| Background and Objective Colorectal cancer is one of common malignant tumors which threaten to human health. The five-year survival rate of colorectal cancer was 32 percent in China. The mortality of colorectal cancer declines in developed countries due to the well-promoted colorectal cancer prevention projects, which are poorly-promoted in China. Because of the improvements in civilian living standard, the adjustment of diet structure, colorectal cancer ranked from 7th place to 5th place among the overall causes of death. Local recurrence, lymph node metastasis and hematogenous metastasis are the common causes of failure in treatment for colorectal cancer. At present, tumor growth and metastasis are considered involving many factors, including the invasion and migration of tumor cells, the anti-apoptosis of tumor cells and the escape of immune surveillance. Studies have shown that, the expression level of Histidine triad nucleotide binding protein 2(Hint2) can affect the biological behavior of tumor cells, which exists close relationship between the expression level and invasion and migration of tumor cells. Hint2 belongs to Hint family in HIT(Histidine triad protein) super family which proteins share the same basic structure domain His-X-His-X-His-X-X. HIT proteins widely exist in prokaryotes and eukaryotes. Hint2 protein located in the mitochondria, which can promote apoptosis and mainly acts on respiration chain. The expression of Hint2 varies in different of human organs. It is reported that Hint2 mainly expressed in liver and pancreas, while poorly expressed in hepatocellular carcinoma. In addition, Hint2 m RNA poorly expressed in many kinds of malignant tumors, its low expression negatively correlated with the prognosis of patients. Currently, the effect of Hint2 expression on the invasion and migration ability of colon cancer cells has not been reported. Our preliminary experimental results have shown that the expression of Hint2 in colon cancer cell line SW620 is relatively low. Thus, we explored the changes of colon cancer cell invasion and migration capability caused by up-regulating the expression of Hint2 and provided a fundamental research for further elucidation of the possible mechanism about how the Hint2 expression influenced the invasion and migration capability of colon cancer cells.Methods 1. Cultured colon cancer cells SW620 in vitro, passaged them, cells in the logarithmic growth phase were taken for experiments. 2. Constructed the Hint2 over-expressed Lentiviral vector p LVX-IRES-Zs Green1-Hint2, then transfected to 293 T cells after identification by bacterial fluid PCR, double enzyme identification and sequencing identification. Cultured and passaged the cells, next transfected back to SW620 cells. Finally, SW620Hint2+ cells were acquired. 3. Took 10 visions under inverted fluorescence microscope, comprehensively evaluated the expression of green fluorescent protein(GFP). 4. The expression of Hint2 protein in SW620Hint2+ cells was detected by Western Blot(using GAPDH as internal control). 5. The capability of cells invasion and migration were detected by Transwell(invasion and migration) and wound healing assay between SW620 group and SW620Hint2+ group. 6. The relationship between Hint2 expression and the capability of cells invasion and migration was analyzed by Spearman correlation.Result 1. The identification of transfection: Full fields of green fluorescence in SW620Hint2+ group were observed under inverted fluorescence microscope, the average green fluorescence protein expression rate is(89.34±3.22) %.2. The expression of Hint2 in SW620Hint2+ group: Greyscale value in SW620 group was set as reference in Western blot detection. Hint2 protein bands greyscale value in SW620Hint2+ group was 3.1 times to that in SW620 group, which proved cells in SW620Hint2+ group overexpressed Hint2. 3. The relation between Hint2 expression and the capability of invasion of colon cancer cells: The number of penetrated cells was lesser in SW620Hint2+ group than that in SW620 group(52.33±5.05 vs 112.83±10.11),(p<0.05), which were examined by Transwell invasion assay after 48 hours. There was negative correlation between the expression of Hint2 and the capability of invasion of SW620Hint2+ cells(r=-1.000, p<0.01). 4. The relation between Hint2 expression and the capability of migration of colon cancer cells: The number of migrated cells was lesser in SW620Hint2+ group than that in SW620 group(51.17±11.64 vs 111.16±11.64),(p<0.05), which were examined by Transwell migration assay after 48 hours. In wound healing assay, the average migrated distance of cells was significantly shorter in SW620Hint2+ group than that in SW620 group(0.37±0.03 vs 0.71±0.04). There was negative correlation between the expression of Hint2 and the capability of migration of SW620Hint2+ cells(r=-1.000, p<0.01).Conclusion The expression of Hint2 can influence the colon cancer cells capability of invasion and migration. High expression of Hint2 can weaken the colon cancer cell invasion and migration capability. There was negative correlation between the Hint2 expression and the capability of invasion and migration of SW620 cells, which indicated that the Hint2 expression had anti-tumor effect. But, the specific role of inhibiting tumor metastasis remain unclear. |
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