1.the Effect And Mechanism Of Prdx2 On The Cycle And Autophagy Of Colorectal Cancer 2.the Effect And Mechanism Of Sestrin2 On The Cancer Stem Cell-Like Phenotype Of Colorectal Cancer

RESEARCH 1 THE EFFECT AND MECHANISM OF PRDX2 ON THE CYCLE AND AUTOPHAGY OF COLORECTAL CANCERObjective: Colorectal cancer is a malignant tumor occurring worldwide.Studies had shown that PRDX2 was associated with tumor function such as proliferation,cell stemness,and chemotherapy resistance in colorectal cancer.However,there were no reports of PRDX2 and cell cycle and autophagy in colorectal cancer,and the relationship between P38 pathway and PRDX2 in colorectal cancer was still unclear.Therefore,this study explored whether PRDX2 affects the cycle and autophagy of colorectal cancer cells through the P38 pathway.Methods:1.The TCGA database data was used to investigate the expression of PRDX2 in various tumors and the relationship between colon cancer andrectal cancer and tumor pathology.2.The data(GSE81429)in GEO database was analysed,which collected high-throughput sequencing results of colorectal cancer cell lines HT-29 and SW480 transfected with Control-siRNA and PRDX2-siRNA,respectively.After converting the data,comparing with the human genome and counting the expression,differentially expressed genes was analyzed and screened and enriched using KEGG pathway analysis.3.The lentiviral system was used to transfect the sh-RNA vector,in order to knock down PRDX2 in HCT116 and HT-29 cells,and stable transfectants were selected.CCK-8 experiment was used to detect cell proliferation;flow cytometry was used to detect cell cycle.Western Blot was used to detect the expression of cell-cycle-related proteins P21 and P27.4.In HCT116 and HT-29 cell lines,immunofluorescence was used to stain the LC3 B protein between the negative control group(NC)and PRDX2 knockdown group(sh-PRDX2).Aggregation of LC3 protein and the formation of autophagosomes were observed.HCT116 was transfected with Cherry-GPF-LC3 plasmid,and the state of intracellular autophagy was observed by red and green fluorescence.Transmission electron microscopy was used to observe the formation of autophagosomes in the NC group and sh-PRDX2 group.Western Blot was used to detect the expression of autophagy-related proteins LC3 B,SQSTM1 / P62 andBeclin1.5.Western blot was used to detect MAPK pathway-related proteins in control group(NC)and PRDX2 knockdown group(sh-PRDX2),including P38,ERK,JNK,and p-P38,p-ERK and p-JNK.After being treated with Dehydrocorydaline chloride(DHC),the P38 agonist,the protein expressions of pathway proteins P38,p-P38;downstream pathway FoxO;cyclin P21 and autophagy protein LC3 were detected again.6.Using colon cancer patient sample data in TCGA database to analyze the mRNA expression relationship between PRDX2 and cell cycle,autophagy-related genes.7.The tumor was subcutaneously implanted into the distal cecum of the nude mice.After 4 weeks,the tumor was removed and Western Blot was used to detect the expression of cyclin P21 and P27;autophagy proteins SQSTM1 / P62,LC3 B and Beclin1.Results:1.In the TCGA database,PRDX2 is highly expressed in colon cancer(COAD)and rectal cancer(READ),and compared with other tumors,the differential expression of PRDX2 in colorectal cancer is more obvious.PRDX2 is also differentially expressed in aggressive breast cancer(BRCA),hepatobiliary carcinoma(CHOL),stem cell adenocarcinoma(LIHC),lung squamous cell carcinoma(LUAD),prostate cancer(PRAD),and endometrial cancer(UCEC).PRDX2 is highly expressed in all pathologicalconditions of patients with colorectal cancer.2.In the GSE81429 dataset of the GEO database,173 genes were found to be significantly differentially expressed after transfection of PRDX2-siRNA in HT-29 and SW480 cell lines.Enrichment analysis suggests that differential genes are enriched in cellular functions such as ribosome(Ribosome)and FoxO signaling.Among them,the FoxO signaling pathway suggests that PRDX2 may affect cell cycle progression and autophagic flow through the P38 MAPK / FOXO signaling pathway.3.In the PRDX2 knockdown group of colorectal cancer cell lines,cell proliferation was inhibited.After knocking down PRDX2,the cell cycle progression was blocked in S phase.In the PRDX2 knockdown group,the expression of cell cycle inhibitory proteins P21 and P27 increased.4.Compared with the control group(NC),the fluorescence accumulation spots of LC3 B autophagosomes in the PRDX2 knockdown group(sh-PRDX2)were reduced.In PRDX2 knockdown group,green fluorescence increased among red and green fluorescence proteins.In the PRDX2 knockdown group,SQSTM1 / P62 protein expression increased,LC3 II / I protein ratio decreased,and Beclin1 protein expression decreased.5.In HCT116 cell,compared with the control group(NC),the protein expression of p-P38,the key molecule of the P38 pathway,was reduced in the PRDX2 knockdown group(sh-PRDX2).In the PRDX2 knockdown group and the control group,after treatment with the P38 activatorDehydrocorydaline chloride(DHC),compared with the untreated group,the pathway protein p-P38,cell cycle P21 protein,and autophagy LC3 II / I protein ratio were all partly restored.6.In the data of the colorectal cancer patient sample set,the mRNA level of PRDX2 is correlated with the mRNA level of the cycle gene CCNA2(r = 0.3367,p = 2.759e-07),CCNB1(r = 0.3964,p = 9.014e-10),CDK1(R = 0.405,p = 3.6e-10),CDK4(r = 0.4098,p = 2.116e-10);and autophagy-related genes BECN1(r =-0.374,p = 8.8846e-09)and ULK1(r=-0.3251,p = 7.336e-07)level correlation.7.In the mouse orthotopic tumor model,PRDX2 knockdown suppressed the formation of colorectal cancer tumors and increased the expression of cell cycle inhibitory proteins P21 and P27,the expression of autophagy promoting protein Belin1 was reduced,and the expression of autophagy inhibitory protein SQSTM1 / p62 increased,LC3 II / I protein ratio decreased.Conclusion: The results of this study suggest that PRDX2 may promote the cycle progression and autophagy of colorectal cancer cells by activating the p38 MAPK pathway.RESEARCH 2 THE EFFECT AND MECHANISM OF SESTRIN2 ON THE CANCER STEM CELL-LIKE PHENOTYPE OF COLORECTAL CANCERObjective: Colorectal cancer is one of the common tumors in China.The studies found that Sestrin2 inhibited cell migration and invasion in colorectal cancer.However,whether Sestrin2 affects the stemness of colorectal cancer cells through the Wnt pathway has not been reported.Therefore,this study explored whether Sestrin2 affects the stemness of colorectal cancer cells through the Wnt pathway.Methods:1.Sestrin2 expression in colon and rectal cancer data was obtained and compared from the TCGA database.2.A lentiviral vector was constructed to overexpress Sestrin2 in colorectal cell lines HCT116 and SW620 and establish a control group(LV-GFP)and Sestrin2 overexpression group(LV-Sestrin2).Stable strains were select.The CCK-8 assays were used to measures cell proliferation.The cloning tests were used to detect cloning ability,and the wound healing assays were used to test tests cell migration ability.3.In colorectal cancer cell lines HCT116 SW620 cells,the sphere formation test was used to detect the sphere-formation ability,and the flow cytometry was used to detect the percentage of CD44 positive cells,q PCR and Western blot was used to detect the stem cell-like phenotype and stemness transcription factors expression,including CD44,Oct4,Sox2 and Cxcr4,between the control group(LV-GFP)and Sestrin2 overexpression group(LV-Sestrin2).4.Western blot was used to detect the protein expression of β-catenin,the key protein in the Wnt pathway,and downstream factor C-Myc.The cells were treated with BML-284,the Wnt pathway activator,and the sphere formation test was used to observe the sphere-forming ability of the cells treated with the Wnt pathway activator.Western blot was used to detect the stem cell-like phenotype protein CD44 and the key Wnt pathwayβ-catenin And downstream factor C-Myc protein expression.5.In the mouse xenograft model,the size of the subcutaneous tumors was compared between the control group and the Sestrin2 overexpression group,and Western blot was used to detect the stem cell-like phenotype Sox2,the Wnt pathway key protein β-catenin and downstream factor C-Myc Protein in subcutaneous transplanted tumor.Results:1.In the TCGA database,the expression of Sestrin2 both in colon cancer and rectal cancer samples was lower than that adjacent in normal tissues.2.In the colorectal cell lines HCT116 and SW620,compared with the control group(LV-GFP),the proliferative ability,clone forming ability,and migration ability in the Sestrin2 overexpression group(LV-Sestrin2)were all reduced.3.Compared with the control group(LV-GFP),the sphere-forming ability was reduced and the proportion of CD44 expressing cells was decreased in the Sestrin2 overexpression group(LV-Sestrin2).The expression of protein and m RNA of stem-related molecules Sox2,Oct4,Cxcr4,and CD44 were reduced.4.In the Sestrin2 overexpression group,Wnt pathway key proteinβ-catenin and downstream factor C-Myc protein were decreased.The Wnt pathway activator BML-284 partially recovered sphere-forming ability.The expression of the stem cell-like phenotype protein CD44,the key protein of Wnt pathway β-catenin and the downstream factor C-Myc were also partially restored.5.In vivo,compared with HCT116-GFP cells,the tumor formation of HCT116-Sestrin2 cells was decreased significantly,while the expression of Sox2,C-Myc,β-catenin protein in Sestrin2 overexpression group was reduced.Conclusion: The results of this experiment suggest that Sestrin2 may suppress the stem cell-like phenotype of colorectal cancer cells by down-regulating the Wnt pathway.