The Study Of Abnormal Expression Of PRRX1 On Promoting Stemness And Chemoresistance Of Colorectal Cancer

Background and Objectives:Colorectal cancer(colorectal cancer,CRC)is one of the most common malignant tumors in the world,and a major disease leading to tumor-related death,seriously endangering people’s lives and health.Chemoresistance is an important reason accouting for the poor prognosis of CRC patients,but its underlying mechanism is still not clear.Paired related homeobox 1(PRRX1)is a member of the cogene-type family of homologous box transcription factors.Studies have reval that PRRX1 play a crucial role in regulating the cell viability and stemness.This study aim to confirm the role and underlying mechanism of PRRX in tumor stemness maintaining and chemoresiscance of CRC.Methods1.Abnormal expression of PRRX1 promoted stemness and chemotherapy resistance in colorectal cancera.The expression of PRRX1 in cancer and adjacent tissues of colorectal cancer patients was detected respectively by collecting clinical tissue samples and paraffin sections of colorectal cancer tissue,and the relationship between the expression of PRRX1 and the development of colorectal cancer was analyzedb.Various methods including cck-8 cell activity detection,EdU incorporation assay,cell cycle analysis and in vivo tumorigenicity experiments were performed to investigate the effect of abnormal PRRX1 expression on the proliferation of colorectal cancer cellsc.Phere-forming experiments and plate cloning experiments were applied to investigate the effect of abnormal expression of PRRX1 on the stem properties of colorectal cancer cells in vitro.d.Western blot and cell immunofluorescence assay were applied to determine the influence of abnormal PRRX1 expression on stemness markers of colorectal cancer cellse.Apoptosis detection,drug IC50 experiment and in situ tumor drug model in nude mice were applied to elaborate the relationship between the abnormal expression of PRRX1 and the sensitivity of colorectal cancer cells to 5-fluorouracil and oxaliplatin.2.PRRX1 activates the IL6/JAK2/STAT3 axis to promote stemness acquirement in colorectal cancer cella.The gene expression profile information of clinical colorectal cancer tissue samples was collected in GEO database,and the enrichment of PRRX1 and IL6/JAK2/STAT3 signaling pathway was analyzed through the enrichment of GSEA signaling pathway.b.Western blot,cellular immunofluorescence and real-time fluorescence quantitative PCR were applied to determine the changes of IL6/JAK2/STAT3 signaling pathway after exogenous changes in the expression level of PRRX1c.Interference with IL6 expression was performed in vitro to determine the role of IL6/JAK2/STAT3 signaling pathway in PRRX1-mediated stemness and chemoresistance of colorectal cancer cells.Results1.High expression of PRRX1 promotes stemness and chemoresistance of CRC cellsAccording to the CRC patients’ clinical informatiom,we found that abnormal expression of PRRX1 was closely related to the tumor metastasis,chemoresistance and poor prognosis of CRC patients.Gain-and loss-function assay in vitro revealed that high expression of PRRX1 promoted the proliferation,stemness properties and chemoresistance of colorectal cancer cells.Further studies also determined that down-regulation of PRRX1 would enhance the chemotherapeutic sensitivity of CRC cells and reduce rate the liver metastasis in vivo.2.PRRX1 enhances tumor stemness and chemoresistance of CRC cells via IL6/JAK2/STAT3 axis.According to the gene set enrichment analysis,the high expression of PRRX1 in CRC cells was positively associated with the activation of JAK2/STAT3 signaling.Further analysis of data from GEO GSE18088,GSE39582 dataset and clinical CRC samples from our hospital suggested that PRRX1 promrted the transcription level of IL6.Further studies revealed that PRRX1 dramatically increased the transcription,intracellular and extracellular expression of IL6,promoting the JAK2/STAT3 signaling and the expression of its downstream(BCL2,CCND1),as well as the cleavage of PARP.The publicly available bioinformatics algorithm JASPAR suggested that IL6 was one target gene of PRRX1.Further luciferase assay confirmed that PRRX1 bound to the promoter of IL6 at Δ(585-592)site.Disturbing the expression of IL6 in PRRX1-high expression SW480 cell line by using vector with short hairpin RNA(shRNA)targeting IL6 dramatically inhibited JAK2/STAT3 signaling and reversed PRRX1-mediated stemness and chemoresistance of CRC cells.ConclusionPRRX1 is tightly associated with the tumor metastasis,chemoresistance and prognosis in CRC patients.High expression of PRRX1 promotes stemness acquirement and chemoresistance in CRC cells through JAK2/STAT3 signaling by targeting 116.Thereby,PRRX1 may be a valid biomarker for predicting the effect of chemotherapy and prognosis of CRC patients.