Study On The Value Of MiR-7-5p And Raf-1 In Early Screening Of Colorectal Adenoma And Adenoma Carcinogenesis

Objective:Colorectal cancer(CRC),as one of the common malignant tumors,has a high morbidity and mortality rate,and is still a major disease threatening human physical and mental health.In addition,the early condition of CRC is hidden and difficult to detect.Studies have shown that most of the development of CRC has undergone the development process of "normal mucosa-adenoma-adenocarcinoma",that is,colorectal adenoma(CA)is considered to be a precancerous lesion,and at least 80%of CRC evolved from CA.Early colonoscopy and removal of colorectal polyps can reduce the incidence of CRC.However,because colonoscopy is an invasive procedure and the population of our country is large,it cannot be a routine screening method for the time being.In addition to colonoscopy,the currently commonly used screening methods include stool occult blood test,stool immunochemical test,blood-borne biomarkers carcinoembryonic antigen(CEA),Septin9,CA199,etc.,but the sensitivity and specificity are slightly poor,And there are few studies on the value of detection in adenoma and early CRC.This research group has proved the potential value of miR-7-5p in the diagnosis and treatment of colorectal cancer in the early stage.However,domestic and foreign studies have not involved miR-7-5p in the process of colorectal adenoma and carcinogenesis.And whether it can play the role of early clinical reminder.Therefore,this study will further explore the application value of miR-7-5p in colorectal adenoma and early cancer screening and targeted therapy.Methods:Collect the intestinal tissues of patients who were examined in the Digestive Endoscopy Center of the First Affiliated Hospital of Kunming Medical University from October 2019 to May 2020 and completed the carcinoembryonic antigen(CEA)routine screening in this hospital at the same time,and divided them into normal groups(20 cases),Adenoma group(20 cases),colorectal cancer group(20 cases);target gene prediction and bioinformatics analysis to explore the potential mechanism of miR-7-5p in colorectal cancer;Real-time fluorescent quantitative PCR(RT-qPCR)and Western blot methods were used to detect the expression levels of miR-7-5p and Raf-1 in normal mucosa,colorectal adenoma tissue,and colorectal cancer tissue.And analyze the expression level of CEA in three tissues;Spearman correlation analysis explores the relationship between miR-7-5p and Raf-1 expression in normal intestinal mucosal tissues,adenoma tissues,and adenocarcinoma tissues;Receiver operating characteristic curve(ROC curve)analyzes the potential ability of CEA,miR-7-5p and Raf-1 single/combination as early diagnosis of colorectal adenoma and canceration markers.Results:(1)Raf-1 is one of the potential target genes of miR-7-5p.KEGG signaling pathway enrichment analysis results show that miR-7-5p target genes are mainly involved in cGMP-PKG signaling pathway,axon guidance,ErbB signaling pathway,mTOR signaling pathway,etc.Among them,the enriched colorectal cancer pathway(number:hsa05210)contains the Raf gene.(2)The results of qRT-PCR and western blot techniques showed that the expression of miR-7-5p in normal intestinal tissues,adenoma tissues,and adenocarcinoma tissues gradually decreased(P<0.01),and Raf-1 expression gradually increased(P<0.01));CEA expression difference between normal group and adenoma group was statistically significant(P<0.5),CEA expression difference between normal group and adenocarcinoma group was statistically significant(P<0.001);adenoma group and adenocarcinoma group did not Statistical difference(P>0.5);(3)The expression of miR-7-5p and Raf-1 in normal intestinal mucosa tissue,colorectal adenoma tissue,and colorectal adenocarcinoma tissue is negatively correlated(r=-0.839,P<0.001);(4)The diagnostic value of ROC curve analysis for colorectal adenoma:the area under the miR-7-5p curve AUC=0.955(P<0.001,95%CI=0.867～1.000)(Figure 5A),the area under the Raf-1 curve AUC=0.920(P<0.001,95%CI=0.812～1.000),the area under the CEA curve AUC=0.743(P<0.05,95%CI=0.577～0.909)(Figure 5B);miR-7-5p and Raf-1 combined diagnosis Area under the curve AUC=0.951(P<0.001,95%CI=0.858～1.000);miR-7-5p and CEA combined diagnosis area under the curve AUC=0.958(P<0.001,95%CI=0.877～1.000);Raf-1 and CEA combined diagnosis area under the curve AUC=0.934(P<0.001,95%CI=0.840～1.000);miR-7-5p,Raf-1 and CEA combined diagnosis area under the curve AUC=0.958(P<0.001,95%CI=0.877～1.000)(Figure 6).(5)The diagnostic value of ROC curve analysis for distinguishing colorectal adenoma from adenocarcinoma:area under the miR-7-5p curve AUC=0.816(P<0.01,95%CI=0.668～0.964)(Figure 7A);under the Raf-1 curve Area AUC=0.865(P<0.001,95%CI=0.747～0.984);Area under the CEA curve AUC=0.633(P<0.05,95%CI=0.452-0.814)(Figure 7B).The area under the curve for the combined diagnosis of miR-7-5p and Raf-1 AUC=0.868(P<0.001,95%CI=0.748-0.989);the area under the curve for the combined diagnosis of miR-7-5p and CEA AUC=0.792(P<0.001,95%CI=0.645-0.940);Raf-1 and CEA combined diagnosis area under the curve AUC=0.898(P<0.001,95%CI=0.799～0.997);miR-7-5p,Raf-1 and CEA Area under the combined diagnosis curve AUC=0.895(P<0.001,95%CI=0.794～0.996)(Figure 8).Conclusion(s):(1)Raf-1 is one of the potential target genes of miR-7-5p.KEGG signaling pathway enrichment analysis results show that miR-7-5p target genes are mainly involved in cGMP-PKG signaling pathway,axon guidance,ErbB signaling pathway,mTOR signaling pathway,etc.Among them,the enriched colorectal cancer pathway(number:hsa05210)contains the Raf gene.(2)The expression of miR-7-5p in normal intestinal mucosal tissue,colorectal adenoma tissue,and colorectal adenocarcinoma tissue is gradually down-regulated,and the expression of Raf-1 is gradually up-regulated,and there are statistical differences in the expression between each group,indicating that both can be used as a specific indicator for early diagnosis of colorectal adenoma and adenocarcinoma.(3)miR-7-5p and Raf-1 may be potential targets for the treatment of colorectal cancer patients including KRAS mutation patients.(4)There were statistical differences in the serum CEA expression levels between normal population and patients with intestinal adenoma and colorectal adenocarcinoma,but there was no statistical difference in CEA expression levels between patients with colorectal adenoma and colorectal adenocarcinoma.(5)ROC curve analysis results show that compared with CEA,miR-7-5p and Raf-1 have higher diagnostic value in the early screening of colorectal adenomas and the detection of adenoma carcinogenesis,and the diagnostic value is higher when multiple indicators are combined.