The Regulation And Mechanism Of PRDX2 On Cancer Stem Cell-like Phenotype In Colon Cancer

Colon cancer is one of the most common tumors all over the world.The incidence of colon cancer increased year by year,threatening more people's life and health in China.According to the statistics of the National Central Cancer Registry of China,new cases of colorectal cancer in 2015 were estimated at 376,300,accounting for 8.7% of the total new tumors in the same year and ranking fifth.The number of deaths from colorectal cancer was estimated at 191,000,Accounting for 6.8% of the total death of cancer cases in the same year and ranking fifth.At present,the treatment of colorectal cancer is still the traditional surgery and adjuvant chemotherapy,but the efficacy of advanced colorectal cancer is not ideal.In order to enhance the curative effect of colorectal cancer,it is necessary to understand the mechanism of tumorigenesis and tumor progression.Such alarming ineffectiveness of standard anti-cancer therapies has been attributed to the existence of relatively rare,highly drug-resistant,quiescent or slow proliferating cells with stem-like properties: cancer stem cells(CSCs).The stem cell concept of carcinogenesis postulates that only a small subset of longlived cells with dual potential(ability to self-renew and ability to produce all the heterogeneous mass of progenitors at different stages of their maturation)can proliferate indefinitely,give rise to macroscopic metastases,and reinitiate tumor formation upon serial transplantation of the low cell number into immunocompromised mice.Like normal stem cells,CSCs seem to depend on a similar,permissive environment,the CSC niche,to retain their exclusive abilities to self-renew and give rise to more differentiated progenitor cells,while staying in an undifferentiated state themselves.Changes in oxidative reduction balance in tumor stem cell niches may have an effect on stem cell characteristics.Peroxiredoxin(PRDXs)is an antioxidant superfamily in cells.PRDX2 is a typical 2-Cys peroxiredoxin in this family.It was found that PRDX2 expression was higher in colorectal cancer than in adjacent normal tissues,and PRDX2 knockdown inhibited colon cancer cell growth and promoted apoptosis.However,the regulation effect of PRDX2,as an important regulator of redox balance,on the characteristics of colon cancer stem cells has not been clear.Aims:To explore the effects and molecular mechanisms of PRDX2 knockdown and overexpression on cancer stem cell-like phenotype in colon cancer.Methods:1)Expression of PRDX2 and CD133 in colon cancer tissue samples were detected by IHC,and the data of 10 samples was analyzed by Pearson correlation.We isolated CD133-and CD133+ cells in colon cancer cells by MACs,detected protein levels of PRDX2 by Western blot.Then we detected expression of PRDX2 and CD133 in colon cancer cells and spheres by immunofluorescence.2)We established PRDX2 knockdown cell line with RNAi technique in SW620,HT29 and HCT116 colon cancer cell lines,examined transfection efficiency with fluorescence microscope,Western blot and QRT-PCR.Stemness changes after PRDX2 knockdown were examined by flow cytometry,Western blot,QRT-PCR,sphere formation,drug toxicity test and tumorigenicity.3)We established PRDX2 overexpression cell line with RNAi technique in SW620,HT29 and HCT116 colon cancer cell lines,examined transfection efficiency with fluorescence microscope and Western blot.Stemness changes after PRDX2 knockdown were examined by flow cytometry,Western blot and sphere formation.4)Signaling pathway was screened in colon cancer cell line with PRDX2 intervention by Western blot.After using Hedgehog pathway inhibitor,CSC marker protein level were examined by Western blot.Results:1)CD133 is linearly dependent on PRDX2 expression.Pearson correlation coefficient is 0.7863,p=0.007.Protein level of CD133+ cells is significantly higher than that in CD133-cells,p<0.05.PRDX2 located in cytoplasm and CD133 located in cytomembrane.2)PRDX2 knockdown reduced CD133+ cell population,CSC marker(CD44,CD133,Lgr5,Nanog,EPCAM)expression and sphere formation in colon cancer cell lines.In addition,CD133+ cells became more sensitive to 5-FU,and CD133+ cells induced smaller tumor in nude mice.3)PRDX2 overexpression promoted CD133+ cell population,CSC marker(CD44,CD133,Nanog)expression and sphere formation in colon cancer cell lines.4)Protein expression of SMO and Gli1 changed along with PRDX2 expression.CD44 and CD133 expression decreased when Hedgehog pathway was inhibited in HT29 colon cancer cell lines,p<0.05.Conclusion: PRDX2 is essential for maintaining cancer stem cell-like phenotype via Hedgehog signaling pathway in colon cancer.