| To detect the expression of NCOA5 in the normal renal epithelial cell lines HK2, 293 and renal carcinoma cell lines ACHN and OS-RC-2, and renal cell carcinoma tissues. To analyze the relationship between NCOA5 and clinical characteristics of patients with renal cell carcinoma. To establish stable cell lines with over-expression of NCOA5. To examine the effects of NCOA5 on cell proliferation, migration and invasion.Methods:(1) Western blot was used to detect NCOA5 expression in renal epithelial cell lines HK2 and 293, and in renal carcinoma cell lines ACHN and OS-RC-2, statistical analysis was applied to show the difference between the expression of NCOA5 in renal epithelial cells and renal cell carcinoma cells.(2) The expression of NCOA5 in renal cell carcinoma of clinical tissue specimens was examined by immunohistochemistry, and the relationship between NCOA5 expression and clinical features was built statistically.(3) The renal cell carcinoma cells ACHN and OS-RC-2 was infected with lentiviral vectors containing NCOA5 gene and blank vectors lentiviral, stable renal carcinoma cells with high expression of NCOA5 were sorted by flow cytometry.(4) The expression of NCOA5 protein in renal cell carcinoma cell line was detected by Western blot before and after lentiviral transfection, the differences were determined via statistical analysis.(5) The CCK-8 method, cell scratch and Transwell assay were used to examine the possible effect of NCOA5 on the cell proliferation, migration and invasion of renal carcinoma individually.Results:(1) The Western blot results showed that the relative expression value of NCOA5 in293, HK2, ACHN and OS-RC-2 cells were 0.52±0.02, 0.34±0.01, 0.11±0.02, 0.04±0.02respectively; the expression of NCOA5 in 293 cell is at highest level, whereas the expression of NCOA5 in OS-RC-2 reached the lowest level. The expression level of NCOA5 in renal cell carcinoma cell lines is lower than in renal epithelial cells.(2) Immunohistochemistry showed that biopsy from 9 out of 14 patients with renal cell carcinoma exhibited NCOA5 positive, the positive rate was 64.3%, however, NCOA5 in the cancer adjacent tissues were all positive(x2=6.087, P=0.041), suggesting that the expression of NCOA5 in renal cell carcinoma was lower than that in adjacent tissues.Analysis also showed that the expression level of NCOA5 in renal cell carcinoma tissues was correlated with patient age, tumor size, lymph node metastasis, clinical staging and nuclear differentiation(x2=7.778, 10.370, 5.833, 5.833, 7.778, p=0.021, 0.003, 0.031,0.031, 0.021, respectively) but it had no correlation with gender, pathological type, tumor location(x2=4.321, 2.121, 0.026, 0.258, 1, p>0.05). Also NCOA5 expression was relatively low in old, large diameter tumor, high grade of tumor differentiation, lymph node metastasis or high clinical stage groups.(3) The prolongation of ACHN, OS-RC-2 cell passage was observed after NOCA5 transfection, and led to a large number of cell death; the empty vector transfected with lentivirus had neither obvious change in passage time, nor a large number of cell death. After change with higher medium of DMEM and culture condition, and using large-scale flow sorting, ACHN cells with stable high expressed NCOA5 in renal cell carcinoma was eventually established. However, OS-RC-2 cells were died after NCOA5 transfection, failing to establish the corresponding stable cell line.(4) Western Blot analysis showed that the relative expression value of NCOA5 in ACHN, NC-ACHN and NCOA5-ACHN cells were 0.13±0.06, 0.16±0.01, 0.81±0.03 respectively. The NCOA5-ACHN was relatively higher than that in NCOA5 group(P<0.05).(5) The CCK-8 test revealed that absorbance of difference between ACHN, NC-ACHN and NCOA5-ACHN group were 0.32±0.04, 0.31 ±0.03, 0.21±0.02 at24 h; 0.54±0.10, 0.58±0.07, 0.34±0.03 at 48 h; and 1.10±0.14, 1.04±0.11, 0.63±0.16 at 72h;Compared with ACHN group, cell proliferation ability in NCOA5-ACHN group was significantly decreased(P<0.05), the NC-ACHN group did not change significantly(P >0.05), indicating a decrease of cell proliferation ability after NCOA5 highexpressing(P<0.05).(6) Cell scratch test showed that the changes of the distance between cell boundaries in ACHN, NC-ACHN, NCOA5-ACHN group were 0.820±0.017,0.785±0.031, 0.482±0.015 respectively at 24 h, there was no statistical difference between ACHN and NC-ACHN group(P>0.05), however, the NCOA5-ACHN group had statistically significant with the other two groups(P < 0.05). Meanwhile the cell number of migration in NCOA5-ACHN group significantly decreased.(7) Transwell invasion assay exhibited that transmembrane cells were 410±14,418±16, 107 ± 9 in ACHN, NC-ACHN, NCOA5-ACHN groups. Among these groups ACHN and NC-ACHN had no significant difference in transmembrane cell number(P>0.05), but there were statistically significant difference between the NCOA5-ACHN cells and the other two groups(P<0.05), suggesting that the renal carcinoma cell line with high expression of NCOA5 decreased the cell invasion capacity signifcantly.Conclusion:(1) This study proved that the expression of NCOA5 in human renal epithelial cells, 293 and HK2 was higher than that in human renal cell carcinoma cell line, ACHN and OS-RC-2, there were significant differences between the expression of epithelial cells and carcinoma cells.(2) NCOA5 in renal cell carcinoma tissue was expressed at a lower level than that in the adjacent area, and it was correlated with age, tumor size, lymph node metastasis, clinical stage and differentiation grade of the nucleus.(3) NCOA5-ACHN stable cell lines were successfully established through the experiments of the stable transfection of NCOA5 gene, these increased the NCOA5 expression of ACHN.(4) Cell proliferation, migration and invasion were inhibited in renal carcinoma cell line ACHN with high NCOA5 expression. |
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