| Our laboratory first discovered and successfully cloned human NDRG2 cDNA from normal human whole brain cDNA library by subtractive hybridization (GenBank accession number AF159092). NDRG2, together with NDRG1, NDRG3 and NDRG4, constitute NDRG gene family. The NDRG genes have relative high conservatism in the evolution process from inferior to high living being, which suggests the gene family play an important role in cell life. Research showed that NDRG2 was evidently expressed in adult brain cerebral gray matter, white matter and nerve corpuscle, as well as in glands epithelial cell and skeletal muscle cell, which are terminal differentiation cell. NDRG2 has a low expression in bone marrow stem cell and sperm cell, and the expression is lower in brain, heart liver, kidney and lung of embryo than adult. There are significant difference in the expression of NDRG2 between glioma, lung cancer, breast cancer, gastric cancer, liver cancer and normal tissues, which reveal a negative correlation between the expression of NDRG2 and generation ability of cell. Zhang Jian induce colon cancer cell line Caco-2 and HT-29 to differentiate, and find the expression of c-Myc is decreased while NDRG2 is increased after differentiation of cell, which indicate NDRG2 is involved in the regulation of generation and differentiation of colon cancer cell,but the exact mechanism os still unknown. So we design this experiment to reveal the relation between NDRG2,c-myc and generation,differentiation of colon cancer cell.We first bought 2 colon cancer tissue arraies, 150 samples in all. Statistical analysis found that there was significant difference among the expression of NDRG2 in variant differentiation colon cancer tissue (P=0.005) by immunohistochemistry,combined basic information of colon cancer patients. And no difference in age, gender, infiltrate depth and Dukes'stage was found.Based on these results, we decide to analyze clinical samples, under the help of department of statistics set power to 0.75 (α=0.05, RMSSE=0.25), and get a reault there should be 70 specimens in each group by power calculation (statistica 6.0). Because we have to devieded all specimens to 3 groups: poorly-differentiated, moderately- differentiated and well-differentiated, the sample size should be 210 at least. We collected colon cancer tissues 213 samples form Oct 2005 to Sep 2007, with matched adjacent tissues and normal tissues. Then analyaed expression of NDRG2 and c-myc in all samples by immunohistochemistry, western blot and real time PCR. We found that the expression of NDRG2 was increased with the differentiation process, while that of c-myc was decreased, whether in mRNA level or in protein level. And an increasing trend was found in the expression of NDRG2 in 136 colon cancer tissues, adjacent tissues and normal tissues, while a decreasing trend in the expression of c-Myc in 151 samples.We used sodium butyrate to induce 3 colon cancer cell lines HT29, SW480 and SW620 to differentiate. The differentiation stage was assessed by transmission electron microscopy (for changes in cell structure and architecture) and alkaline phosphatase activity (AP, as a marker of differentiation). Collect cell samples each day in the differentiation process from 1st to 6th day, isolates total RNA and protein to detect the expression of NDRG2 and c-Myc. We found the expression of NDRG2 was increased with the differentiation process, while c-myc was decreased.To identify the causation relationship NDRG2 involving in differentiation control of colon cancer cell. We designed NDRG2 micro interfere RNA to detect the effect of NDRG2 to colon cancer cell HT29. We transfected NDRG2 siRNA and control siRNA into colon cancer cell HT29 respectively. Induced cell to differentiate by sodium butyrate after western blot assessment. Differentiation stage was assessed by alkaline phosphatase activity, we found AKP level of NDRG2 siRNA group was lower than control siRNA group (P<0.05).Conclusions:1. The expression of NDRG2 was higher in well-differentiated cancer tissues than poor-differentiated, and lower in cancar tissues than in adjacent and normal tissues, while an opposite trend was found in c-MYC by large clinical colon cancer sample analysis.2. Indued colon cancer cell to differentiate by sodium butyrate, the expression of NDRG2 was increasing, while the expression of c-myc wan decreasing.3. After transfected NDRG2 siRNA into colon cancer cell HT29, the differentiation process retard significantly,which demonstrated NDRG2 was an important gene involved in colon cancer differentiation.
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