, compared with control cells (C) and negative control cells (NC), miRNA-143 overexpression significantly inhibited proliferation of cells of colon carcinoma cell line RKO but not cells of the normal colon cell line CCD-1Co ( p !0.05).
Discussion
Items
Groups
O50 (years) !50 (years) Gender Male Female Primary tumor O2 cm diameter !2 cm Tumor distant Yes metastasis No Age
HighLowCases expression expression 34 44 42 36 49 29 33 45
18 21 18 21 19 20 15 24
16 23 24 15 30 9 18 21
c2
p
0.21 0.65 1.86 0.17 6.64 0.01 0.47 0.49
Items
Groups
O50 (years) !50 (years) Gender Male Female Primary tumor O2 cm diameter !2 cm Tumor distant Yes metastasis No Age
HighLowCases expression expression 34 44 42 36 49 29 33 45
16 23 19 20 20 19 14 25
18 21 23 16 29 10 19 20
c2
p
0.21 0.65 0.83 0.36 4.46 0.03 1.31 0.25
MiRNA-143 Inhibits Colon Cancer
5
Figure 3. Overexpression of miRNA-143 inhibited glucose uptake and downregulated GLUT-1 expression in colon cancer cells. Before transfection, miRNA143 level was significantly higher in cells of colon carcinoma cell line RKO than in cells of the normal colon cell line CCD-1Co After transfection, miRNA143 overexpression was reached in cells of both cell lines (A). This figure also shows the effects of miRNA-143 overexpression on glucose uptake (B) and GLUT-1 expression (C) in cells of colon carcinoma cell line RKO and normal colon cell line CCD-1Co. *p !0.05.
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Zhao et al./ Archives of Medical Research
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(2019)
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Figure 4. Overexpression of miRNA-143 inhibited proliferation colon cancer cells. miRNA-143 overexpression significantly inhibited proliferation of cells of colon carcinoma cell line RKO (A) but not cells of the normal colon cell line CCD-1Co (B) (p ! 0.05).
both revealed that downregulation of miRNA-143 is likely involved in the pathogenesis of colon cancer and low miRNA-143 expression level may has potential diagnostic value for this disease. In our study we observed that miRNA-143 is significantly correlated with tumor size but not distant tumor metastasis, indicating the potential involvement of miRNA-143 in the regulation of tumor growth but not tumor metastasis in colon cancer. Our in vitro cell proliferation experiments also proved that overexpression of miRNA-143 caused accelerated proliferation of colon cancer cells. Accelerated glucose metabolism is common in different types of cancers and is required for the abnormal growth of tumors. Overexpression of glucose-regulated proteins has been observed in patients with colon cancer (18). In addition, the significant correlation between highglucose diet and increased risk of colon cancer has also been reported (19). MiRNAs are key regulator of glucose metabolism in different cancers including colon cancer (20). In our study, we proved than miRNA-143 is a negative regulator of glucose uptake in colon cancer cells, and the inhibitory role of miRNA-143 on glucose uptake is likely achieved through the downregulation of GLUT-1, which is critical for glucose translocation (21). Therefore, miRNA-143 may inhibit tumor growth in colon cancer by inhibiting tumor growth through the downregulation of miRNA-143. Our study also revealed a miRNA-143- GLUT-1 signaling in glucose uptake in colon cancer. Whether the interaction between miRNA-143 and GLUT-1 is direct or indirect is unknown. In addition, the regulatory role of miRNA-153 on glucose uptake and GLUT-1 expression was not observed in normal colon cancer cells. Therefore, the regulatory role of miRNA-153 on glucose uptake and GLUT-1 expression is likely mediated by disease or colon cancer related factors. Our future studies will focus on the identification of those factors. In conclusion, our study further confirmed the downregulation of miRNA-143 in colon cancer. Our study
identified the involvement of miRNA-143 in colon cancer, which is achieved through the inhibited glucose uptake and GLUT-1 expression.
Acknowledgments We thank the financial support from Shenyang Technology and Science Project in 2018 (18-014-4-69).
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