PART Ⅰ:Development Of Novel DNAJB6/KIAA1522/p-mTOR Three-protein Prognostic Prediction Models For CRC PART Ⅱ:Effects Of High Expression Of MAP4 On The Malignant Phenotype Of Colorectal Cancer Cells And Related Mechanisms

Background:Globally,the incidence and mortality of colorectal cancer(CRC)are at the forefront of malignant tumors,and accurate prognosis judgment is the key to improve the survival rate of patients.At present,TNM stage system is widely used for tumor staging and prognosis prediction of CRC.However,CRC is highly heterogeneous at the molecular level,and there may be significant differences in the prognosis of patients with the same clinicopathological features,so molecular markers are expected to play an important role in improving the accuracy of prognosis in patients with CRC.This study aims to evaluate the value of expression levels of DNAJB6,KIAA1522,and p-mTOR proteins in the prognosis of CRC and to establish a valid prognostic model for patients with CRC.Methods:Firstly,the expression of DNAJB6,KIAA1522,and p-mTOR(Ser2448)proteins in 329 CRC specimens was detected by immunohistochemistry,and the relationship between their expression and clinicopathological parameters was analyzed.Next,the patients were randomly divided into a training set(n=230)and validation set(n=99),and the prognostic value of the three proteins in the training set was evaluated by the Kaplan-Meier method,univariate and multivariate Cox proportional hazard regression models,and the prognostic nomograms model integrating the three proteins and TNM stage system were constructed.Subsequently,we used the Receiver operating characteristic(ROC)curve,The concordance index(C-index),the calibration curve,and the Decision curve analysis(DCA)to evaluate the predictive power of the nomogram in the training and validation cohorts.Results:The expression of DNAJB6,KIAA1522,and p-mTOR in CRC tissues was significantly upregulated(P<0.01),and their expression levels were independent prognostic factors for overall survival(OS)and disease-free survival(DFS)(both P<0.05).The area under the ROC curve and the C-index values are approximately 0.7.The findings of the calibration curve demonstrate that the model’s projected values and actual measurements coincide rather well.The DCA curve indicates that the nomogram model has a higher clinical benefit than the TNM stage system.Overall,the predictive model has satisfactory consistency,sensitivity,specificity,and clinical practicability.Conclusion:High expression of DNAJB6,KIAA1522,and p-mTOR are independent influencing factors for the poor prognosis of colorectal cancer.Nomograms integrating DNAJB6,KIAA1522,p-mTOR,and TNM stage systems can accurately provide prognostic information for colorectal cancer patients and are expected to become new monitoring tools for colorectal cancer prognosis.Background:Colorectal cancer(CRC)is a world-wide epidemic malignant tumor of the digestive system,with the highest incidence and mortality.The invasion and metastasis of tumor cells to surrounding tissues is an important reason for the poor prognosis of advanced patients,and the discovery of key genes that regulate colorectal cancer invasion and metastasis and the analysis of their biological functions have become a research hotspot in the field of CRC.Objective:To investigate the effect of microtubule-associated protein 4(MAP4)on the malignant phenotype of colorectal cancer cells and related molecular mechanisms.Methods:Immunohistochemistry was used to detect the change of MAP4 protein expression in CRC tissues,and Chi-square test and Fisher precision test were used to explore the correlation between MAP4 protein expression and clinicopathological parameters in CRC patients.The Kaplan-Meier method and Cox risk proportional regression model were used to explore the correlation between MAP4 expression level and the prognosis of CRC patients.Transfect small interfering RNA(siRNA)and short hairpin RNA(shRNA)to colorectal cancer cell DLD1 and HCT116 by silencing MAP4.Recovery experiments were performed in HCT116 and DLD1 that stably knocked down MAP4 with an overexpressed plasmid vector(labeled MAP4-R)synonymously.The effects of MAP4 on the malignant phenotype of DLD1 and HCT116 cells were detected by CCK8 cell proliferation and viability experiments,cell colony formation experiments,wound healing experiments,and Transwell experiments.Western blot was used to detect changes in the expression of MAP4 and related downstream molecular proteins.Results:Tissue microarray and immunohistochemical testing in 496 colorectal cancer patients showed significant upregulation of MAP4 in colorectal cancer tissues(P<0.001).We found that MAP4 expression was associated with local lymph node metastasis(P=0.009),local invasion depth(P<0.001),and histopathological grade(P<0.001)and pathologic TNM stage(P=0.002)were closely related,and Cox proportional hazards regression analysis suggested that MAP4 may be an independent prognostic factor in colorectal cancer patients(overall survival,P=0.015;disease-free survival,P=0.020).Next,we selected DLD1 and HCT116 colorectal cancer cell lines with high expression of MAP4 to explore the effect of MAP4 on the malignant phenotype of colorectal cancer,and found that the proliferation ability of cells did not change significantly after silencing MAP4,the wound healing and invasion migration ability of tumor cells were significantly reduced.The response experiment showed that after exogenous overexpression of MAP4 in cells with stable knockdown of MAP4,the invasion and migration ability of cells was restored.Molecular level testing has found that interference with MAP4 can lead to downregulation of phosphorylation levels of ERK and S6.Conclusion:MAP4 is abnormally high expressed in CRC and is an independent predictor of OS and DFS in patients,and its high expression may enhance the invasion and migration capacity of CRC cells by activating ERK and S6-related signaling pathways.