The Effect Of DDR1 On The Evolution Of Colorectal Cancer And Its Molecular Mechanism In Metabolic Reprogramming

Background: Colorectal cancer is one of the most common malignant solid tumors of the gastrointestinal system,which seriously affects people's health.It has been observed that the progression from inflammatory bowel disease to adenoma and then to adenocarcinoma in clinical research and practice.This evolution from normal cells to cancer cells is necessarily accompanied by changes in the expression of multiple genes and changes in cellular metabolic pathways.Previous studies have demonstrated that discoid domain receptor 1(DDR1)is a member of a novel receptor type protein tyrosine kinase family,which plays an important role in regulating cell proliferation,metabolism,migration and other biological behaviors.Objective: The purpose of this study was to reveal the relationship between DDR1 and cell grade and clinical stage of colorectal cancer,and to explore the molecular mechanism of DDR1 affecting energy metabolism programming and key enzymes of energy metabolism in tumor cells at cytological level and animal experimental level,so as to lay a foundation for targeted therapy of colorectal cancer.Methods: In this study,clinical characteristics and tissue samples of 126 patients with colorectal cancer were collected.The expression of DDR1 protein in cancer and adjacent tissues was detected by immunohistochemistry,and the expression score was calculated.DDR1 knockout mice were constructed using CRISPR/Cas9.Induction of a murine model of colorectal cancer by a dosing pattern of azoxymethane sequential dextran sodium sulfate.The disease activity index of the model mice was calculated.The number of neoplasms in the intestinal wall was recorded,and the histological score and Ki67 index were determined.Serum levels of inflammatory cytokines IL-1?,TGF-? and TNF-? were detected by ELISA.Western blot was used to detect the expression of proinflammatory and protumoral proteins TGF-?,?-SMA and COL1A1.Lo Vo cell lines with overexpression and low expression of DDR1 a were constructed by lentivirus mediated overexpression and knockdown techniques.The cell proliferation activity of the two groups was measured by CCK-8 method,and growth curves were drawn to detect the sensitivity of the two groups of Lo Vo cells to5-fluorouracil.Mito Tracker CMTMRos staining was used to show the fluorescence intensity changes of DDR1 a overexpression and low expression Lo Vo cells.Oxygen consumption rate and extracellular acidification rate were measured by Seahorse XF extracellular flow detector.The activity of three key metabolic enzymes(phosphofructokinase,hexokinase,and pyruvate kinase),as well as the intracellular lactic acid content,were determined by colorimetry.The expression levels of PI3 K,p AKT,PKM2 and other proteins related to energy metabolism were detected by Western blot,and the correlation between DDR1 and energy metabolism programming of tumor cells was analyzed.Results: The results showed that the expression score of DDR1 in colorectal adenocarcinoma was significantly higher than that in adjacent tissues(P<0.01).The positive expression rate of DDR1 in poorly differentiated colorectal cancer was significantly higher than that in moderately differentiated and well differentiated adenocarcinoma.The high expression group of DDR1 had high p T and p N stage,vascular invasion and high clinical AJCC stage.In the mouse model of colorectal cancer,DDR1 knockout mice had lower disease activity index,number of intestinal wall tumors,histological score and Ki67 index than wild-type mice.The levels of IL-1?,TGF-?,TNF-? in serum and the expression of protumoral proteins TGF-?,?-SMA and COL1A1 in DDR1 knockout mice were lower than those in wild-type mice(P<0.01).Cell proliferation experiments showed that the lentivirus-mediated DDR1 a knockdown of Lo Vo cells decreased proliferation rate and increased sensitivity to 5-fluorouracil-induced apoptosis.Mitotracker staining intensity of mitochondria was nearly 3 times higher than that of controls.The oxygen consumption rate and extracellular acidification rate of Lo Vo cells knocked down by DDR1 a were reduced by mitochondrial pressure and glycolytic pressure.The activity of pyruvate kinase in DDR1 a knockdown Lo Vo cells decreased(P<0.01),while the activity of phosphofructokinase and hexokinase did not change significantly(P>0.05).The expression levels of PI3 K,p AKT,PKM2 and other proteins in DDR1 a knockdown Lo Vo cells decreased,and the intracellular lactic acid content in this group was significantly higher than that in the control group(P<0.01).Conclusion: The expression level of DDR1 is related to the clinical prognosis of colorectal cancer patients.DDR1 promotes the progression of inflammatory-adenoma-colorectal cancer.DDR1 a knockdown inhibited the proliferation of Lo Vo cells and increased their sensitivity to 5-fluorouracil.The expression of DDR1 a not only affects the proliferation-apoptosis balance of Lo Vo cells by mediating the PI3K/AKT/Bcl2 signaling pathway,but more importantly,inhibition of DDR1 a can reduce the activity of pyruvate kinase,promote intracellular lactic acid accumulation and reduce the energy supply of tumor cells by down-regulating the PI3K/AKT/PKM2 signaling pathway.DDR1 a affects Lo Vo cell proliferation and energy supply patterns by reprogramming energy metabolism.