EGFL6 Promotes The Development Of Colorectal Cancer Through AKT/ERK Signaling Pathway

BackgroundColorectal cancer is the most common malignant tumor of digestive system in clinic.According to statistics,the incidence and mortality of colorectal cancer in the United States has ranked third in all malignant tumors.the symptoms of many patients are not obvious,often found in the middle and late stages of cancer,and accompanied by a large degree of metastasis and infiltration?colorectal cancer is currently the fourth leading cause of cancer death worldwide,and cancer metastasis is the key and important cause of death of patients with colorectal cancer.Reducing the invasive and metastatic ability of tumors through effective targets in the early stage of tumorigenesis is also the key to the current research of medical workers.In recent years,the occurrence of colorectal cancer in China has shown an obvious upward trend,which may be related to changes in the dietary structure of Chinese people and the aging of the population of the First Fund for Colorectal Cancer Census.It is of great significance to study the molecular mechanism of occurrence and regulation in patients with colorectal cancer for the treatment and diagnosis of colorectal cancer.the cause of colorectal cancer is not clear,which may be related to the patient's heredity,colorectal polyps,carcinogens damage,intestinal inflammatory damage,radiation damage and other adverse factors,but how these factors cause the occurrence and development of colorectal cancer remains unclear.Many studies have reported that the abnormal expression of oncogenes or tumor suppressor genes is an important regulator of the occurrence and development of colorectal cancer.These genes are involved in the regulation of the growth,invasion and other functions of cancer cells.Their abnormal expression is often related to the prognosis,progress and metastasis of cancer patients.At present,medical workers have been working to find molecular markers closely related to the malignant progression and occurrence of colorectal cancer,and to find effective molecular targets for the treatment of colorectal cancer by analyzing its role in the function of colorectal cancer cells.This is of great significance for the molecular mechanism of colorectal cancer and the early diagnosis and treatment of colorectal cancer patients.The occurrence and progression of malignant tumors is a complex multi-step process,which is regulated by a network of multiple genes in tissue cells.These genes together affect the growth,cloning,cycle progression,invasion,apoptosis and other biological functions of cancer cells.Currently,surgical resection is the main treatment for cancer.Radiotherapy and chemotherapy are adjuvant therapy.In recent years,with the continuous progress of molecular biology,molecular targeted therapy has gradually become a hot spot in cancer treatment.It has high accuracy and little side effects.Actively searching for effective target genes is a hot research topic for medical workers.EGFL6 is a hot research molecule in oncology,it is a member of the EGF duplicate superfamily,the repetitive motifs of EGF are generally 30 to 40 amino acid residues,it contains the conserved structure of cysteine and glycine,it is believed to be involved in the interaction between proteins and proteins.At present,about 70 different kinds of EGF superfamily proteins have been found,which are involved in blood coagulation,fibrinolysis,cell adhesion and so on.EGFL6 contains two N-linked glycosylation sites,three hydroxylation sites and ATP/GDP binding sites in the MAM domain,and one tyrosine phosphorylation site,which can be used as a kinase substrate to phosphorylate EGFL6 to regulate its expression.EGFL6 is related to the progression of melanoma.It has been proved that EGFL6 silencing can effectively inhibit the proliferation and invasion of melanoma cells.EGFL6 promotes the migration and invasion of breast cancer cells,and can also increase the growth rate of breast cancer in vivo,and blocking EGFL6 can significantly reduce the occurrence and migration of cancer cells.The expression of EGFL6 in tumor-related endothelial cells was higher than that in normal ovarian tissues.EGFL6 was involved in the formation of tumor-related blood vessels,and the activation of EGFL6 was related to the activation of AKT signaling pathway.The expression of EGFL6 was not only abnormally high in cancer tissues,but also higher in plasma of patients with 7 oral squamous cell carcinoma than that of healthy controls.The expression of EGFL6 was positively correlated with distal metastasis and TNM stage of patients with oral squamous cell carcinoma.At present,there is no study of EGFL6 in colorectal cancer,and the role of EGFL6 in the biological characteristics of colorectal cancer cells is not clear.The involvement of oncogenes or tumor suppressor genes in cancer progression is a very complex process,which results from the co-transduction of multiple genes and signals.Oncogenes can often induce tumorigenesis by activating downstream signal transduction that promotes tumorigenesis,while anti-oncogenes often play an anti-tumorigenic role by downregulating downstream signal transduction that promotes tumorigenesis.It is of great significance to study the molecular mechanism of oncogenes or tumor suppressor genes in tumorigenesis for the treatment of tumors.At present,the mechanism of EGFL6 has been studied,EGFL6 is a multifunctional regulator,which can play a variety of biological roles by regulating the transduction of various intracellular signals.EGFL6 can affect the angiogenesis and migration of endothelial cells by activating ERK signaling pathway.Overexpression of EGFL6 can provide phosphorylation levels of ERK and AKT proteins in cells.EGFL6 can positively regulate the activation of ERK and AKT signals.In the progress of malignant tumors,it is not clear what mechanism EGFL6 affects the malignant tension of cancer cells.MAPK is a family of protein kinases widely existing in eukaryotic cells.It contains many signal transduction pathways,among which ERK pathway is involved in cell proliferation,differentiation and invasion.ERK contains two subfamilies,ERK1 and ERK2,and is a dual specific protein kinase.Activated ERK can activate downstream target proteins by acting and existing with substrates in cytoplasm or nucleus,thus regulating cell proliferation,apoptosis and differentiation.ERK signaling pathway can activate growth factors,cell stress,cytokines and so on,which affect the activation and phosphorylation of protease by regulating these factors,thus participating in the normal physiological or pathological process of the body.In normal physiological state,ERK mostly exists in the cytoplasm,and it is activated to enter the nucleus after being stimulated by the outside world to play a biological role.AKT signaling pathway is a regulator with many functions in human tissues.There are many effector molecules downstream of AKT signaling pathway.AKT phosphorylation can activate downstream molecules,thus affecting cell apoptosis,growth and metastasis.Current studies on AKT have found that AKT is over-activated in cancer tissues,and targeting inhibition of AKT signaling pathway is also an effective way to treat cancer so far.Activation of AKT can promote angiogenesis,metastasis and cell cycle.ObjectiveThis experiment explored the expression difference of EGFL6 in colorectal cancer tissues and cells,and down-regulated or up-regulated the expression of EGFL6 through cell transfection.We also explored the role of EGFL6 in the growth,invasion,cycle,apoptosis and angiogenesis of colorectal cancer cells,and its role in the growth of transplanted tumors in nude mice,and preliminarily explored its mechanism for targeted treatment of colorectal cancer,to provide new target genes for targeted therapy of colorectal cancerMethods(1)Cancer tissues and corresponding adjacent tissues were collected from 54 patients with colorectal cancer.The adjacent tissues were taken about 10 cm away from the cancer tissues.The patients did not receive radiotherapy and chemotherapy before operation.Thirty-six of the patients with colorectal cancer were male and eighteen were female,of which 29 were over 50 years old,25 were under 50 years old,23 were in TNM stage I-II and 31 were in stage III-IV.The expression of EGFL6 in cancer tissues and adjacent tissues was detected by qRT-PCR.The expression of EGFL6 protein in cancer tissues and adjacent tissues was determined by immunohistochemistry.The correlation of EGFL6 expression level with age,sex,TNM stage,and histological grade was analyzed.(2)Human colorectal cancer cell lines SW480,SW620,HT29,SW1116 and normal human intestinal epithelial cell line FHC were selected,and the expression of EGFL6 in cells was determined by qRT-PCR.(3)pcDNA3.1-EGFL6 and EGFL6 siRNA were transfected into colorectal cancer cells.Western blot was used to detect the expression of EGFL6 in cells.Cell proliferation was measured by MTT.Cell cycle distribution was measured by flow cytometry with PI monochrome staining.Plate cloning assay was used to determine cloning formation ability.Cell invasion and migration were measured by Transwell chamber.Angiogenesis was measured by angiogenesis test.Annexin V-FITC/PI double staining flow cytometry was used to detect apoptotic changes.(4)Western blot was used to detect the expression of p-AKT,p-ERK,AKT and ERK in human colorectal cancer cell lines SW620,HT29 and normal human intestinal epithelial cell lines FHC.(5)Colorectal cancer cells were treated with AKT signal inhibitor MK-2206 and ERK signal inhibitor U0126.Western blot was used to detect the expression of p-AKT,p-ERK,AKT and ERK proteins.Cell proliferation was measured by MTT and cell cycle distribution was determined by PI monochrome flow cytometry.Plate cloning assay was used to determine cloning formation ability.Cell invasion and migration were measured by Transwell chamber.Angiogenesis was measured by angiogenesis test.Annexin V-FITC/PI double staining flow cytometry was used to detect apoptotic changes.(6)Western blot was used to detect the expression of p-AKT,p-ERK,AKT and ERK in colorectal cancer cells after overexpression and downregulation of EGFL6.(7)Colon cancer cells were infected with EGFL6 shRNA lentivirus and inoculated subcutaneously into nude mice.The growth volume and quality of transplanted tumors in nude mice were measured.(8)The transplanted tumors of nude mice were harvested,the expression of EGFL6,p-AKT,p-ERK,AKT and ERK was detected by Western blot.Results(1)Immunohistochemistry showed that the expression of EGFL6 in colorectal cancer was higher than that in normal adjacent tissues(P<0.05).The expression level of EGFL6 in cancer tissues was significantly higher than that in adjacent normal tissues(P<0.05).The expression of EGFL6 was related to TNM stage and histological grade in patients with colorectal cancer.It has nothing to do with the age and sex of the patients.(2)The level of EGFL6 gene in human colorectal cancer cell lines SW480,SW620,HT29 and SW1116 was significantly higher than that in normal human intestinal epithelial cell line FHC(P<0.05).(3)The level of EGFL6 protein in colorectal cancer cells transfected with pcDNA3.1-EGFL6 increased significantly(P<0.05).Transfection of EGFL6 siRNA could significantly inhibit the expression of EGFL6 protein in cells(P<0.05).The proliferation,cloning,invasion,migration and angiogenesis of colorectal cancer cells overexpressing EGFL6 were significantly increased,the proportion of S phase was significantly increased,and apoptosis was significantly reduced(P<0.05).The proliferation,cloning,invasion,migration and angiogenesis of colorectal cancer cells with EGFL6 down-regulated were significantly reduced,the proportion of G0/G1 phase cells was significantly increased,and the apoptosis was significantly increased(P<0.05).(4)The levels of p-AKT and p-ERK in human colorectal cancer cell lines SW620 and HT29 were significantly higher than those in normal human intestinal epithelial cell lines FHC(P<0.05).(5)The levels of p-AKT and p-ERK decreased,cell proliferation,cloning,invasion,migration and angiogenesis decreased,the proportion of G0/G1 phase cells increased and cell apoptosis increased in colorectal cancer cells after AKT signal inhibitor MK-2206 and ERK signal inhibitor U0126(P<0.05).(6)The levels of p-AKT and p-ERK in colorectal cancer cells overexpressing EGFL 6 increased(P<0.05).Downregulation of EGFL6 decreased p-AKT and p-ERK levels in colorectal cancer cells(P<0.05).(7)The growth volume and quality of subcutaneous transplanted colorectal cancer cells infected with EGFL6 shRNA lentivirus were significantly reduced(P<0.05).(8)The levels of EGFL6,p-AKT and p-ERK in subcutaneous transplanted colorectal cancer cells infected with EGFL6 shRNA lentivirus decreased(P<0.05).Conclusions(1)EGFL6 is highly expressed in colorectal cancer tissues,and its expression level is related to TNM stage and histological grade of patients with colorectal cancer.(2)EGFL6 can positively regulate the activation of AKT/ERK signaling pathway in colorectal cancer cells.(3)Inhibiting AKT and ERK signaling pathways reduces the proliferation,cloning,invasion,migration and angiogenesis of colorectal cancer cells,and promotes cell apoptosis.(4)Downregulation of EGFL6 can inhibit the growth of transplanted colorectal cancer cells in nude mice by inhibiting AKT/ERK signaling pathway.