| Colorectal cancer(CRC) is a common malignant tumor in the digestive tract, and its incidence is increasing every year. CRC is the third most common cancer deaths disease in the world with more than one million new cases of CRC and 600,000 dead patients every year. 5-Fu is the most commonly used chemotherapy drug for CRC, but its precise resistance mechanism has not yet been clearly elucidated. That CRC could start DNA damage repair mechanisms, and antagonize the 5-Fu–induced DNA strand breaks in cells is important reason for 5-Fu resistance in CRC. Our preliminary results showed that the co-stimulatory molecule B7-H3 over-expression can significantly reduce the sensitivity of CRC to 5-Fu. Therefore, this study from the level of B7-H3-regulated DNA damage repair, investigate the role of B7-H3 in 5-Fu resistance in CRC.In this study, we constructed two CRC stable transfected cell lines, B7-H3 over-expression cell SW480-B7-H3 and the control cell SW480-NC, B7-H3 knockdown expression of HCT-8-shB7-H3 cells and the control HCT-8-NC cells. By comparing the sensitivity of the two cell lines to 5-Fu using MTT assay, we found that the resistance index in B7-H3 over-expressed cell is significantly higher than that in the B7-H3 low-expressed cell lines. The result was further verified by clony formation experiment. B7-H3 over-expression cell line has stronger cell proliferation ability compare with the control cells. Since one role of 5-Fu is leading to cell DNA damage, comet assay method was used to detect DNA damage. The results showed that 5-Fu-caused DNA damage in the B7-H3 over-expressed CRC cell line was significantly weaker than that in the B7-H3 low-expressed cell lines. Thus, these data reveals that B7-H3 inhibit 5-Fu sensitivity by increasing the repair ability of CRC cells to 5-Fu-induced DNA damage. The next task is to explore how B7-H3 plays a role in DNA damage repair.Early results of microarray revealed that B7-H3 over-expression also raised BRCC3 expression. Therefore, to verify this phenomenon, we performed further experiments, qRT-PCR results showed that B7-H3 over-expression indeed increased BRCC3 expression, whereas B7-H3-silenced CRC cell line HCT-8-shB7-H3 showed decreased BRCC3 expression, Western blot data confirmed the same results. Confocal immunofluorescence results showed that B7-H3 expression was positively correlated with BRCC3, and BRCC3 translocated from cytoplasm to nuclear after 5-Fu treatment. Since BRCC3 is a key DNA damage repair protein, B7-H3 may antagonize 5-Fu resistance by regulate BRCC3.These results proved that B7-H3 regulate the expression of BRCC3 in 5-Fu-treated CRC cells. However, it is unclear whether BRCC3 is a key protein by which B7-H3 antagonises 5-Fu. A BRCC3-specific siRNA was used to suppress the BRCC3 expression, Western blot and immunofluorescence confocal analyses proved that BRCC3 was down-regulated in SW480-B7-H3; and subsequent BRCC3 was knocked down in SW480-B7-H3 and then cell proliferation was significantly weakened in these cells by cloning formation experiment. Cell cycle analysis showed that the ratio of G2/M phase cells significantly increased after BRCC3 knockdown, reducing checkpoint to check the DNA damage; The results of comet assay showed that the DNA damage repair capacity of SW480-B7-H3 cell was decreased after BRCC3 knockdown with increased sensitivity to 5-Fu. Therefore, all the above results prove that B7-H3 increase DNA repair capacity and thus cause 5-Fu resistant through regulating BRCC3.Our study for the first time prove that B7-H3 regulate the expression of BRCC3, increase the ability to repair DNA damage, antagonize 5-Fu-caused DNA damage, and reduce the sensitivity of CRC cells to 5-Fu. These results suggest a new approach to reverse the drug resistance in CRC. |
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