Study On Clinical Significance And Pathway Molecules Of ARF6 In Colorectal Cancer

Background:Colorectal Cancer(CRC)is a common malignant tumor of the digestive system,which is a serious threat to human health.Although the survival period of CRC patients has been effectively prolonged with advances in technologies such as surgery,radiotherapy,chemotherapy,and molecular targeted therapy,but in general,the long-term efficacy is not satisfactory,and the 5-year survival is still only 50%.Therefore,the study of its malignant progression mechanism has always been a hot spot in the field of tumor research.ADP ribosylation factor 6(ADP ribosylation factor 6,ARF6)is a small GTPase binding protein in the ras-related G protein family.In recent years,many studies have found that ARF6 can mediate a variety of ways to regulate the proliferation of tumor cells,closely related to the invasion and metastasis of a variety of malignant tumors.However,there is currently no research report on ARF6 in CRC.In order to clarify the role of ARF6in the malignant progression of CRC,this topic is divided into three parts to explore the expression and clinical significance of ARF6 in CRC,preliminary exploration of Arf6upstream regulatory factors in CRC,and further through online database analysis ARF6passage molecules prognostic significance in the CRC.Part Ⅰ The expression of ARF6 in colorectal cancer and its clinical significance1.Objectives:To clarify the expression of ARF6 in colorectal cancer and its relationship with clinicopathological characteristics and prognosis of colorectal cancer.2.Methods:Changhai Hospital collected 136 cases of colorectal cancer with complete data and clear clinical pathological diagnosis.All cases were treated with paraffin embedded tissue.Observation of CRC pathological morphological characteristics and invasive transfer conditions using Hematoxylin-eosin staining;According to the standard,determine histological grade,TNM staging,and tumor budding degree.The expression of ARF6 and Ki-67 protein was detected by immunohistochemistry.The postoperative and treatment status of patients were collected by telephone follow-up.3.Results:The expression of ARF6 in CRC tumor tissues:ARF6 is selectively and highly expressed in CRC tumor tissues,mainly in the cytoplasm and cell membrane,and is positively expressed in yellow to brown particles.The expression level of ARF6 in CRC tumor tissue is closely related to lymph node metastasis and TNM stage(P<0.05),and there was no significant correlation between the expression level of ARF6 with the patient’s age,gender,tumor site,Ki67 proliferation index,concurrent adenoma,family history,tumor size,gross tumor type,tumor differentiation,tumor mucus composition,invasion depth,accompanied by distant metastasis,tumor budding,and cancer nodules(P>0.05).The survival curve of ARF6 high expression cases and the low expression group did not find significant differences,and Log-Rank test showed no statistical significance between the two groups.(χ~2=2.608,P=0.106).4.Conclusion:The high expression of ARF6 in colorectal cancer tissues is significantly correlated with lymph node metastasis and TNM staging.Part Ⅱ:Study on Upstream Regulatory Factors of ARF6 in Colorectal Cancer1.Objectives:Exploring the possible mechanism of promoting the expression of ARF6 protein in CRC.2.Methods:The expression of mutant P53 protein in 136 cases of CRC was detected by immunohistochemistry,and the K-Ras,N-Ras,and BRAF gene mutation were detected by fluorescence quantitative PCR.The difference in expression of Arf6 protein between K-RAS,N-RAS,BRAF gene mutation and wild-type CRC cases was analyzed.The transcription factor,binding site,and bonding element(Motif)combined with an EPD eugenic promoter database predicts the ARF6 promoter region.CRC cell lines infection with KLF4 adenovirus,the regulation effect of KLF4 on ARF6 expression was detected by Western blot.3.Rresults:(1)Among 136 cases of CRC patients,there were 20 cases of wild-type P53expression(14.71%),59 cases of missense mutant expression(43.38%),and 57 cases of nonsense mutant expression(41.91%).There was no statistical significance between ARF6protein expression level and P53 gene mutation(χ~2=0.258,P=0.611).(2)Among 136 cases of CRC tumor tissue,50 cases(36.76%)of RAS gene mutation were detected,including 47 cases of KRAS gene mutation and 3 cases of NRAS gene mutation.There was no statistical significance between the expression level of ARF6protein and the mutant status of RAS gene(χ~2=0.563,P=0.453).(3)Among 136 cases of CRC tumor tissues,7 cases(5.15%)of BRAF gene mutation were detected.There was no statistical difference between the expression level of ARF6protein and the mutation status of BRAF gene(χ~2=0.007,P=0.653).(4)Bioinformatics analysis showed that there are 3 KLF4 binding sites in the promoter region of ARF6,and the binding element sequence is AGGGTGGGGC.In CRC tissues,KLF4 inhibits ARF6 protein expression.4.Conclusion:In CRC tissues,KLF4 inhibits ARF6 protein expression,and the molecular mechanism may be that KLF4 binds to the ARF6 promoter region and plays a transcriptional inhibitory role,thereby inhibiting ARF6 protein expression.Part Ⅲ Prognostic Significance of ARF6 Pathway Molecules in Colorectal Cancer1.Objectives:To explore the relationship between family molecules,activating molecules,inhibitory molecules and effector molecules in ARF6 pathway and prognosis of CRC patients.2.Methods:ARF6 pathway molecules are divided into five categories:family genes(ARFs),Guanine nucleotide exchange factors(GEFs),GTPase-activating proteins(GAPs),effectors and ARF6 Inhibition of ligand-receptor pairs(SLITs-ROBOs).Use String online tool to construct protein-protein interaction(PPI).By analyzing the overall survival rate or disease-free survival rate of the GEPIA database,and further verifying it with the Human Protein Atlas database,to verify the protein expression in CRC tissues.3.Rresults:(1)GEPIA database analysis showed that ARFs family molecules have no significant correlation with the prognosis of CRC patients.(2)GEPIA database analysis shows that IQSEC3/GEP100 in the ARF6 GEFs family is related to the overall survival rate of patients with colorectal cancer;Human Protein Atlas online database does not have IQSEC3/GEP100 data information.(3)GEPIA database analysis shows that SMAP1 and AGAP3 in the ARF6 gap family are related to the overall survival rate of colorectal cancer patients,and AGAP1 is related to the disease-free survival rate of colorectal cancer patients;Human Protein Atlas online database further confirms that SMAP1 and AGAP3 are effective in colorectal cancer patients The prognosis has an important influence on the molecule.(4)GEPIA database analysis shows that MAPL8IP3/JIP3 in the ARF6 effector family is related to the disease-free survival rate of patients with colorectal cancer;Human Protein Atlas online database further confirms that MAPL8IP3/JIP3 is an important prognostic molecule for CRC patients.(5)GEPIA database analysis showed that SLIT1 is related to the overall survival rate of CRC patients in the SLITs-ROBOs family;Human Protein Atlas online database does not have SLIT1 data information.4.Conclusion:SMAP1,AGAP3,MAPL8IP3/JIP3 in ARF6 pathway molecules are prognostic markers for CRC patients.