The Effect And Mechanism Of PRDX1 In The Occurrence And Progressing Of Colorectal Cancer

Background and objectiveColorectal cancer is one of the common malignant tumors in digestive system.With the changes of life style and diet structure,the incidence of colorectal cancer showed a rising trend in the world in recent years.The incidence and mortality were in the top three of all tumors.The age of onset tended to be younger.Metastasis is the primary reason of death with rectal cancer.In the initial diagnosis and clinical treatment process,approximately 40-50%of patients are detected with metastases.Lymph node metastasis is one of the main ways of colorectal cancer metastasis.The liver is the main target organ metastasis of colon cancer.Liver metastasis rate was high as 45%?71%in the death cases with colorectal cancer.Therefore,which is important for improving the cure to strengthen the basic research on colorectal cancer and metastasis of colon cancer for finding the molecular markers which provides a new target for rectal cancer because its high specificity and sensitivity.Redox status is one of the basic elements to maintain homeostasis balance.More and more studies have confirmed that the internal redox state imbalance of tumor cells is closely related with tumor.In the tumorigenesis and progression of tumor,reactive oxygen species?ROS?in the tumor microenvironment cluster increased dramatically because the increasing energy demand,the intermittent hypoxia in the growth process and the accompanying infection,inflammation of tumor cells,which resulting in oxidative stress which influence cell metabolism pathway and signal transduction pathway,and then cause the tumor biological behavior changes.Based on the theory above,regulating the intracellular redox status may influence tumor metastasis.Peroxide redox protein 1?peroxiredoxins1,PRDX 1?is a newly discovered important intracellular antioxidant protein.PRDX1?Peroxiredoxinl?gene is located on chromosome 1p34.1,15Kb length.It has four transcripts,all encoding PRDX1 protein.The oxidation of PRDX1 is involved in cell redox regulation through the thioredoxin system rather than through glutaredoxin to reduce peroxides and reducing equivalents which may play an important role in the reduction of superoxide generation.It plays a key role in the cellular antioxidant defense process against ROS mainly through the thioredoxin reduction,scavenging peroxide or superoxide,and or through regulating the intracellular H₂O₂ accumulation to participate in the signal amplification effect of growth factors and tumor necrosis factor.Research has showed that PRDX1 acts as intracellular harmful active oxygen scavenger which has a dual role in tumor cells:On the one hand,PRDX1 can hinder the normal cells to malignant transformation from DNA damage caused by reactive oxygen species to suppress the occurrence of tumor,on the other hand,PRDX1 can also inhibit apoptosis induced by a large amount of active oxygen of the transformed tumor cells which promotes the survival of tumor cells and support and protect the tumor cells.In addition,PRDX1 may also play an important role in tumor cell proliferation and gradual progress through other ways.The previous research shows the action mechanism of PRDX1 is not the same in the evolution of different tumors.The conclusions are larger differences.Therefore,the further research which is also the hotspot of research in recent years is necessary.At present,the studies of PRDX1 in colorectal cancer were rare,and limited to the superficial identification of histology.The correlation with colorectal cancer metastasis has not been reported.Target point of PRDX1 to explore the correlation between the imbalance of redox state and colorectal cancer metastasis is expected to become a new research field which to elucidate the regulatory mechanism for the prevention,the node metastasis of rectal cancer to provide ideas and new targets for new treatmentMaterials and Methods1.The different stage of CRC paraffin samples was collection from happening to progressing such as hyperplastic polyp-adenoma-atypical hyperplasia-carcinoma-tumor lymph nodes metastasis-liver metastasis for immunohistochemistry detection?IHC?.24 fresh colorectal cancer and matched normal tissues were collected to detect the expression of PRDX1 by Western blot?Western Blot?and real-time quantitative PCR?RT-qPCR?.The collection of colorectal cancer paraffin samples was prepared for The relationship between differentiation,stage and metastasis clinical pathological parameter and PRDX1 expression of colorectal cancer tumors would be analyzed.2.Establishing PRDX1 stable expression cell line SW620-PRDX1 and interference of the colorectal cancer cell line SW480-shRNAi-PRDX1.MTT,Colony-forming,transwell and scratch assay was used for detect cell proliferation and migration movement;transplanted subcutaneously in nude mice model was established to detect PRDX1 expression on cell proliferation in in vivo;Organelles change after overexpression or knockdown PRDX1 was observed by transmission electron microscopy?TEM?.3.In order to construct oxidative stress model of CRC cell lines,Different concentrations?0,20,40,60,80,100,120?M?act on the cell lines to ensure the optimum H₂O₂,and different reaction times?30min,3h,6h,12h,24h,48h?of the H₂O₂ act on SW480 and SW620 cells to determain the optimum time.After that,we detect cell apoptasis and cell cycles by FCM?flow cytometry?.5.Gene chip technology to detect the change of downstream target genes of RNAi PRDX1 cell to find the related pathway.To detect the expression of some pathway target genes and signal pathways by CO-IP,Western Blot.7.The statistical methods:Statistical analysis was performed using SPSS 17.0 statistical software.The correlation between the expression of PRDX1 and clinical pathological features in CRC were determined by Chi-square test;MTT assay were analyzed by factorial design analysis of variance.RT-qPCR,colony formation assay,transwell assay,transplanted subcutaneously in nude mice model assay were determined by two independent t-test.P<0.05 considered stasistically significant.Results1.The expression of PRDX1 1 in colorectal cancer1.1 immunohistochemistry showed the positive expression of PRDX1 was mainly localized in the cell cytoplasm.It can be divided into negative,pale yellow,yellow,deep yellow,brown four colors and scores were recorded as 0,1,2,3,4.The number of positive cells were divided into five stages,<5%,5-25%,26-50%,56-75%,76-100%and the scores were recorded as 0,1,2,3,4.Multiplication is the total score.Interestingly,PRDX1 expression were dynamic in CRC different development stages.In the early stage of CRC happening,PRDXl expression was step by step encresed?F=58.054,P<0.001?;however,after tumor tumor lymph nodes metastasis,majority samples Appears PRDX1 expression receding?P<0.001?.1.2 Western blot and RT-qPCR showed that the expression of PRDX1 protein and mRNA in colorectal cancer tissues were higher than in normal colorectal tissue?P<0.001?.2.The impaction of PRDX1 on colorectal cancer cell biological behavior2.1.PRDX1 expression of protein and mRNA detection in rectal cancer cell lines SW480,SW620,HT29,HCT15,HCT116,LOVO,LS-174T,DLD-1,M5 showed the high expression of PRDX1 in SW480,lower expression in the SW620 by Western blot and RT-qPCR.2.2 Using lentiviral vector to construct stable over expression PRDX1 cell linesSW620-PRDX1 and stable interference PRDX1 cell line SW480-shRNAi-PRD-X1.Western blot and RT-qPCR proved the over expression and interference effect?P<0.001?.2.3 MTT assay shows:compared with control group,the growth of SW620-PRDX1 cell with PRDX1 overpression decreased markedly?P<0.001?;but the growth of SW480-shRNAi-PRDX1 cell with PRDX1 knockdown increased dramaticAlly?P<0.001?,2.4 As demonstrated in colony formation assay the colony number was 20±6.689 in SW620-PRDX1 and 34±14.850 in SW620-Vector?P<0.05?,however the colony number was 27±7.806 in SW480-shRNAi-PRDX1 and 63±10.185 in S W480-shRNAi-NC?P<0.001?,2.5 The results from Xenograft model in nude mice exhibited that,the growth of tumor from CRC cells with PRDX1 overexpression decreased?P<0.05?,Ki67 positive rate of tumor?31.2±6.301%?was lower than that in the control group?69.6±9.787%??P<0.05?;but the growth of tumor from CRC cells with PRDX1 knockdown increased?P<0.05?,Ki67 positive rate of tumor?42.2±11.882%?was higher than control group?22.2±6.496%??P<0.05?.2.6 The results from transwell migration assay showed that,the migration cell numbers of CRC cells with PRDX1 overexpression was 73.6±24.453,ecreased markedly compared with the control group 120±24.453?P<0.001?;but the migration cell numbers of CRC cells with PRDX1 knockdown was 71.6±29.497 increased dramatically compared with the control group 26.3±9.036?P<0.001?.2.7 The results from scratch wound healing assay displayed that the speed of wound healing of CRC cells with PRDX1 overexpression slower than that in the control group,the speed of wound healing of CRC cells with PRDX1 knockdown faster than that in the control group.2.8 transmission electron microscopy results shows:compared with control group,CRC cells with PRDX1 overexpression mitochondrial swelling were obvious improved;otherwise,CRC cells with PRDX1 knockdown mitochondrial swelling were obviously compared with control group.2.9 H₂O₂ oxidative stress model of CRC cells Constructed to observe the useness of PRDX1 in oxidative stress progressingFirstly,we confirmed that the 80?M/L?100?M/L was the optimum concentration for SW620,SW480 cells;then,optimum reaction time 18h 24h for each cells were confirmed by MTT.Then,flow cytometry detected the cell cycle distribution revealed that overexpression PRDX1 SW620 cell displayed an increased percentage of cells in G1-phase and cells in G2-phase were decreased compared with the control group?P<0.05?,similarly,when exposed to H₂O₂,SW620 cells in G1-phase keeping increased and cells in S-phase decreased significantly?P<0.001?;on the other hand,knockdown PRDX1 SW480 cell displayed an decreased percentage of cells in G1-phase and cells in S,G2-phase increased compared with control group?P<0.05?;after exposed to H₂O₂,the results are consistent.All data shows that whether exposed to H₂O₂ or not,oevrexpress PRDX1 might induced retardation of cell G1-phase,further more suppress the proliferation of CRC cells.At last,flow cytometry detected the cell apoptosis revealed that PRDX1 mainly influenced early apoptosis?Q4?but not late apoptosis?Q2?of CRC cells SW620 and SW480.3.The gene chip results showed that the number of difference genes of stable interference PRDX1 colorectal cancer cell line was 1096 compared with control group FC?Fold change?>1.5,p<0.05,including 452 up-regulated genes,644 down-regulated genes.Enrichment analysis was implemented according to the information of KEGG and BIOCARTA all pathway genes with the P value sorting,and top ten major tumor related pathway were tumor related pathway?Pathway in cancer?,actin cytoskeleton pathway?Actin cytoskeleton?,cell invasion and migration?Transedothelial magratration?,cells cycle?Cell cycle?,cell adhesion?Focal adhesion?,oocyte maturation related pathways?Oocyte meiosis?pathway etc.Four genes BIRC5,FN1,FOS,MAPK9 were selected in the largest correlation pathway cancer gene,and Western blot tests validate that three differences genes BIRC5,FOS,MAPK9 protein expression consistent with the gene chip,reminds that the results of gene chip is reliable.4.Co-IP and western blot results displayed that MAPK9 and PRDX1 can combine together further more activate MAPK signaling pathway and then influence the tumor progressing of CRC cells.Conclusion1.The high expression of PRDX1 existed in colorectal cancer tissues,and related with the evolution process of colorectal cancer and pathological staging and grading.2.Over expression of PRDX1 can inhibit the proliferation and invasive ability of colorectal cancer;knockdown PRDXlcan promote proliferation and invasiveness of colorectal cance.3.PRDX1 can influence the progression of tumor cells through its antioxidant effect.4.PRDX1 involves in multipe cancer related signaling pathway,and play an important role via combine with MAPK9 and activate MAPK signaling pathway.