Mitochondrial Uncoupling Protein 2 Expression In Colon Cancer And Its Clinical Significance

Colon cancer is one of the malignant tumors threatening to human health, and the mortality of colon cancer patients ranks second among all malignant tumors in developed countries. In recent years, its incidence has been increas- ing in China, particularly in the economically developed coastal cities. Its etiology and pathogenesis remain unclear.Uncoupling protein-2 (UCP2) is a mitochondrial membrane protein, which negatively regulates the production of reactive oxygen species (ROS). Adaptive mechanisms in cancer cells include resistance to tumor growth inhibition and evasion of apoptosis, and cellular events that are appreciably affected by oxidative stress. The UCP2 expression level is significantly higher in colon cancer tissue than in its adjacent tissue and UCP2 may play a role in intestinal epithelial cells from benign to malignant transformation. However, the role of UCP2 in development of colon cancer is unclear. In this study, the expression of UCP2 in normal human colon tissue and colon cancer tissue was detected, and the relation between UCP2 expression in colon cancer tissue and clinical pathological features of colon cancer wasn analyzed. What's more, Effect of RNA interference-based silencing of uncoupling protein-2 gene on the expression of glycolysis associated genes in colon carcinoma cells was also studied.AIM:To detect the expression of mitochondrial uncoupling protein 2 (UCP2) in colon cancer and analyze the relation between UCP2 expression and clinical pathological features of colon cancer. METHODS:1. Fifteen colon tissue samples and 15 its adjacent tissue samples were obtained from colon cancer patients during surgical interventions. Quantitative reverse transcription polymerase chain reaction and Western blotting were used to detect UCP2 expressions in colon cancer tissue samples and its adjacent tissue samples.2. UCP2 expression was detected with immunohistochemical method in 10 normal controls, 10 hyperplastic polyp patients, 20 tubular adenoma patients and 78 colon cancer patients. Patients with rectal cancer were excluded. Relation between UCP2 expression and clinical pathological features of colon cancer was also analyzed.3. UCP2 and glycolysis associated genes expression was detected with immunohistochemical method in 10 normal controls, 10 tubular adenoma patients and 15 colon cancer patients. Patients with rectal cancer were excluded. Quantitative reverse transcription polymerase chain reaction and Western blotting were used to detect UCP2 expressions in colon cancer tissue samples and its adjacent tissue samples. Relation between UCP2 expression and clinical pathological features of colon cancer was also analyzed.4. Caco2, Caco2/Neo, Caco2/shRNA cells were incubated under normoxic conditions for different durations (0, 12, 24, 36, 48 and 72 hours); MTT was used to test the proliferation of cells.5. Caco2, Caco2 + 5-FU (100μg/mL), Caco2/Neo, Caco2/shRNA +5-FU (100μg/mL) cells were incubated under normoxic conditions for 24h. Then determine the apoptosis of cells.6. Caco2, Caco2/Neo, Caco2/shRNA cells were incubated under normoxic and hypoxic conditions for 12h. Then spectrophotometry were employed to determin the content of lactic acid (LA) in the culture supernatant.7. Caco2, Caco2/Neo, Caco2/shRNA cells were incubated under normoxic and hypoxic conditions for 72 h. Then the expression of glycolysis associated genes (LDH 5, HK-Ⅱ) was measured on protein level by Western blot and RT-PCR.RESULTS:1. The UCP2 mRNA expression level was fourfold higher in colon cancer tissue samples than in its adjacent tissue samples. The UCP2 protein expression level was three-fold higher in colon cancer tissue samples than in its adjacent normal tissue samples.2. The UCP2 was mainly expressed in cytoplasm. The UCP2 was not expressed in normal colon mucosa. Strong positive staining for UCP2 with a diffuse distribution pattern was identified throughout the mucosa in colon cancer tissue samples with a positive expression rate of 85.9% (n = 78) and 86.7% (n = 15).3. The UCP2 expression level was higher in colon cancer tissue samples at clinical stagesⅢandⅣthan in those at stageⅠ+Ⅱ. Univariate analysis showed that the high UCP2 expression level was significantly correlated to colon cancer metastasis (hazard ratio = 4.321, confidence interval = 0.035-0.682, P = 0.046).4. The expression of glycolysis associated genes (LDH5, HKII, PFKFB3, TU2-MPK) was also mainly expressed in cytoplasm. The LDH5 and HKII was srong positively expressed in a in colon cancer tissue samples, compared with they hardly expressed in normal colon mucosa. However, the expression of PFKBK3 and TUMPK2 between colon cancer tissue samples and normal colon mucosa was not very obvious.5. The expression of UCP2 was hardly detected in UCP2 silenced cells.6. The proliferation of the Caco2/shRNA cells is more slowly than the Caco2 and Caco2/Neo cells.7. The apoptosis of cells: Caco2/shRNA +5-FU (100μg/mL) cells was increased than the other cells with significant differences (P < 0.05).8. The lactic acid content in the culture supernatant was obviously decreased in UCP2 silenced cellswith significant differences (P < 0.05).9. The expession of LDH-5 mRNA and protein expression were decreased greatly in UCP2 silenced cells than Caco2 and Caco2/Neo cells (P < 0.05), and the expression of HKII mRNA and protein expression in Caco2, Caco2/Neo, Caco2/shRNA cells with no significance (P > 0.05).CONCLUSION:1. UCP2 is highly expressed in human colon cancer tissue and may be involved in colon cancer metastasis.2. RNA interference targeting the UCP2 gene is able to efficiently silence the expression of UCP2 gene in human colon carcinoma cells (Caco2).3. The expession of LDH-5 were down-regulated obviously when interferencing UCP2 gene.4. Silenceing UCP2 gene can inhibit the expression of glycolysis associated genes and the content of lactic acid in the culture supernatant. The expression of UCP2 was closely related to tumor glycolysis.