硫氧还蛋白过氧化物酶2在人结直肠癌的表达及其对HCT116细胞增殖、迁移的作用

Expression of peroxiredoxin 2 in colorectal cancer and its effect and mechanism on proliferation and migration of HCT116 cells

目的探讨硫氧还蛋白过氧化物酶2（PRDX2）蛋白在结直肠癌、腺瘤及正常黏膜组织中的差异性表达及其与结直肠癌的生物学关系；利用RNA干扰（RNAi）技术，特异性沉默PRDX2基因的表达，观察沉默PRDX2对结直肠癌细胞HCTJ16增殖与迁移的影响并探讨其机制。方法采用Westernblot、免疫组织化学检测并分析PRDX2蛋白的表达与结直肠癌发生及预后的关系。构建稳定沉默PRDX2细胞株，设立阴性对照组（shContr01）和沉默PRDX2实验组（shPRDX2-F3），通过细胞计数试剂盒（CCK-8）、平板克隆形成实验检测细胞增殖能力，划痕实验和Transwell小室检测体外迁移能力，WesternNot检测E-钙黏蛋白（E—cadherin）、N．钙黏蛋白（N—cadherin）、波形蛋白（Vimentin）、Snail、Slug蛋白表达。结果PRDX2蛋白在结直肠癌、腺瘤和正常黏膜组织中的表达阳性率依次减低（P〈0．01）。其表达与结直肠癌的TNM分期（P〈0．01）、肿瘤分化程度（P〈0．01）有关，PRDX2蛋白阳性表达者生存率（71．90％）明显低于阴性表达者（86．28％，P〈0．05）。沉默PRDX2基因后能抑制HCTI16细胞的增殖生长；PRDX2沉默后HCT116细胞克隆数量[（69．53±5．20）个]少于空载对照组[（98．33±10．69）个，P〈0．05]；沉默PRDX2的细胞迁移能力增强，且E—cadherin蛋白表达下降，N—cadherin、Vimentiu、Snail、S1ug蛋白表达增强。结论PRDX2蛋白表达参与结直肠癌的发生、发展，并与结直肠癌的预后有关。沉默PRDX2可抑制结直肠癌细胞HCT116增殖，促进迁移，其机制可能与转录因子Snail、Slug表达有关。

Objective To investigate the differential expression of peroxiredoxin 2 （PRDX2） pro- tein in coloreetal cancer, adenoma and normal mucosa tissues, the biological relationship between PRDX2 protein and coloreetal cancers, and the effects of small interfering RNA （siRNA） - mediated silencing of PRDX2 gene expression on colorectal cancer HCT116 cells proliferation and migration. Methods Using Western blotting and immunohistochemistry, the expression of PRDX2 was detected, and its relationship with occurrence and prognosis of colorectal cancer was analyzed. Three shRNA sequences targeting PRDX2 and one negativecontrol （NC） were designed and infected into the cultured cancer cells HCT116. ShPRDX2 - F3 with the highest silencing efficiencies was selected for experimental analyses ＇after puromy- cin screening. Cells infected with empty vector were used as controls and shPRDX2 - F3 was identified as experiment. Two groups were used for experimental analyses of proliferation [ by the cell counting kit - 8 （CCK -8 ） assay, and plate colony formation assay], and cell migration （would healing test, Transwell）. In addition, Western blotting was also used to detect the protein expression of E - cadherin, N - cadherin, Vimentin, Snail and Slug. Results The positive expression rate of PRDX2 protein was decreased gradually from colorectal cancer [83.96% （157/187） 1, adenoma [64. 52% （40/62） ] to normal tissues [ 16. 67% （25/150） ] （P 〈 0. 01 ）. PRDX2 protein expression was correlated with colorectal cancer TNM stage （P 〈 0. 01 ）, and tumor differentiation grade （P 〈0. 01 ）. The survival rate in patients positive for the PRDX2 protein expression （71.90% ） was significantly lower than in those negative for the expression （86. 28% ） （ P 〈 0. 05 ）. Western blotting indicated that ShPRDX2 - F3 significantly inhibited PRDX2 protein expres- sion. After silenced, the clone number of cell line HCT116 in shPRDX2 - F3 （69. 53 ± 5.20） cells was significantly lower than shControl （98.33 ± 10. 69 ） cells （ P 〈 0. 05 ）. As compared with control group, proliferation of colorectal cancer cell line HCT116 was significantly inhibited and the migration was signifi- cantly increased （ P 〈 0. 05 ）. The results of Western blotting showed that E - cadherin expression level was reduced, and the expression levels of N - cadherin, Vimentin, Snail and Slug were increased due to PRDX2 gene interference. Conclusion PRDX2 protein may involved in the occurrence, development and prognosis of colorectal cancer. As the biological indicator, PRDX2 protein has the potential ability to be used in colorectal cancer screening and early diagnosis. PRDX2 gene silencing could inhibit the prolifera- tion and increase the migration of colorectal cancer cells HCT116, and it may be related to the Snail and Slug expression.