Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
World J Gastroenterol. 2012 Sep 14;18(34):4704-13. doi: 10.3748/wjg.v18.i34.4704.
AIM: To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS: Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide (PI) using Annexin V/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS: Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION: These findings suggest that RRM2 may be a facilitating factor in colorectal tumorigenesis and UV-induced DNA damage repair.
目的：探讨核苷酸还原酶 M2（RRM2）亚基在结直肠癌（CRC）和紫外线（UV）诱导的 DNA 损伤修复中的作用。
方法：采用组织微阵列免疫组织化学染色检测 RRM2 的表达。培养 7 株 CRC 细胞系，选择 3 个人结肠癌细胞系 HCT116、SW480 和 SW620，分别通过逆转录聚合酶链反应和 Western blot 检测 RRM2 的 mRNA 和蛋白表达水平。进行细胞增殖试验、细胞周期分析。用 Annexin V/PI 凋亡试剂盒通过异硫氰酸荧光素（FITC）-缀合的 Annexin V 和碘化丙啶（PI）双重染色评估细胞凋亡。用 Transwell 室测定 CRC 细胞的迁移和侵袭。用 254nm UV-C 灯照射细胞，检测 RRM2 耗竭后对 UV 的敏感性。
结果：免疫组织化学染色显示 CRC 组织中 RRM2 水平升高。RRM2 过表达与浸润深度（P < 0.05）、低分化类型（P = 0.0051）和肿瘤淋巴结转移分期（P = 0.0015）呈正相关。HCT116 细胞转染后 RRM2 表达下调，与对照组相比，HCT116 细胞增殖明显受到抑制（P < 0.05）。在侵袭试验中，RRM2-siRNA 组穿过室的细胞数为 81 ± 3，低于阴性对照组（289 ± 7）和空白对照组（301 ± 7.2）。这些差异具有统计学意义（P < 0.01）。我们的数据表明，RRM2 过表达可能与 CRC 进展有关。用 siRNA 沉默 RRM2 可能抑制 CRC 细胞的过度增生和侵袭，提示 RRM2 可能在 CRC 的浸润和转移中发挥重要作用，这是 CRC 的一种潜在治疗策略。此外，RRM2 耗竭增加了 UV 的敏感性。
结论：这些发现表明，RRM2 可能是结直肠肿瘤发生和 UV 诱导的 DNA 损伤修复的促进因素。
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